NOŢIUNI DE SĂNĂTATE PUBLICĂ, EPIDEMIOLOGIE ŞI …...1“Lucian Blaga” University of Sibiu...

INTERNATIONAL CONFERENCE

“THE BIENNALE SIBIU – CHIŞINĂU”,

organized by

Pediatric Clinic Sibiu, Faculty of Medicine,

“Lucian Blaga” University of Sibiu, România

and

IMSP, Mother and Child Institute,

“Nicolae Testemiţanu” State University of Medicine and

Pharmacy,

Chişinău, Republic of Moldova

September 8-9th 2017

Sibiu, Pediatric Clinic, 2-4, Pompeiu Onofreiu street

ACTUALITIES IN PEDIATRICS

TABLE OF CONTENTS


“THE BIENNALE SIBIU-CHISINAU”

ORGANIZERS:

Ministry of Health, Romania;

LBUS -“Lucian Blaga”University Sibiu,

Pediatric Clinic, Sibiu, Romania;

SUMP - “Nicolae Testemiţanu” State

University of Medicine and Pharmacy,

PMSI-Mother and Child Institute,

Chisinau, Republic of Moldova;

National Public Health Institute - Public

Health Centre of Sibiu;

Center of Research and Telemedicine in

Neurologic Diseases in Children Sibiu

ORGANIZING COMMITTEE:

Honorary President – Professor Florian-

Dorel Bodog, Ministry of Health, Romania;

Vice-presidents:

Professor Claudiu Kifor, vice-

rector of LBUS;

Professor Mihai-Leonida Neamţu,

vice-president of Romanian

Society of Pediatrics;

Professor Ninel Revenco, vice-head

of Research, Transfer of

Technology and Innovation

Department, PMSI-Mother and

Child Institute, President of

Republic Society of Pediatrics,

Republic of Moldova;

Professor Carmen Domnariu, Head

of Public Health Centre of Sibiu;

ORGANIZING COMMITTEE MEMBERS:

Assistant professor Corina Cazan,

Pediatric Clinic Hospital Sibiu, Chief of II

Pediatric Department, LBUS;

Assistant professor Luminiţa Dobrotă,

Pediatric Clinic Hospital Sibiu, Chief of I

Pediatric Department, LBUS;

Assistant professor Bogdan Neamţu,

Pediatric Clinic Hospital Sibiu, Chief of

Research Department, LBUS;

Raluca Costea, MD, researcher, Pediatric

Clinic Hospital Sibiu;

Instructor Ioana Octavia Mătăcuţă-

Bogdan, MD PhD, senior specialist,

Pediatric Clinic Hospital Sibiu, LBUS;

Assistant professor Berghea-Neamţu

Cristian-Stefan, Pediatric Clinic Hospital

Sibiu, Chief of Pediatric Surgery

Department, LBUS;

Professor Mihaela Maria Cernuşcă

Miţariu, Dean, Faculty of Medicine, LBUS

SCIENTIFIC COMMITTEE MEMBERS:

Professor Carmen Domnariu, Head of Public

Health Centre of Sibiu;

Professor Mihai-Leonida Neamţu, LBUS;

Professor Ninel Revenco, SUMP;

Assistant profesor Corina Cazan, LBUS;

Assistant professor Luminita Dobrotă, LBUS;

Assistant professor Bogdan Neamţu, LBUS;

Assistant profesor Berghea Neamtu Cristian Stefan,

LBUS.

SPONSORS: Nestlé Romania, SunWave

Pharma Romania.


“THE BIENNALE SIBIU – CHIŞINĂU” 1. HEALTH PROMOTION THROUGH

INTERVENTIONS REGARDING HEALTHY

FOOD AND PHYSICAL EDUCATION IN

CHILDREN

Carmen Daniela Domnariu

……………………………...1

2. GUT MICROBIOTA, TYPE 2 DIABETES

MELLITUS, LOW-GRADE SYSTEMIC

INFLAMMATION. ETIOPATHOGENIC,

CLINIC AND THERAPEUTIC

CORRELATION

Cristian-Ştefan Berghea-Neamţu

………………………...3

3. THE ROLE OF GUT MICROBIOTA IN

CARBOHYDRATES DEGRADATION

Luminiţa Dobrotă

……………………………………….10

4. CLINICAL PECULIARITIES OF

CEREBROVASCULAR ACCIDENTS IN

NEWBORNS, INFANTS AND CHILDREN OF

SMALL AGE

Svetlana Hadjiu, Mariana Sprincean, Nadejda Lupusor,

Cornelia Calcii, Ramina Pasari, Vladimir Iacomi, Alina

Bantas, Andriana Gruzinschi, Ninel Revenco

………....13

5. GUT MICROBIOTA AND CENTRAL

NERVOUS SYSTEM IN HEALTH AND

AUTOIMMUNE DISEASES REVIEW

Mihai Bogdan Neamţu, Andreea Barbu, Ionela

Maniu.....17

6. TETANY OR DIAGNOSTIC PITFALL

TABLE OF CONTENTS

Raluca Maria Costea, Bogdan Neamţu

…………………22

7. GUT MICROBIOTA AND FOOD ALLERGY

Corina Cazan

…………………………………………...25

8. MICROBIOTA AND THE IMMUNE SYSTEM,

A COMPLEX LIAISON

Ioana Mătăcuţă-

Bogdan.………………………...............28

9. RISK FACTORS FOR BRONCHIOLITIS IN

INFANTS AND THE ROLE OF FECAL

MICROBIOTA PROFILES

Olga Cîrstea, Oxana Turcu, Ninel Revenco

…………….32

10. GUT MICROBIOTA AND IRRITABLE

BOWEL SYNDROME

Corina Cazan

………………………………………….…35

11. THE IMPACT OF ANTIBIOTICOTHERAPY

ON GUT MICROBIOTA IN CHILDREN

Oxana Turcu, Olga Cirstea, Ala Holban, Ninel Revenco

…………………………………………………………..

38

12. LEFT INGUINAL HERNIA AND

CRYPTORCHIDISM. PARTICULARITIES OF

TREATMENT AND EVOLUTION. CASE

REPORT

Cristian-Ştefan Berghea-Neamţu

…………….………....41

INTERNATIONAL CONFERENCE “THE BIENNALE SIBIU – CHIŞINĂU”


AMT, vol. 22, no. 3, 2017, p. 1

HEALTH PROMOTION THROUGH INTERVENTIONS

REGARDING HEALTHY FOOD AND PHYSICAL EDUCATION

IN CHILDREN

CARMEN DANIELA DOMNARIU

1

1“Lucian Blaga” University of Sibiu

Keywords: health

education, health

promotion, children, interventions

Abstract: Childhood obesity is one of the most serious public health issues of the 21st century. The

problem is global and prevalence has increased at an alarming rate. According to a report issued by the

World Health Organization, in 2015, the number of overweight children under the age of five was estimated to be over 42 million worldwide.(1) In response to these alarming rates, the World Health

Organization member states have agreed on strengthening and coordinating their efforts to implement

strategies to improve nutrition and physical activity at states’ level. Romania, as well, has understood

the importance of increasing the percentage of children and adolescents who live a healthy lifestyle.

1Corresponding author: Carmen Daniela Domnariu, Str. Luptei, Nr. 21, Sibiu, 550330, România, E-mail:[email protected], Phone +40269

212812

ACTA MEDICA TRANSILVANICA- SUPPLEMENT September 2017;22(3):1-2

INTRODUCTION

The importance of healthy nutrition and regular

physical activity has been well established.(2) Children who are overweight and obese during their childhood are most likely to

remain obese into adulthood, too and more likely to develop

noncommunicable diseases, such as diabetes and cardiovascular

diseases at a younger age. Overweight and obesity, as well as their related diseases, can be prevented. Therefore, prevention of

childhood obesity needs high priority.(1)

Strategies targeted to behaviour change include:

increase physical activity, promote breastfeeding, increase fruit and vegetable consumption and reduce television viewing-time.

Such strategies have been elaborated and revised regularly by

the health authorities at every state’s level. The USA have

issued the Dietary Guidelines for Americans, which are a science-based diet and physical activity recommendations to

promote health and prevent illnesses.(3)

At European level, there has been drawn up a School-

based Nutrition Education Guide, which places healthy nutrition education in the health-promoting school.(4)

In Romania, in 2016, the Ministry of Health, through

the Presidential Administration, has launched the Multiannual

Integrated Plan to Promote Healthcare and Education for Health, which aims to constantly increase in the next 5 years the

proportion of the population with healthy behaviours, especially

among children. The document reflects the Health 2020

principles that form the basis of the Romanian National Health Strategy 2014–2020, thus strengthening the cooperation between

Romania and WHO/Europe.(5)

One of Romania’s experiences in increasing primary

prevention services in the field of healthy eating and physical activity among children and adolescents has been materialized

in a national level strategic project, “The increasing access to

primary medical prevention services for children and adolescents from Romania. The healthy nutrition and physical

activity among the children and adolescents from Romania”.(6)

The project was earned through competition within the

Norwegian Cooperation Programme with Romania and Bulgaria, funded by the Norwegian Government through

Innovation Norway and co-financed by the Ministry of Health

and ran from August 2009 and April 2011.

The aim of the project was to build capacity for implementing interventions for healthy nutrition and physical

activity in children and adolescents (age: 3 – 19) at national and

local level.

The project had has as main partners the Romanian Ministry of Health – as the project promoter – and the National

Institute of Public Health through the Regional Centre of Public

Health of Sibiu – as the implementation unit. The institutional

partners were: the Public Health Institute of Oslo, Norway, the Education, Research, Young People and Sports Minister from

Romania and the Romanian Association of Health Psychology.

In order to accomplish the objectives of the European

Strategies on Nutrition, Physical Activity and Health, the project targeted many levels, from the individual one – the child, to

family, school, health professionals and the community (figure

no. 1).

Figure no. 1. Targeted levels in changing behaviours

The project was aimed at children aged between 3 and

19 years old and addressed the field of promotion of healthy eating and physical activity.

The project has four main components.



AMT, vol. 22, no. 3, 2017, p. 2

The first component, the research component, started

from the analysis of the current situation regarding the school

children behaviours, which was accomplished through the Health Behaviour in School-aged Children (HBSC) study, following the methodology established by the World Health

Organization and validated for Romania in the previous research

carried out in 2005.(7) This component also included a social marketing

analysis, targeting children and families, to understand why they

are acting like this and what was wrong. In this research, there

have been identified four unhealthy behaviours among children: lack of breakfast, reduced intake of fruits and vegetables,

drinking juice instead of water and low physical activity level.

There have also been pilot interventions and schools and

kindergartens to test the appropriateness of the tools developed within this project.

The targeted behaviours were: to drink water instead

of juice; to eat 5 portions of fruits, vegetables daily; to eat

breakfast daily and to have at least one hour of vigorous physical activity /day.

The second component aimed at public awareness

campaign, and included TV and radio spots, printed materials, a

website of the project etc. All these actions developed within this component aimed at empowering the child, the family,

physician, the teacher to lead a healthy lifestyle. The most

important campaign, LIFE, is a model of an interdisciplinary

and interinstitutional approach to make aware the population of the importance of changing the unhealthy behaviours.

The third component, capacity building, related to the

institutional development at local levels through the eight

facilitators employed within the project. There have been signed local partnerships among stakeholders, and there have been

developed local action plans for healthy nutrition and physical

activity in children. Also, this component targeted micro-

projects which took place in schools and kindergartens, as well as coaching and training for local stakeholders.

The capacity building component has issued a

communication tool, namely the Intervention Guidelines at

Community Level, designed to be a model of good practice for

defining the implementing the changing behaviours campaigns

regarding healthy nutrition and physical activity (figure no. 2).

Figure no. 2. Intervention guidelines at community level –

communication tool

The fourth component, the legislative component, addressed

proposals of amendments to the legislative framework to support the sustainable implementation of changing behaviour

patterns at local, county and national levels.(6)

The actions and activities developed within this

project have been materialized in 42 partnership agreements signed among stakeholders, county actions plans developed in

all the counties of the country and endorsed by the local

stakeholders, and more than 500 micro-projects that started in

schools and kindergartens with a small financing from the project and a local contribution.

The project was a success for our country

taking into account the target population, the good subject, the

good international evidence and the good transfer of results to the institutions. Also, it was awarded (Best Poster Award),

following the presentation in Budapest, Hungary at the

Conference “Action for Prevention”, held on the occasion of the

Hungarian Presidency of the European Union, in May 2011 (an Expert Level Conference on Member States’ Activities on

Nutrition, Physical Activity and Smoking Related Health Issues,

Jointly Organized and Co-Financed by The Hungarian

Presidency of the Council of The European Union and the European Commission May 30-31, 2011). But, as any other

projects of such size, it also had weaknesses: the project

duration, of only 20 months, no time to implement the micro-

projects, no outcome evaluation, just process evaluation. This project represented a first step in approaching

and developing primary prevention politics regarding the

healthy nutrition and physical activity in children in Romania.

REFERENCES 1. World Health Organization. Global Strategy on Diet,

Physical Activity and Health. Childhood overweight and

obesity. http://www.who.int/dietphysicalactivity/childhood/en/

Accessed on 17.07.2017.

2. Macera CA. Division of Nutrition and Physical Activity

National Center for Chronic Disease Prevention and Health

Promotion Centers for Disease Control and Prevention.

Promoting healthy eating and physical activity for a

healthier nation. https://www.cdc.gov/healthyyouth/publications/.../pp-ch7.pd.Accessed on 16.07.2017.

3. Stang J, Story M, Kossover R. Guidelines for Adolescent

Nutrition Services Stang J, Story M (eds) Guidelines for

Adolescent Nutrition Services (2005) http://www.epi.umn.edu/let/pubs/adol-book.shtm.

Accessed on 18.07.2017.

4. Dixey R, Heindl I, Loureiro I, Pérez-Rodrigo C, Snel J,

Warnking P. Healthy Eating For Young People In Europe. School-Based Nutrition Education Guide; 1999.

5. http://www.euro.who.int/en/countries/romania/news/news/

2016/02/who-regional-director-visits-romania. Accessed on 10.07.2017.

6. Domnariu CD, Furtunescu F. Intervention pattern regarding

healthy nutrition and physical activity in Romania. Acta

Medica Transilvanica. 2011;2(1):143-144. 7. http://insp.gov.ro/sites/1/wp-

content/uploads/2014/11/Raport-HBSC-Romania-bun.pdf.

Accessed on 17.07.2017

https://www.cdc.gov/healthyyouth/publications/.../pp-ch7.pd.Accessed

https://www.cdc.gov/healthyyouth/publications/.../pp-ch7.pd.Accessed

http://www.epi.umn.edu/let/pubs/adol-book.shtm.%20Accessed%20on%2018.07.2017

http://www.epi.umn.edu/let/pubs/adol-book.shtm.%20Accessed%20on%2018.07.2017

http://www.euro.who.int/en/countries/romania/news/news/2016/02/who-regional-director-visits-romania.%20Accessed%20on%2010.07.2017



http://insp.gov.ro/sites/1/wp-content/uploads/2014/11/Raport-HBSC-Romania-bun.pdf

http://insp.gov.ro/sites/1/wp-content/uploads/2014/11/Raport-HBSC-Romania-bun.pdf



AMT, vol. 22, no. 3, 2017, p. 3

GUT MICROBIOTA, TYPE 2 DIABETES MELLITUS, LOW-

GRADE SYSTEMIC INFLAMMATION. ETIOPATHOGENIC,

CLINIC AND THERAPEUTIC CORRELATION

CRISTIAN-ŞTEFAN BERGHEA-NEAMŢU1

1“Lucian Blaga” University of Sibiu, Sibiu Pediatric Clinical Hospital

Keywords: gut

microbiota, metabolic

diseases, low-grade

inflammation, microbiome,

therapeutic

manipulation

Abstract: The gut microbiota plays an essential role in health promotion. Any kind of gut microbiota

ecosystem changes can trigger the low-grade inflammation, loss of insulin sensitivity, type 2 Diabetes

Mellitus. The low-grade inflammation is the common link for obesity, insulin resistance. On the other

hand, metabolic endotoxemia triggers the low-grade inflammation by lipopolysaccharides mechanism, which in turn plays a major role in the obesity linked diseases. Based on these metabolic interactions a

various studies have approached the concept of therapeutic manipulation of the microbiome with the

final scope of combating and preventing the metabolic diseases in humans. Although it is acknowledged

that the pre~ and probiotics use improves the gut microbial equilibrium and glucose homeostasis, this relationship needs to be extensively investigated. The further studies will allow identifying the

population at risk of metabolic diseases and the basic therapy focus needs to be adapted on individual

habits and predispositions.

1Corresponding author: Cristian-Ştefan Berghea-Neamţu, Str. Gh. Baritiu, Nr. 1-3, Sibiu, Romania, E-mail: [email protected] Phone:

+40722 641331


1. The essential role of gut microbiota in human health

In human gut are living trillions of microbes making

up what is known as gut microbiota (GM);(1) the colonization

with these microbes begins in the prenatal period, by mother to fetus transmission, which continues after birth, being modulated

by gestational age, the type of birth (natural/caesarean section),

nutrition (natural/artificial), hygiene and antibiotics exposure.(2)

GM community genes are involved in encoding of the basic functions too, such as digestion and degradation of

indigestible nutrients, immune system and host digestive tract

development and stimulation.(3-7)

GM generate signaling molecules, pharmacologically active ones, which interacts with host metabolism;(8-10) e.g.,

short chain fatty acids (SCFA) produced by dietary fiber

fermentation by intestinal bacteria coupled with G proteins

receptors (GPCRs) affects insulin sensitivity in adipocytes and peripheral organs too, thereby adjusting the energy

metabolism.(11) These transient changes of the intestinal

ecosystem take place all of the time, and can lead to GM-host

symbiosis interruption, sometimes.(12) Also, the intestinal ecosystem change can trigger some

disturbances, like: low-grade inflammation, metabolic disorders

(excessive lipids accumulation, loss of insulin sensitivity),

which create the risk of metabolic disease.

2. Type 2 Diabetes Mellitus (T2D) and gut microbiota

The recent advances in DNA sequencing allowed the

microbial communities collection related to human intestine.(13)

The GM changes are associated with different states, e.g., obese/weak people.(14) GM related to T2D has been recently

described, the specific bacterial sequencing being characteristic

to hyperglycemia.(15) Amar and collab. (16) concluded that 16S rSNA is

considered as a marker of T2D risk, reinforcing the concept

according to which the tissue bacteria are involved in human

diabetes mellitus onset, (16) also demonstrating that in the case of hyperglycemia due to lipids and insulin resistance the gut

bacteria are transposed in adipose tissue, as well as in blood,

where bacteria can induce the inflammation.(17) This bacterial

transposition can be reversed by probiotic treatment, with the

improvement of the global inflammatory status, finally.(17)

3. The gut microbial ecosystem changes in obesity GM is modified in obesity, proven fact by animal

studies and animal models, also. GM in weak wild mice vs.

obese ones (obese mice/leptin deficiency obese mice) attests the

Bacteroidetes/Firmicutes differences, Firmicutes/Bacteroidetes ratio being positively correlated with “obese” phenotype, diet

independent one.(18)

Turnbaugh and collab. (19) compared the GM of mice

vs. weak mice with obesity due to diet. They found the increase of Firmicutes, Mollicutes respectively, and the complete

reversibility of the compositional changes after returning of

normal diet. It has been suggested that the diet represents the

main factor contributory to GM changes related to obesity. The studies of Murphy and collab. (20) revealed the

increase of Firmicutes/Bacteroidetes ratio at obese mice/obese

mice fed with fat-rich diet vs. weak mice. This increasing was

significant in mice fed with fat-rich diet than obese mice. Ridaura and collab. (21) proved the existence of a

causal relationship between GM and obesity by feces samples

transplant from obesity co-discordant twins to separate mice

groups without germs, thereby demonstrating that: - the mice colonized with feces microbiota from obese co-

twins have a greater increase in weight and adipose tissue

vs. mice colonized with feces microbiota from weak co-

twins; - the obese mice held together with weak mice showed a

lower weight gain than mice held together with obese mice,

conjunctively with a GM compositional changes in the sense of a poor state of GM, related with Bacteroidetes

improvement, also with a protein expression improvement,

those involved in branched-chain amino-acids catabolism,

and SCFA production, finally.(21) The intestinal production of SCFA (although a calories

source for host) was especially associated with low-grade



AMT, vol. 22, no. 3, 2017, p. 4

systemic inflammation, and with the global positive metabolic

effects. (22,23)

These results show: - weak or obese murine model GM is largely influenced by

diet, and to a lesser extent by weak with obese mice

habitation;

- the effects of weak-obese habitation were highly transferable in mice without germs, with a big contribution

for a weak model preservation or for obesity onset.

In humans, the results of the studies of GM changes related

to obesity are controversial. Turnbaugh and collab. (19) observed: differences

between distal GM in obese vs. weak people, respectively

Bacteroidetes relative abundance together with weight loss of

individuals subjected to low-fat diet or low-carb one, with fewer calories (reduced Bacteroidetes/Firmicutes ratio in obese vs.

weak people due to more effective hydrolysis of undigested

polysaccharides along intestinal lumen, and, finally, with the

removal of more food calories and fats in obese).(11) Other studies, which compared GM structure in weak

people vs. obese, failed to compared obesity and

Bacteroidetes/Firmicutes ratio partnership.(24,25)

Recently, it has been suggested that GM in obese and weak people has a different response to the calories content.(26)

The nutrients absorption has caused a change in gut microbial

composition, only in weak people, not in obese, increasing the

Firmicutes relative abundance and decreasing the Bacteroidetes relative abundance simultaneous.(25)

Other study indicated the changes in GM related to

obesity and correlated with microbial genotype. Its decreasing

could play a role in host inflammatory status, also linked to obesity:

- the obese people with a great number of bacterial genes

have a higher proportion of species related to inflammatory

status (e.g. Firmicutes prausnitzii) and a lower proportion of species related to pro-inflammatory status (e.g.

Bacteroides spp.);

- the number of bacterial genes related to oxidative stress

was greater in people with a few number of bacterial genes

than people with a high number of bacterial genes;(26)

- because the difficulty of performing an interventional diet-

controlled study in humans the complex interaction

between diet, host age, environment and host genetic status is not fully understood.

Nevertheless, a recent report suggests that GM

alterations related with behavioral changes, eating habits

inclusively (27) and antibiotics use could be the main stimulus in pandemic obesity.(28,29)

GUT MICROBIOTA AND INSULIN RESISTANCE.

METABOLIC ENDOTOXEMIA (LPS HIGH SERUM

CONCENTRATION) AND LOW-GRADE

INFLAMMATION

The change in GM induces the low-grade

inflammation (LGI), which is the common link for chronic non-communicable diseases, obesity, insulin resistance and T2D,

inclusively. The metabolic endotoxemia trigger the LGI by

lipopolysaccharides (LPS) mechanism, LPS playing a major role

in the obesity linked diseases, (30-36) also in T2D onset, and CD14/toll like (TLR) dependent mechanism.(37)

The mice deprived by CD14 activity resist to fat or

LPS-rich diets. The binding of LPS to mCD145-TLR4 complex

on the surface of the innate immune cells activates the inflammation.(38,39) The pro-inflammatory stimulators (LPS,

lipids, fatty acids, chemokines) activates the intracellular

pathways of c-Jun N-terminal kinase (JNK) and lkβ kinase

(lKK-β):

- the lKK-β activation stimulates the transcription of nuclear

factors (NF-kB), increasing at the same time the expression of the inflammation mediators that can cause the insulin

resistance;

- the JNK activation induces the phosphorylation of the

insulin receptor substrate from serum sites (IRS-1) that inhibits the signal transduction through insulin receptor

axis, thus generating the insulin resistance.(40)

Therefore, the LPS accumulation (metabolic endotoxemia)

is related to inflammation and insulin resistance

CHRONIC INFLAMMATION BINDS INTESTINAL

MICROBIOTA TO OBESITY AND INSULIN

RESISTANCE In humans, the circulating endotoxin levels have 20 %

grown in obese and glucose intolerance people and 125 % in

T2D people by comparison with weak people.(41)

The circulating endotoxin values have also been

associated with high levels of adipocyte TNF-α and IL-6.(42) The LPS secretion, also the expression of TLR4, NF-

kB and 3 cytokine suppressor (SOC), are activated by fat or

carbohydrates-rich diet (not by fiber or fruits-rich ones), getting

involved in the pathways that regulate the insulin secretion.(42) Together, all these results show the important role of

LPS as inflammatory pathways mediator in obesity and obesity

related diseases. The aromatic amino-acids metabolites

(tyrosine, tryptophan, phenylalanine) can interact with the host signaling pathways, in this way affecting the host immunity.

The indole is one of the main tryptophan metabolites,

(43) produced by the action of bacterial tryptophanase (present

in Bacteroides thetaiotamicron, Proteus vulgaris, Escherichia coli).(44) The indole metabolism implies the hepatic sulphation,

with 3-indoxilsulphate production, consequently, or additional

metabolism, with related compounds production (indol-3-

piruvate, indol-3-lactate and indol-3-acetate).(45) All of whom give a broad perspective to tryptophan bacterial metabolism

impact on health damage and human diseases:

- 3-indoxylsulphate and indol-3-propionate interact with the

inflammation linked process in human host;(46)

- 3-indoxylsulphate activates the aryl hydrocarbon receptor

(AhR), with the regulation of IL-6 transcription and also

with the regulation of a several enzymes, compounds of

P450 complex (e.g. CYP1A1, CYP1A2, CYP2S1);(47) - 3-indol-propionate is a X pregnane receptor agonist (PXR),

with a benefic role in intestinal barrier function, either by

up-regulation of junctional protein expression or by

regulation of enterocytes TNF-α productivity; by reducing the intestinal barrier permeability, indol-3-propionate limits

the antigen and pathogen agents translocation, also the LPS

infiltration in blood stream; therefore, can reduce the

metabolic endotoxemia and the host inflammation.(48) Thereupon, an unaffected gut microbiota maintain the

host intestinal metabolic health by intestinal physiology

modulation mechanism; a disbiotic gut microbiota affects the host intestinal metabolic health by LPS infiltration; caloric

input, fat-accumulation and insulin reaction are also very

important. A health GM comprises an equilibrate symbionts

(health promotion bacteria) and pathobionts (disease promotion bacteria) representation.

As a partial conclusion, LGSI is the common element of obesity, insulin resistance and T2D. (49) LPS (also called endotoxins), derived by the membrane cells of gram-negative bacteria, initiate the inflammation related with the onset of obesity and insulin resistance, passes the intestinal mucosa at the level of junctional intestinal cells, and finally, by infiltration of



AMT, vol. 22, no. 3, 2017, p. 5

chylomicrons responsible for triglyceride and cholesterol intestine-plasma absorption, (50-52) reach in plasma: - plasmatic LPS infiltrates the liver and adipocyte tissues,

with the secondary innate immune response; (50)

- LPS link plasma LPS-binding protein (LBP), with the

secondary CD14 protein receptor activation in macrophages plasmatic membrane;(52)

- thus generated, the complex binds to toll-like 4 receptor

(TLR4) at the macrophages surface, triggering the

transduction signals which act the genes expression which encode the inflammatory effectors cells, such as NF-kB and

1 activator protein (AP-1);(52,53)

- LPS also adjust the nucleotide oligomerization`s type

receptors (NOD) from macrophage and dendritic cells, which collaborate with TLR for inducing NF-kB;

- LPS participate to the recruitment of other effector

molecules, such as leucine-rich protein (NLR), adaptor

protein (ASC) and 1-caspase (inflammasom compounds), and all, finally, activate the innate immune system (54)

After bacterial translocation in other tissues, LPS from

blood stream and some organs act inflammatory macrophages

signals and their link with insulin pathways transcription.

THERAPEUTIC POTENTIAL OF MANIPULATION

GUT MICROBIAL ECOLOGY

The studies of metabolic interaction, or of the

immunity between gut microbiota and host, also the way in which these interaction modulate the brain, muscle, liver and

intestine host activity motivate the concept of the

THERAPEUTIC MANIPULATION of the MICROBIOME,

with the final scope of combating and preventing the diseases in humans.(4,10) The selection of specific intestinal strains

and the improvement of the intestinal microbial ecology

represent a promising therapeutic approach for energy input control and for reducing the obesity and metabolic syndrome

prevalence. The pro~ and prebiotics use in the improvement of GM and obesity (and other metabolic diseases) relationship has been extensively investigated.(55) The probiotics are live microorganisms, used like food supplements, improve the gut microbial equilibrium and change the gut microbiota composition.(56) Some bacterial species, such as Bifidobacterium spp., have demonstrated that improve glucose homeostasis, with the following effects: weight loss, lipid-mass loss, glucose mediate insulin secretion restoration.

The probiotics are food ingredients which have a

good host influence, by selective growth stimulation and/or

limited number of gut bacteria activities. The prebiotics are oligosaccharides or short-chain polysaccharides, are found in

ordinary diet (vegetables, integral grain) or can be added in milk

and yogurt.

Fructosyl-oligosaccharides (FOS), inulin, galactosyl-oligosaccharides (GOS), inclusively, are transformed, via gut

microbiota, in SCFA which stimulate the selection of intestinal

commensal bacteria proliferation.(57-60) For example, inulin

stimulates the bifidobacteria, reduces the caloric input and fat-mass in experimental animals.(58)

The prebiotic stimulation regarding bifidobacteria

growth is correlate with increasing glucose tolerance, the

improvement of the insulin secretion induced by glucose and the normalization of murine inflammation.(61)

GOS also modulate the intestinal monosaccharides

absorption by stimulation of host monosaccharides transporters,

which in their turn stimulate glycolytic pathways, (59) reduce hepatic, renal and plasmatic lipids level in rodents.(57,58)

In healthy mice, GOS supplement decrease the hepatic

triglycerides level by some mechanisms: low lipogen enzyme

activity, fatty acids synthesis and microsomal triglycerides

transfer proteins involved in VLDL synthesis. (58,62)

Therefore, prebiotic input may decreases the lipogenic activity and increase the lipolytic one.

The increasing of lipolytic activity. In rodents, the effects of pre~ and probiotics on inflammation pathways, weight gain, glucose metabolism were largely attributed to SCFA production.(63)

SCFA interact with GPCR41 and GPCR43 from intestinal immune cells and promote the specific chemokine expression in intestinal epithelium.(64,65) SCFA repress NF-kB and affect the production of leukocytes inflammatory markers (Il-2, IL-10), (66) increase the satiety by increasing PYY synthesis, increase the epithelial cells pro-glucagon secretion by decreasing leptin expression.(67)

Other studies show that the probiotic effects on intestinal health and inflammation, too, are mediated by glucagon type proteins secretion (GLP-1 and GLP-2) in L enteric-endocrine cells.(60,68) Cani and collab (48) demonstrate that the obese mice/obese mice fed with carbohydrates-rich diet with oligofructose supplement present an increase of bifidobacteria and lactobacillus in intestine, an improvement of close junctions interconnection, a reduction of intestinal permeability, a reduction of systemic endotoxemia and systemic/hepatic inflammation; at the opposite pole stand obese mice/obese mice fed with carbohydrates-rich diet. These physiologic changes were correlated with GLP-2 levels and disappeared when the mice was treated with GLP-2 antagonist.(48) Another study stressed that a symbiotic treatment which combined polydextrose with B420 Bifidobacterium lactis decreased the abundance of Porphyromonadaceae in mice fed with fatty-rich diet.(69) This later food supplement is considered an effector of 17 T helper cells inhibition in the small intestine, and a marker of metabolic inflammation and T2D prevention.(69)

In humans, the probiotic intervention studies show a positive effects on glucose metabolism.(70) For example, a randomized, placebo-controlled, 6 weeks study shows in 60 healthy (Indians) subjects a decrease of glucose level and systemic insulin after ingestion of VSL #3 probiotic mixture.(71) However, the evidences of anti-obesity effects of prebiotics remain to be demonstrated. In humans, many studies show only moderate or not weight losses after prebiotics interventions.(72) The randomized controlled studies have identified surrogate markers of prebiotics treatment (plasmatic PYY, GLP-1, ghrelin), negative correlation with weight gain, impaired inflammation and glucose metabolism, which support the observed mechanisms in rodents.(73,74) On the other hands, does not exist evidences that can suggested if prebiotics supplement in infant formula improves growth or causes long-term side effects in infants, because the studies in children, adults or elders varies regarding quality and results. However, has been shown that probiotics modulate feces microbiota and the immune system in elders, and reduce metabolic syndrome markers level in overweight adults.(75-78) The effect of pre~ and probiotics concerning obesity and its pathology in humans requires further investigations; the studies using adequate doses of pre~ and probiotics, and controlled diets need to be carefully designed to be truly valuable to support the individual answers to different types of interventions and its dependence by microbial, genetics, environment and intestinal factors.

CONCLUSIONS

The proofs of strong contribution of gut microbiota at the appearance of obesity and metabolic diseases, in rodents and humans, are growing. The changes in gut microbiota ecology due to nutrient factors, antibiotics, pre~ and probiotics, observed both in rodents and humans, have been further highlighted the essential role of modulation of gut microbiota. Noting the fact of some host metabolic disorders related to inflammation-like composition of gut microbiota. However, the way in which the



AMT, vol. 22, no. 3, 2017, p. 6

extern factors (such as, diet, stress, age, drug use and circadian cycles) affect the intestinal microbial composition and the efficacy of microbial functions in rodents and humans is still unclear. It is necessary that future studies to promote analytic approaches from up to down on an epidemiological scale, and to integrate into questionnaires dietetic data, relevant environment factors (stress, circadian cycles influential factors), drug and antibiotics use history.

Need to be deepened those studies of intestinal bacterial functions related to physio-pathogeny of human obesity. In combination with animal experiments, the integration of epidemiological analysing will facilitate deciphering missing links of the metabolic chain that binds intestinal microbes to the host, and optimizing the therapeutic strategies for remodelling intestinal microbial ecology.

The further knowledge will allow to identify the population at risk of occurrence of metabolic diseases, and to concretize some perspectives for personalized medical assistance which will permit the adaptation of basic therapy focus on individual habits and predispositions.

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AMT, vol. 22, no. 3, 2017, p. 10

THE ROLE OF GUT MICROBIOTA IN CARBOHYDRATES

DEGRADATION

LUMINIŢA DOBROTĂ

1

1“Lucian Blaga” University of Sibiu, Pediatric Clinic, Research and Telemedicine Center in Neurological Diseases in Children,

Sibiu Pediatric Clinical Hospital

Keywords: gut

microbiota, carbohydrates

fermentation, energy

gain

Abstract: The gut microbiota can be considered as a “new organ” inside the body. Many carbohydrates

are fermented by gut microbiota, with energy gain and with the impact on health via its metabolites. This review focused on gut microbiota and responsible mechanisms for degradation, also fermentation

of polysaccharides. Gut microbiota will impact the glucose or lipid metabolism. It is a strong

relationship between gut microbiota, diet and metabolic diseases. The carbohydrates research open the

opportunities for treatment of obesity, type 2 diabetes mellitus and other metabolic disorders.

1Corresponding author: Luminiţa Dobrotă, Str. Pompeiu Onofreiu Street, Nr. 2-4, Sibiu, România, E-mail: [email protected], Phone:

+40722501145


INTRODUCTION

In humans, the gut microbiota is considered a “new

organ” inside the body, because its important role in the diseases preventing and in the maintaining health.(1)

The complex carbohydrates are degraded and fermented

by the human gut microbiota in sense of increased the whole

capacity of energy.

Figure no. 1. Energy intake and expenditure. Flint HJ.

Obesity and the gut microbiota. J Clin Gastroenterol 2011;

45:Suppl S128 - 32; http://dx.doi.org/10.1097/MCG.0b013e

31821f44c4; PMID: 21992951 [CrossRef], [PubMed], [Web

of Science ®], [Google Scholar]

On the other hand, the question about how much is the

impact of gut microbiota in the metabolism of energy in human

host is increasingly attractive. This question is relevant mostly in highly developed countries, where fat-rich diet in simple

sugars predisposes to metabolic disorders, such as obesity,

insulin resistance, and type II Diabetes mellitus.(2)

The polysaccharides are the most important class of components derived from diet which are metabolized by the gut

microbiota. The fermentation of carbohydrates originated from

diet produces short-chain of fatty acids (SCFA) which mediate

in turn the repercussions of gut microbiota on host energetic metabolism.(3)

Several recent studies have demonstrated the

relationship between gut microbiota-induced obesity and the

increased output of energy provided by diet.(2,4) Coming back to SCFA, butyrate is certainly an

important SCFA component, as well as the others (propionate

and acetate), which contribute as substrate in liver and intestinal

gluconeogenesis, and lipogenesis, too. Starting from here, liver lipogenesis plays a major role in fatty storage regulation.(5)

The same SCFA components are also a signaling

molecules by coupled to the receptors of fatty acids, known as

Gpr41, respectively Gpr43. Gpr41 helps adiposity process of human host.(6,7) The above mentioned couple, SCFA-receptors

of fatty acids, is involved in very important hormones secretion

(glucagon-like, GLP-1 and YY peptide, PYY), including insulin

secretion.(8) Therefore, the mechanism of microbiota influence

about host metabolism of energy contains three major aspects:

- the increasing of energy provided by diet;

- the fatty storage regulation; - gut hormones synthesis.

Over them all, stands the digestion of carbohydrates.

1. The increasing of energy provided by diet. Starting from

the studies with focus on the weight gain in the colonized mice

vs. germ-free ones, the linked mechanisms are: accessible-

carbohydrates degradation and fermentation of gut microbiota

(MAC), SCFA absorption and its liver transportation, adipocytes lipids storage.(4)

The obese microbiota seems to be more productive to

extract diet-induce energy than weak people microbiota.



AMT, vol. 22, no. 3, 2017, p. 11

In healthy people the dominant species of bacteria are:

Bacteroidetes spp., Firmicutes spp, and Actinobacteria. This

normal composition of gut microbiota have a short, and maybe a long-term implications.(9,10)

In obese individuals microbiota, an altered microbiota,

is about 50 % abundant in Firmicutes species, and less

Bacteroidetes, consecutively, than weak individuals microbiota. The Firmicutes species are the major producers of

SCFA; at the opposite pole, a microbiota abundant in

Bacteroidetes species leads to weight loss.(11)

The chromatography analysis of obese microbiota revealed a significant concentration of butyrate and acetate,

whose metabolism pathways including active enzymes, also

called CAZ, derived from families of glycoside hydrolase.(2)

2. The fatty storage regulation. The studies show that the weight gain occurs despite of the food intake reduction, because

of the host gene modulation due to polysaccharides from diet.

This kind of process affect adipocytes energy

storage,(4) and leads to liver new triglyceride production and its adipose tissue incorporation.

The propionate component of SCFA act to liver

lipogenesis.(12)

Some factors, as Fiaf (fasting-induced adipose factor), regulate the liver lipogenesis. The gauge of storage energy is

activated by a protein kinase (AMPK) and the final effect is

catabolic one, to restore the level of energy.

But, an altered microbiota causes the AMPK inactivation.(12,13)

3. The gut hormones synthesis. GLP-1 produce secretion of

insulin, and the process is more important than the glucose

does.(14) This GLP-1 insulin secretion continued despite of levels of glucose or glucagon.(14)

PYY is negative correlated with body mass indices,

and suffered some action changes in obese individuals. For

example, a PYY deficiency leads to obesity, with a good response at substitution therapy, fortunately.(15)

The receptor ligands of fatty acids, especially FFAR3

(active in adipocyte tissues) become a new research area in the

treatment of obesity and type 2 Diabetes Mellitus.(16)

Regarding the digestion of carbohydrates, two aspects

are very important: the synthesis of butyrate and β-

porphyranases use in Bacteroidetes plebeius.

The pathway of synthesis of butyrate is modified by the higher amount of Firmicutes species in gut microbiota.(17)

The Japanese individuals gut microbiota present the

Bacteroidetes plebeius specie able to release porphyran, an

unaccessible/non-digestible source of carbon.(18,19) It insists on the idea that the type, balance, and amount

of diet components, namely carbohydrates, have an important

impact on microbiota.

The SCFA, butyrate, acetate, propionate, are the end-mainly products from the microbial carbohydrates degradation

in the gut microbiota. The SCFA have many effects on human

host (see, their oxidation and energy release). Macfarlane & Macfarlane(20) show in their study that

a neutral pH in the distal part of colon creates a good conditions

for Bacteroidetes species growth.(21)

Certain intake carbohydrates from diet cannot directly digested, and, in this way, represent an important source of

energy, and can be referred like prebiotics.

Prebiotics can stimulate the growth of bifidobacteria,

oligofructose and fructooligosacharides.(22,23). Some recent studies have observed that a diet

supplementing with SCFA had modified the composition of

microbiota, the changes being linked with weight and glucose

control, for example.

These changes were connected with gut gluconeogenesis via butyrate and propionate metabolism.

The prebiotic stimulation regarding bifidobacteria

growth is correlate with increasing glucose tolerance, the

improvement of the insulin secretion induced by glucose and the normalization of murine inflammation.

Understanding these source of carbon and its

microbial degradation is important for promoting the health by

dietary improvement (by understand how kind of bacteria is linked by dietary or host origin carbohydrates).(24)

The studies of metabolic interaction, or of the

immunity between gut microbiota and host, also the way in

which these interactions modulate the brain, muscle, liver and intestine host activity motivate the concept of the therapeutic

manipulation of the microbiome, with the final scope of

combating and preventing the diseases in humans.

It is observed an inter-individual diversity of microbiota in individuals by fermentation in vitro studies. It

makes to put a question about the efficacy of dietary microbiota

in dietary and drug application. Or, about the manipulation of

gut microbiota to defined a nutritional pattern. And, not the end, the question about how many of bacteria are required to obtain

some benefits? And, what happen when the intervention in diet

stops?(25)

It is necessary that future studies to promote analytic approaches from up to down on an epidemiological scale, and to

integrate into questionnaires dietetic data, relevant environment

factors (stress, circadian cycles influential factors), drug and

antibiotics use history.

Acknowledgement: Part of this work has been conducted in the Pediatric

Clinical Hospital Sibiu, within the Research and Telemedicine

Center in Neurological Diseases in Children - CEFORATEN project (ID 928 SMIS-CSNR 13605) financed by ANCSI with the

grant number 432 / 21. 12. 2012 thru the Sectoral Operational

Programme “Increase of Economic Competitiveness”.

CONCLUSIONS The Bacteria of the gut microbiota have larger genes

diversity, and also an amount of degradation enzymes, having as final the carbohydrates fermentation.

The Bacteroidetes species possess a lot of number of CAZymes.

Together with Firmicutes species, these components of gut microbiota must be a future research priority.

The carbohydrates from diet must be put in a relationship with gut microbiota and metabolic diseases, as a complex interaction group.

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AMT, vol. 22, no. 3, 2017, p. 13

CLINICAL PECULIARITIES OF CEREBROVASCULAR

ACCIDENTS IN NEWBORNS, INFANTS AND CHILDREN

OF SMALL AGE

SVETLANA HADJIU

1, MARIANA SPRINCEAN², NADEJDA LUPUSOR

3,

CORNELIA CALCII4, RAMINA PASARI

5, VLADIMIR IACOMI

6, ALINA BANTAS

7,

ANDRIANA GRUZINSCHI8, NINELI REVENCO

9

1,2,3,4,5,6,7,8,91„Nicolae Testemițanu” State University of Medicine and Pharmacy, Chișinău, Republic of Moldova,

1,2,3,4,9Institute for Maternal and Child Healthcare, Chisinau, Republic of Moldova

Keywords: cerebrovascular accident, hemoragic

stroke, acute ischemic

stroke, neonatal,

pediatric

Abstract: Cerebrovascular accident (CVA) in children is one of the most complicated pathologies due to

nonspecific clinical manifestations and remote neurological sequelae. The aim: to study causes and symptoms suggestive for clinical preventive diagnosis of CVA in children depending on their age.

Material and methods: retrospective analysis of a group of 216 children with CVA during the years 2010

– 2017. Medical records were obtained from Department of Neurology, Institute of Mother and Child.

For statistic analysis were used software Statistica 7.0 (Statsoft Inc.) and MS EXCEL. Results. The results of analysis of clinical symptomatology of CVA by method of logistic regression allowed

demonstration of the most important symptoms suggestive for CVA in children depending on their age.

Conclusions. CVA in children is evolving with the subtle and nonspecific symptoms, being initially

attributed to other causes. Recognition of clinical symptoms which are characteristic for different ages improves diagnosis of CVA.

1Corresponding author: Svetlana Hadjiu, Str Burebista, Nr. 93, MD-2060, Chișinău, Republic of Moldova, E-mail: [email protected], Phone:

+37369142479


INTRODUCTION

Cerebrovascular accident (CVA) is defined as "a clinical syndrome with rapid evolution, manifested by global or focal

disorders of cerebral functions, which lasts longer than 24 hours

or which lead to death without any obvious nonvascular

causes".(1).Another definition denotes a correlation between the clinical and imaging aspects of CVA as "CVA is a clinical

syndrome characterized by a neurological deficit related to the

area of a cerebral artery perfusion and with neuro-radiological

evidences of an ischemic lesion".(2) Being a rare neurological disease in children, CVA may

be hemorrhagic, ischemic or mixed origin, caused by occlusion

or rupture of cerebral blood vessels.(3) The overall incidence of

CVA is estimated from 2 to 13 for 100000 of population.(4) Other studies estimated the incidence as 1 – 6 to 100000 of

population, with well shown impact on infant mortality, along

with the results and effects of CVA on the quality of life of

patients and their families.(5) Ischemic CVA is more frequent in prenatal period and during the first 28 days after birth, with a

frequency of 1:4000 of live newborns. Hemorrhagic CVA

(HCVA) sharing about half of all CVA revealed in

children.(6,7) Risk factors for CVA in children differ significantly from those found in adults are related with

predisposing conditions, but CVA can be considered as an

independent nosologic entity.(5) Clinical manifestations of CVA in children vary according to age, artery involved and

cause.(3,4) Focal symptoms, especially hemiplegia, are most

commonly present in hemorrhagic CVA (HCVA) and vary in

different authors.(3) Ischemic CVA (ICVA) manifests by various clinical symptoms that can be registered also in HCVA,

e. g., disorders of conscience asyou may encounter, seizures of

various types, etc.(8)

Diagnosis of CVA in children is often delayed because

the signs and symptoms can be subtle and nonspecific, treatment options are often limited.(9) Several authors showed that the

causes of the majority cases of pediatric CVA, especially

neonatal, have not been established, and large-scale case-control

studies are necessary to understand the early clinical manifestations and pathogenesis, where the results should be

improved.(10)

PURPOSE The preliminary study was to make a retrospective

analysis of causes and primary clinical symptoms, significant for

preventive diagnosis of CVA in children in dependence from the

age, of a group of 216 children with CVA, hospitalized departments of Neurology of Newborns and Neuropsychiatrics

of Small Children, Institute of Mother and Child, in the years

2010 – 2017.

MATERIALS AND METHODS The study was carried out at the Department of

Pediatrics of the „Nicolae Testemițanu” State University of

Medicine and Pharmacy during the years 2017 – 2018, as a part of the project "Evaluation of the incidence, prevalence, and risk

factors, studying of clinical and neuro-imaging and

neurophysiological aspects of CVA in children and methods of neurotrophic treatment" being conducted under the aegis of the

State program "The system of risk factors, nursing service

optimization, sustainable evaluation and mathematical modeling

of cerebrovascular accident". One of the compartments of the study consists in evaluation of the causes and clinical

particularities of CVA in children depending on the age.

Retrospective analysis was made on a group of 216 children, of



AMT, vol. 22, no. 3, 2017, p. 14

whom 202 newborns (93.5%, 95 CI 91,82 – 95,18), which

suffered from CVA during the years 2010 – 2017. Medical

records were selected in the Departments of Neurology of the Institute of Mother and Child. Were determined and classified

causes and clinical symptoms characteristic of CVA. The work

contains preliminary data. For the statistical analysis of the

obtained data have been used software Statistica 7.0 (Statsoft Inc.) and MS EXCEL. It has been calculated the arithmetic

mean, the standard error, the method of logistic regression.

RESULTS The sharing of CVA in studied children was as

follows: ICVA in 134 cases (62%, 95 CI 58,7 – 65,3), HCVA in

68 cases (31,5%, 95 CI 28,34 – 34,66), mixed CVA in 18 cases

(8,3%, 95 CI 6,42 – 10,18). Neonatal CVA was determined in 202 cases (93,5%; 95 CI 91,82 – 95,18), and CVA in early

childhood in 14 cases (6,5%, 95 CI 4,82 – 8,18). Of the total

number of children included in the study 15 cases (6,9%, 95 CI

5,17 – 8,63) children had heart congenital anomalies. In some children who have suffered from CVA were diagnosed genetic

syndromes: Marfan syndrome in one case, tuberous sclerosis in

one case, homocysteinuria in one case. Systemic diseases were

common at 56 newborns (27,7%, 95 CI 24,55 – 30,85). Among children with neonatal CVA 123 cases (60.9%, 95 CI 57,47 –

64,33) were boys, 20 (9,9%, 95 CI 7,8 – 12) were premature.

The pregnancies were complicated with placenta pathology in

22 cases (10,9%, 95 CI 8,81 – 13,09) or pathology of amniotic membranes in 25 cases (12,4%, 95 CI 10,08 – 14,72). Detailed

results of the causes responsible for the CVA will be presented

in another study. Clinical picture in CVA in newborns differs

from the pathology in other ages. The pathology was symptomatic and manifests by more generalized symptoms

(Table 1), related to the features of child development

characteristic for this age.

Table no 1. Clinical manifestations of CVA in newborns

(n=202)

Clinical

symptoms Abs.

P±SE

(%) 95CI P

Seizures 158 78,2 75,3-81,1 0,001

Non-focal

neurological

signs

136 67,3 64,0-70,6 0,001

Disorders of

mental status 147 72,8 69,67-75,93 0,001

Irritability 55 27,2 24,07-30,33 0,05

Breathing

disorders 76 37,6 34,19-41,01 0,01

Tremor 44 21,8 18,9-24,7 0,05

Generalized

motor disorders 128 63,4 60,01-66,79 0,001

Need in

resuscitation 78 38,6 35,17-42,03 0,05

Imaging data also allowed determining the affected part of the brain. In newborns enrolled in the study predominant

regions were anterior and medium areas in 129 cases (63,9%;

60,52 – 67,28), and the left hemisphere in 137 cases (67,8%; 95 CI 64,51 – 71,09). Multifocal lesions were determined in one

third of all the cases (67) (33,2%, 95 CI 29,89 – 36,51). Lack of

focal symptoms often led to errors in diagnosis, as it was

determined in 112 cases in our study (55,4%, 95 CI 51,9 – 58,9),

being assumed as the "neonatal encephalopathy". However, in

the presence of generalized symptoms, it must be considered the

diagnosis of CVA, which requires confirmation by visualization

and continuous EEG monitoring, especially in intensive care

units. Clinical manifestations of pediatric CVA also differ

according to age, involved vessel and cause. In young children

the symptoms are commonly symptomatic, and in older children, resemble those of adults. Retrospective analysis was

made of a group of 14 children (6.5%, 95 CI 4,82 – 8,18),

selected from the general group of 216 children aged from 28

days to 3 years, suffered from pediatric CVA (boys 10 – 71.4%, 95 CI 59,33 – 83,47). The most common symptoms recorded in

pediatric CVA were focal neurological deficits as hemiplegia or

severe focal motor deficit. CVA was developed in 5 cases

(35,7%, 95 CI 22,89 – 48,51) up to age one year and manifested by many clinical symptoms (Table 2). Often the pathology

manifested on one side of the body, often on the right in 4 cases

(80%, 95 CI 62,11 – 97,89).

Table no 2. Clinical manifestations of CVA in children of

small age (n=14)

Clinical symptoms Abs

.

P±ES

(%) 95CI P

Age: 28 days – 1 year (n=5)

Focal neurologic

deficit 5 35,7 22,89-48,51 0,05

Decreasing of

muscular force on the

one side of the body

3 60 38,09-81,91 0,001

Preferential using of

one hand 2 40 18,09-61,91 0,01

Fist squeezing 3 60 38,09-81,91 0,001

Foot squeezing 2 40 18,09-61,91 0,01

Breathing disorders 2 40 18,09-61,91 0,01

Seizures 4 80 62,11-97,89 0,001

Disorders of

conscience 3 60 38,09-81,91 0,001

Age: from 1 to 3 years (n=9)

Decreasing of

muscular force on the

one side of the body

5 55,6 39,04-72,16 0,01

Hemiparesis 3 33,3 17,59-49,01 0,05

Focal disorder of

movement 6 66,7 50,99-82,41 0,001

Sensorial disorders 7 77,8 63,94-91,66 0,001

Visual disorders 3 33,3 17,59-49,01 0,05

Speech disorders 5 55,6 39,04-72,16 0,01

Seizures 7 77,8 63,94-91,66 0,001

Alert status 4 44,4 27,84-60,96 0,01

Disorders of

coordination 2 22,2 8,34-36,06 0,05

Nystagmus 1 11,1 0,62-21,58 0,05

Breathing disorders 3 33,3 17,59-49,01 0,05

Tremor 2 22,2 8,34-36,06 0,05

Vomiting 2 22,2 8,34-36,06 0,05

In 9 cases (64,3%, 95 CI 51,49 – 77,11) over 1 year were

assessed neurological symptoms in acute period of CVA (Table

2). Neuroimaging has allowed the determining of artery involved, often middle cerebral artery (55,6%, 95 CI 39,04 –

72,16) and in left hemisphere (77,8%, 95 CI 63,94 – 91,66).

Other tests allowed for the assessment the cause of CVA, i. e., 4 cases of heart congenital anomaly (44,4%, 95 CI 27,84 – 60,96),

in 3 cases were genetic syndromes (33,3%, 95 CI 17,59 –

49,01), in 1 case of blood disease (11,1%, 95 CI 0,62 – 21,58).

The simultaneously presence of clinical symptoms, imaging data and age reported significant results of logistic

regression. Results of the analysis by modeling the relationship

between a lot of independent variables and a dichotomous

dependent variable allowed demonstration of the most important symptoms suggestive for the diagnosis of CVA in children



AMT, vol. 22, no. 3, 2017, p. 15

according to age:

1. In newborns: seizures (p=0,000; OR=5.118), non-focal

neurological signs (p=0,002; OR=2.861), disorder of conscience (p=0,006; OR=2.909), generalized motor

disordes (p=0,004; OR=3.963).

2. In children from 28 days to one year: seizures (p=0,006;

OR=3.377), decreasing the force on the one side of the body (p=0,000; OR=4.324), squeezing the fist (p=0,004;

OR=8.588), disorders of conscience (p=0,003; OR=1.143).

3. In children from one to three years: decreasing the force on

the one side of the body (p=0,003; OR=3.438), seizures (p=0,000; OR=3.348), sensorial disorders (p=0,004;

OR=4.163), focal disorders of movements (p=0,002).

Thus, diagnosis of CVA in children often delayed

because the clinical symptoms are subtle and unspecific, initially being conferred on other causes. Recognition of symptoms

suggestive of CVA, attributed to the category of age, improve

the share of correct diagnosis from 66 to 99,7%.

DISCUSSIONS

CVA manifests with a focal disturbance of cerebral

circulation secondary to rupture or embolisation of arteries or

veins, confirmed by neuroimaging or neuropathological investigations. Children who suffer from CVA often develops

persistent dramatic consequences for the rest of his life. Despite

the clinical and socioeconomic significance, there are no clinical

strategies effective to treat this disease. Strategies of using neuroprotective measures that can be derived from the

pathophysiological mechanisms of CVA are in progress of

clinical and experimental trials. Pathogenesis and early clinical

manifestations of CVA are questionable anyway. CVA, regardless of age of first manifestations, can be

hemorrhagic (subarahnoidal or intracerebral) or ischemic. The

type of CVA varies depending the age and etiology, which is

different in children and in adults. Are described more than 70 potential risk factors of CVA in children and adults. However

the ischemies are the result of vascular pathology which is not

related to atherosclerotic process, of cardiac emboli, or

coagulopathies.In children CVA can be result of congenital

heart diseases, acquired heart diseases, infectious or

inflammatory diseases, vascular disorders, haematological

diseases, cerebrovascular anomalies or brain trauma; however,

in a third of all cases of CVA etiology remains undetermined.(11)

Perinaral ICVA is the most common form of cerebral

infarction in children, with an incidence from 1 to 2800 up to 1

to 5000 live births. One of the main causes of perinatal cerebral ICVA is embolus, which comes from the placenta through the

foramen ovale. The majority of risk factors in newborn related

to vascular placental pathology and trauma, i.e., injuries of

cervical cerebral arteries, leading to cerebral embolus formation during delivery.(12)

Some authors assume that maternal-fetal inflammation

leads to vasculitis selective affecting carotid system and promotes a focal thrombosis and a subsequent CVA. Obtained

preclinical results suggest that the combination of prothrombotic

stress and selective intracranial arteriitis caused by immune

activation of pregnant women at the end of pregnancy seems to play a role in patophysiology of ICVA.(13)

However, in the study conducted within our

institution, most of the causes which lead to perinatal CVA and

its mechanisms often are unknown and remain to be identified. Similarly, as part of an international study was carried

out clinical and imaging analysis of neonatal symptomatic

ICVA. Study of the clinical presentations summarized different

clinical manifestations, risk factors, investigations, methods of

treatment and immediate results of the ICVA. The most

important clinical symptoms are: seizures (72%) and neurological non-focal signs (63%).(10) Some authors showed

that in 60% of children occur early symptoms, expressed by

recurrent focal seizures within the first 3 days of life.(12) In our

study were noted in the presence of epileptic seizures and non-focal neurologic signs the presence of symptoms such as: altered

mental status, and generalized motor disorders.

However, some studies state that about 40% of

children have no specific symptoms in the neonatal period and are recognized later with the appearances of motor disorders,

developmental delay, cognitive deficiency or seizures.(12) Our

study showed delay in diagnosis in 55,4% of cases.

Prenatal diagnosis of cva is difficult, but it can be easily confirmed by ecoencephalographic examination and

cerebral MRI carried out on early stages. MRI has a key role in

diagnosing the ICVA, and also has a significant prognostic

value to predict the outcome of neurological development of the child. In most children who have suffered CVA develops the

following neurological sequelae: cerebral palsy, epilepsy,

cognitive or behavioral disorders.(12) Children with a history of

unilateral neonatal CVA are at increased risk for cognitive skills deficits during development. Many of them will develop

evolving speech disorders and other higher cognitive deficits

that occur later in the pre-school and school age.(3) Some

authors showed that it is important to find the predictors of outcome and measures of preventing or improving these

problems. In such cases there are different groups of newborn

with CVA: birth at term or premature newborn, symptomatic

newborns and children with delayed manifestations. However, at present there is no evidence for the preventative use of

anticoagulants for the purpose of neonatal protection.(12)

The results of several studies showed that pediatric

CVA leads to significant morbidity and mortality. Among children with pediatric CVA approximately 10 – 25% will die,

up to 25% will have a recurrence and up to 66% will have

persistent neurological deficiency, or develop epilepsy, learning

problems or developmental problems.(3,15,16,17) Neurological

impairment during childhood will have a major impact on the

quality of life of the child and the family, as well as lead to

emotional and economic burden for the state economy.(3)

Regarding infants and small children there are current studies related to the question how the symptoms appear is of

great importance, and up to one-third of children who suffered

from ICVA have a history of recent events which can be

attributed to transient ICVA.(14) Early diagnosis of pediatric CVA includes timely

neurological consultation, hospitalization of the child in

appropriate departments of hospital, to provide diagnostic

assistance, i. e., brain imaging, and for adequate etiological and pathogenetic treatment, in order to improve the results.

Diagnosis of pediatric CVA is based on recognition of

the clinical picture depending on the age and the vessel involved. Symptoms of CVA in early childhood are usually

nonspecific, and in older children are often manifests by focal

neurological deficits, such as acute hemiplegia.(14) Neurologic

examination should reveal the most subtle symptoms suspicious for a CVA, including monitoring of vital parameters, to identify

the neurological damage and to make the presumed diagnosis

with determining the topography of the vessel involved. Usually

it is necessary to exclude some systemic diseases that increase risk of CVA.(4-18) Symptoms suggestive of vascular lesions on

some area of cerebral perfusion are the following: (1) internal

carotid artery – hemiparesis, aphasia and hemianopsia; (2) the



AMT, vol. 22, no. 3, 2017, p. 16

anterior cerebral artery – hemiparesis, especially of the lower

limbs; (3) middle cerebral artery – hemiparesis of upper limb,

hemianopsia and aphasia; (4) posterior cerebral artery – hemiparesis, hemianopsia, ataxia and dizziness; (5) basilar

artery – breathing difficulties, sensory or balance disorders,

ataxia, nystagmus, opisthotonus, tremor and vomiting; (6)

cerebellar artery – sensory difficulties, headaches, fever, vomiting and cerebellar signs.(18)

It is known that acute neurological deficits may be

caused by tumors, central nervous system disorders, including

acute disseminated leukoencephalitis, cerebellitis, posterior reversible leukoencephalopathy, alternating hemiplegia,

metabolic disorders, epilepsy and psychogenic diseases. Thus,

the differential diagnosis of CVA in children must exclude all

pathologies associated with acute neurological deficits. In the present study were carried out selection the most important

symptoms for diagnosis of suspected CVA using logistic

regression. However neuroimaging is crucial in defining the

diagnosis, other tests are needed depending on the clinical picture. We are plan the following study of important prognostic

factors within the our project.

Pediatric CVA, while it is becoming increasingly

recognized among practitioners as an clinically significant entity, remains challenging for clinicians and researchers.(19)

CONCLUSIONS

Neonatal and pediatric CVA represents one of main causes of severe handicaps throughout the life: epilepsy, motor

and cognitive consequences. Diagnosis of CVA in newborn and

young child is difficult, caused by scarce symptomatology and

the presence of specific symptoms common to other pathologies. Disease history and clinical examination have a primary role in

confirmation or exclusion of disease and factors predisposing

for CVA. Logistic regression method allowed highlighting the

most important symptoms suggestive for CVA, depending the age, i. e., in newborns (seizures, neurological non-focal signs,

altered state of conscience, generalized motor disorders), in

infants (seizures, decreased force on the one side of the body,

fist squeezing, disorders of conscience) and small children

(decreased force on the one side of the body, seizures, sensorial

disorders and focal movement disorder). On the basis of the

presence of generalized symptoms a possible diagnosis of CVA

must be considered, which require confirmation by imaging and EEG monitoring.

Future studies represent a priority direction and should

determine major clinical syndromes are related to risk factors

and predictors of neonatal and pediatric CVA, to advance preventive treatment and improve the neurological outcome of

survivors.

REFERENCES 1. Aho K, Harmsen P, Hatano S. Cerebrovascular disease in

the community: results of a WHO collaborative study.

Bulletin of the World Health Organization.

1980;58(1):113-130. 2. Hunter JV. New radiographic techniques to evaluate

cerebrovascular disorders in children. Seminars in

Pediatric Neurology. 2008;7(4):261-277.

3. Tsze DS, Valente JH. Pediatric Stroke: a review. J Emerg Med Int. 2011;2011:73406.

4. Manea M, Golea G, Mălăescu R. Accidentul vascular

cerebral arterial ischemic la copii. Revista de Neurologie

şi Psihiatrie a Copilului şi Adolescentului din România. 2014;17(3):27-38.

5. Rosa M, De Lucia S, Rinaldi VE, et al. Paediatric arterial

ischemic stroke: acute management, recent advances and

remaining issues. Ital J Pediatr. 2015;2(41):95.

6. Earley CJ, Kittner SJ, Feeser BR, et al. Stroke in children and sickle-cell disease: Baltimore-Washington cooperative

young stroke study. J Neurology. 1998;51(1):169-176.

7. Carvalho KS, Garg BP. Arterial strokes in children. J

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11. Nica S, Davidescu I. Accidentele vasculare cerebrale la

copil și adultul tânăr. Revista Română de Neurologie. 2006;2:67-73.

12. Chabrier S, Husson B, Dinomais M, et al. New insights

(and new interrogations) in perinatal arterial ischemic

stroke. J Thromb Res. 2011;127(1):13-22. 13. Guiraut C, Cauchon N, Lepage M, Sébire G. Perinatal

Arterial Ischemic Stroke Is Associated to Materno-Fetal

Immune Activation and Intracranial Arteritis. Int J Mol

Sci. 2016;17(12). pii: E1980. 14. DeVeber G. Stroke and the child's brain: an overview of

epidemiology, syndromes and risk factors. Current

Opinion in Neurology. 2002;15(2):133-138.

15. Lanthier S, Carmant L, David M, et al. Stroke in children: the coexistence of multiple risk factors predicts poor

outcome. J Neurology. 2000;54(2):371-378.

16. DeVeber G. In pursuit of evidence-based treatments for

paediatric stroke: the UK and Chest guidelines. The Lancet Neurology. 2005;4(7):432-436.

17. Kolk A, Ennok M, Laugesaar R, et al. Long-term

cognitive outcomes after pediatric stroke. Pediatr Neurol.

2011;44(2):101-109.

18. Lopez-Vicente M, Ortega-Gutierrez S, Amlie-Lefond C,

Torbey MT. Diagnosis and management of pediatric

arterial ischemic stroke. Stroke and Cerebrovascular

Diseases. 2010;(3):175-183. 19. Cárdenas JF, Rho JM, Kirton A. Pediatric stroke. Childs

Nerv Syst. 2011;27(9):1375-90.

https://www.ncbi.nlm.nih.gov/pubmed/?term=C%C3%A1rdenas%20JF%5BAuthor%5D&cauthor=true&cauthor_uid=21336993

https://www.ncbi.nlm.nih.gov/pubmed/?term=Rho%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=21336993

https://www.ncbi.nlm.nih.gov/pubmed/?term=Kirton%20A%5BAuthor%5D&cauthor=true&cauthor_uid=21336993

https://www.ncbi.nlm.nih.gov/pubmed/21336993

https://www.ncbi.nlm.nih.gov/pubmed/21336993



AMT, vol. 22, no. 3, 2017, p. 17

GUT MICROBIOTA AND CENTRAL NERVOUS SYSTEM IN

HEALTH AND AUTOIMMUNE DISEASES

REVIEW

MIHAI BOGDAN

NEAMŢU

1, ANDREEA BARBU

2, IONELA MANIU

3

1,3”Lucian Blaga” University of Sibiu, 1,2,3 Research and telemedicine center of neurological diseases in children, 2Clinical Pediatric Hospital Sibiu,

2University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca

Keywords: Human

microbiota, Central

Nervous System

Disorders, Gut-Brain Axis

Abstract: Gut microbiome is highly connected with the central nervous system (CNS) in a bidirectional

manner by top-down and bottom-up signalling systems. We present a review with an update on the most

important mechanisms and pathways by which probiotic strains modulate autoimmune CNS diseases

such as Multiple Sclerosis(MS), Neuromyelitis optica (NMO), Guillain-Barre Syndrome (GBS). Lactobacillus, Bifidobacterium, Bacteroides probiotic species were intensively studied for their

beneficial effects on metabolism and regeneration (butyric, propionic acids), neurotransmission

(tryptophan, serotonin, γ-aminobutiric acid, dopamine, norepinephrine, acetylcholine, histamine,

agmantine), antiinflammatory effects with a decrease in specific cytokine levels. In autoimmune diseases, pathogenic strain antigens were found to trigger proinflammatory Th1, Th17 cells and elevate

proinflammatory biomarkers (TNF-α IL-1, IL-6, IL-12, IL-17, MIP-10, MCP-1, IFN-γ). Probiotic strains

reduced the inflammatory outcomes stimulating Treg cells subsets and IL-10 profiles both in humans

and in experimental animal models.

1Corresponding author: Neamtu Mihai Bogdan Str. Pompeiu Onofreiu, Nr. 2-4, Sibiu, România, E-mail: [email protected], Phone: +40773

994375


INTRODUCTION

In the last 30 years, numerous studies concerning

human microbiota have shown a special interest in network links between various pathophysiological phenomena and commensal

bacteria. Whether orally administered or manipulated with

specific diet nutrients (the so-called gut microbiota shaping),

probiotic bacteria are associated with impressive pleiotropic effects: anti-inflammatory, metabolism modulation,

antihypertensive, anticancer and neurodevelopmental to mention

the most important of them. There is a complex interaction

between gut microbiota, intestinal epithelium, GALT(Gut Associated Lymphoid Tissue), gut endocrine tissue

(enterochromaffin cells) and gut-associated nervous system.

Considerable attention is addressed to nervous system

interaction with human microbiota. The gut microbiome is highly connected with the central nervous system(CNS) in a

bidirectional manner.(1,2,3) It is a network that functions in

both directions: top-down and bottom-up signalling. For this

reason, many hypotheses and research designs aimed to study this interrelationship between human microbiota and CNS

autoimmune or neurodegenerative disorders such as Multiple

Sclerosis(MS), Neuromyelitis optica(NMO), Guillain-Barre

Syndrome(GBS).

The purpose of our review was to present an update on

the most important mechanisms and pathways by which

probiotic strains modulate autoimmune CNS diseases evolution.

MATERIAL AND METHOD

We performed an analysis of PUBMED database and

Google Scholar using criteria like “Human microbiota, CNS, Gut-Brain Axis”. Seventy-nine articles have emerged. Further

on, we considered the most important references for probiotic

and/or gut microbiota potential to modulate neural pathways and

their impact on autoimmune CNS disorders (MS, NMO, GBS).

A special consideration was given to microbiota capacity to

produce antiinflammatory responses corelated with the disease

specificity.

RESULTS AND DISCUSSIONS

Human microbiota has a dynamic architecture

throughout the lifespan. Human gut is colonised starting with intrauterine life (placenta and amniotic fluid). Its organisation is

modulated up the 3rd year of age.(4-9) The most important

species mentioned in the literature in the prenatal period are

Propionibacterium, Streptococcus, Staphylococcus. The proposed mechanism is the translocation of mother’s gut

microbiota using placenta and umbilical cord blood.(10) At birth

and during the first months infant's gut is populated mainly with

bifidobacteria. Both Bifidobacteria and Lactobacillus species were identified in human milk.(11,12) After 4-6 months a

multiplication of Clostridial species takes place. At 2-3 years of

age, the architecture is mainly composed of Bacteroidaceae,

Lachnospiraceae, and Ruminococcaceae and a relative equilibrium is preserved until adulthood.(13) In adults, we refer

to a steady state status with Bacteroides and Firmicutes the most

prominent phyla.(14,15) Poor diversity in composition was

incriminated in many diseases(15). In elderly people, the

Bacteroides are more numerous than Firmicutes which is an

inverted trend related to the adult period.(16,17)

Human gut microbiota contains thousands of species

(between 500 and 3000) counting more than 7000 strains with an outstanding gene network. This network is approximately

150 times bigger than the human genome.(18-21) Our body cells

are 10 times outnumbered by an approximately 105 billions of cells within the microbiota.(20,21,22) Therefore microbiota is

considered as a true organ with complex implications in

neurodevelopment, endocrine pathways, metabolism, immune

modulation.(19) Gut-brain axis (GBA) is a system that



AMT, vol. 22, no. 3, 2017, p. 18

integrates these connections in a bottom-up and top-down

signalling pathways.(4,23,24)

Bottom-up signalling Microbiota modulates through its biomolecules

metabolic, endocrine or immune functions but most importantly,

a part of nervous system activity. Probiotic strains of

Bacteroides, Bifidobacterium, Lactobacillus turn nondigestible fermented carbohydrates into short-chain fatty acids(SCFAs).

Acetate, propionate or butyrate SCFAs are of paramount

importance for intestinal epithelium integrity in neonates (25-

27) and epithelial cell signalling pathways.(28-30) SCFAs actions were associated with host gene expression (epigenetic

control), stimulation of leptin production by adipocytes through

enterochromaffin cells (31), suppression of pathogens expansion

(19), modulation of lymphocytes and neutrophil functions or Treg subset anti-inflammatory activity.(32-35) It is hypothesised

that SCFAs enter the systemic circulation and influence blood-

brain barrier permeability (36,37) and CNS functions including

sympathetic nervous system (38,39). Moreover, it seems that SCFAs could affect neuropeptide regulation.(40,41) In neonates,

probiotic microflora synthesises lower concentrations of SCFAs

than in adults.(42) Interesting experimental designs revealed

autistic-like features in direct animal brain tissue exposure to propionate while butyrate synthesis had an opposite effect

suggesting a complex and poorly understood role for SCFAs

pathways in brain activity.

Other metabolites such as conjugated linoleic acids (CLAs) were described to possess a substantial role in obesity,

diabetes and immune function (43) but also in CNS

development. Different types of commensal bacteria can

produce CLAs. In newborns, Bifidobacteria, Lactobacillus, Propionibacterium, Lactococcus, Enterococcus are capable of

yielding CLAs necessary for growth.(19) In experimental

models, CLAs producing Bifidobacterium breve strains

administered daily for 8 weeks to male mice were responsible for an increased concentration of brain fatty acids.(44) The

mechanism remains unclear.

Besides SCFAs and CLAs synthesis, microbiota can

exert neurological effects by neuroactive biomolecules:

tryptophan, serotonin, γ-aminobutiric acid, dopamine,

norepinephrine, acetylcholine, histamine, agmantine.(9,45,46)

Tryptophan, an essential aminoacid is crucial for CNS

development in neonatal behavioral traits. Probiotic strains modulate its levels presumably by kynurenine pathway.

Serotonin is the metabolite of tryptophan controlling appetite,

mood, sleep, anxiety.(19) It is released from intestinal

enteroendocrine cells. Bifidobacterium longum ssp infantis administered in rat models induced an increment in tryptophan

levels, the inhibition of proinflammatory actions and

antideppresive effects.(47,48,49) Strain specific influences were

hypothesized to be directly on vagal terminals and/or enteric nervous system.(50)

Applied research using germ free mice reported

anxious and augmented motor behavior with a decline in levels of serotonin receptor in the hippocampus and N-methyl D-

aspartate receptor in amygdale.(51,52) In addition, a raise in the

expression of the brain-derived neurotrophic factor receptor

(BDNFR) was observed in the hippocampus as a consequence of decreased levels for BDNF.(51,52) After repopulating the gut

with Bifidobacterium infantis these changes seemed to be to a

certain degree changeable at an early period of life and

changeless at a later phase.(51-55) Another notably anxiety-like behavior effect was remarked in relation to synaptogenesis for

different gut colonisations in mice with a diminished expression

for synatophysin and pSD-95 proteins in the striatum.

Consequently, there were increased local levels of different

neurotransmitters such as serotonin, dopamine and

noradrenaline.(56) Conversely, Lactobacillus acidophillus and fermentum and Bifidobacterium lactis strains were found to

enhance synaptic transmission in the hippocampus after an

eight-week treatment.(57,58)

Enterochromaffin or enteroendocrine cells (EECs) are considered veritable biosensors for aminoacids, SCFAs, LCFAs

(long chain fatty acids). They are organized in a large gut

associated endocrine network. Microbiota modulating EECs can

activate hormonal pathways (leptin and insulin production) or regulate neural circuits (serotonin release).

Enteric nervous system (ENS) has more than 500

million neurons.(40,59,60-63) By far, the most important

bidirectional signaling route for ENS is vagal.(64-68) It seems that behavioral effects after vagal stimulation are strain specific.

Pathobiots such as Clostridium jejuni activate vagal ascendings

inducing an anxious behavior while probiotic strains like

Lactobacillus rhamnosus present an opposite, beneficial effect reducing the deppresive behavior. The proposed mechanism for

Lactobacillus rhamnosus strains actions was connected with the

transcription stimulation of γ-aminobutiric acid(GABA)

receptors. The vagal component represented the key factor for this actions because the vagotomized mice couldn’t reveal the

same pattern.(60,69-72)

Lastly, a speculated mechanism for bottom-up

signaling involves the immune system. In this theory, there is an activation in the gut for specific subsets of immune cells and

another one in the CNS, after these cells cross the blood-brain

barrier and make the contact with local DC (dendritic

cells).(73,74) Evidence based research in neonates showed a strong impact from gut microbiota on brain development in a

specific developmental window.(73-77) The mediators might be

TNF-α, IL-1, IL-6, and HMGB-1 as well as immune cells (DCs,

macrophages, T, B cells).

Top-down signaling

The prominent component is represented by HPA axis

(hypothalamus-pituitary-adrenal axis). Human brain undergoing

a stressful event stimulates the sympathetic system which in turn

release IL-6.(73-77) Therefore, HPA axis is activated increasing

gut permeability to bacterial metabolites and antigens and the

mucosal immune response. The outcome is an extended period

rise in serum levels inflammatory biomarkers in experimental models.(73-78) Furthermore, catecholamine high concentrations

in the gut can stimulate pathobiots aggressiveness. This is the

case of Campylobacter Jejuni and norepinephrine testing in vitro

but this phenomenon was also noticed for other pathogenic strains (Escherichia Coli).(78) A cholinergic route using vagal

pathway has been confirmed with a decline in immune cells and

cytokines in inflammatory states after stimulation.(75,78)

Multiple sclerosis (MS) Multiple sclerosis is an autoimmune disorder affecting

brain and spinal cord. The main incriminated pathophysiological

process implies demyelinating events with the damage of nerve cells insulating covers. The clinical picture is dominated by

muscle weakness, double vision, sensory and coordination

problems, dizziness. To this suite of symptoms, cognitive,

memory deficits and behavioural problems are frequently described. The most common findings at the cellular and

molecular levels indicate an inflammatory process with CD4,

CD8 T-cells, B-cells and activated monocytes, microglia,

macrophages and elevated levels of TNF-α, IL-1, IL-6, IL-12, IL-17, MIP-10, MCP-1, IFN-γ. Neurodegeneration is claimed to

be a consequence of prolonged oxidative stress.(32,78)

Important risk factors were mentioned concerning this



AMT, vol. 22, no. 3, 2017, p. 19

disease: vitamin D deficiency in patients with haplotype HLA-

DR, vitamin A deficiency, obesity in early life, diet, smoking,

infections with herpetic family viruses and genetic variants (100 types of minor risk alleles, HLAs, IL2R, IL7RA, CD6,40,80,86,

genes). A mild disbiosis has been reported in MS with a lower

number for Bacteroides species.(32-35,78)

In experimental models, susceptible mice were exposed to oligodendrocyte glycoproteins while the gut was

colonised with segmental filamentous bacteria. An inflammatory

response was induced by Th1, Th17 T-cell subsets proliferation

and CD40-CD154 expression, axonal loss, and gliosis, mimicking the events in MS. This experimental autoimmune

encephalomyelitis model (EAE) has been proposed to test anti-

inflammatory effects of specific strains. Hence, research

approaches using Lactobacillus casei Shirota, Bifidobacterium Animalis and Bacteroides Fragilis were associated with a

decline in TNF-α IL-1, IL-6, IL-12, IL-17, MIP-10, MCP-1,

IFN-γ levels. An explanation for the changes referred to SCFAs

(butyric and propionic acids) binding to the G-protein coupled receptors (GPCRs) stimulating Treg activity and IL-10

production.(78)

Another particular finding in EAE was recorded after

antibiotic treatment with a decrease in the proinflammatory biomarkers and improvements in symptoms.

Neuromyelitis optica(NMO)

NMO appears to be similar to MS, but there are

specific pathophysiological hallmarks. It belongs to the same family (CNS autoimmune disorder), but the inflammation and

demyelination processes are due to astrocyte impairment

(astrocytopathy or astrocytic channelopathy). A T-cell-mediated

immune response against aquaporin 4 (AQP4) marks the pathogenesis. Anti-AQP IgG positive or negative states were

discovered. Monophasic or recurrent forms include optical

neuritis with blindness and spinal cord injury (myelitis) with

different degrees of weakness or paralysis in arms, legs, bladder and bowel dysfunction. So far, the relationship with gut

microbiota dysfunctions seemed to be both direct and indirect.

AQP4 sensitised T-cells were shown to react also against

Clostridium perfringens proteins (molecular mimicry) while

Anti-AQP IgG positive forms revealed antibodies against

dietary proteins.(78)

Guillain-Barre Syndrome(GBS)

GBS is considered an acute autoimmune polyneuropathy affecting myelin insulation with a rapid onset.

The main complaint is muscular weakness involving the upper

and lower limbs and the upper body. Life threatening events can

ensue, naming weakness of breathing muscles, heart rate and blood pressure anomalies. Again molecular mimicry mechanism

leading to neuronal damage is alleged. A prior viral, atypical

germ or even bacterial infection sets up the inflammatory paths.

Different strains of Campylobacter were mentioned to correlate with these abnormal immune responses. At least three Th

subsets were identified in GBS evolution: Th1 with TNF-α,

IFN-γ and IL-1 as triggers for the acute inflammation, Th17 subset with IL-2,12,17,22 TNF-α/β as proinflammatory

populations and the last but not the least Th2 type cells with IL-

10 and TGF-β in the remission phases.(78,79)

CONCLUSIONS

There is a complex interplay between gut microbiota

and brain development at different life stages. A crucial role in

brain maturation is already acknowledged for gut synbiots in a particular time window after birth. The messengers for these

data transfers are metabolites, cytokines, neurotransmitters, and

hormones. In the autoimmune diseases affecting the nervous

system, a molecular mimicry mechanism and a pathogenic strain

were invoked in most of the instances triggering

proinflammatory cells and biomarkers whereas probiotic strains proved to alleviate the disease pathogenesis both in human and

in experimental animal models.

Acknowledgment:

This work has been conducted in the Pediatric Clinical Hospital Sibiu, within the Research and Telemedicine

Center in Neurological Diseases in Children - CEFORATEN

project (ID 928 SMIS-CSNR 13605) financed by ANCSI with the

grant number 432 / 21. 12. 2012 thru the Sectoral Operational Programme “Increase of Economic Competitiveness”.

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TETANY OR DIAGNOSTIC PITFALL

RALUCA MARIA COSTEA

1, BOGDAN NEAMŢU

2

1Clinical Pediatric Hospital Sibiu, 2Research and Telemedicine Center of Neurological Diseases in Children

Keywords: congenital

myotonia, tetany,

muscle stiffness

Abstract: The authors present the case of a female patient, aged 17 years, diagnosed with congenital

myotonia. The patient with a history of painless intermittent muscle stiffness and restriction in executing

rapid movements for about 7-8 years addressed our outpatient clinic tardly. The patient described a

slow progression of symptoms and was diagnosed with autoimmune thyroiditis and treated for two years for, hypocalcemia and tetany, with no improvement. The athletic allure, age at onset, percussion

myotonia, EMG confirmation of the myotonic discharge and the lack of significant progression of the

symptoms, represented pathognomonic criteria for congenital myotonia. In the absence of suggestive

family history and with a borderline phenotype no assignation in the Becker or Thomson form could be done. The screening of the CLCN1A mutations remains the “gold standard” for establishing the

congenital myotonia type. Under membrane stabilizer treatment with Carbamazepine moderate

improvement of the myotonic phenomenon was obtained.

1Corresponding author: Raluca Maria Costea, Str. Pompeiu Onofreiu, Nr. 2-4, Sibiu, România, E-mail: [email protected], Phone:

+40728981091


INTRODUCTION

Myotonia is defined by continued active contraction

and impaired muscle relaxation following cessation of voluntary

effort or stimulation. Several clinical disorders of different

etiologies are characterised by myotonia. The inheritance

pattern, clinical history and assessment of systems are all

important in establishing the different causes. Clinically the age

of onset, temperature effects, food composition effects, associated transient or predominant weakness are essential in

working out the underlying illness. The electromyography and

molecular genetic testing are able to confirm the final

diagnose.(1,2,3,4,5) Congenital myotonia is a rare hereditary

musculoskeletal disorder characterised by myotonia- muscle

stiffness, muscular hypertrophy, sometimes muscular weakness,

with onset in childhood. Both dominant (Thomson) and recessive (Becker) variants are considered to be chanellopathies.

There is a high variability in the mutations involving the

chloride channel gene (CLCN1), located on 7q35.

This mutations reduce the stabilising chloride conductance causing a electrical instability of the muscle fibre

membrane expressed in myotonia and muscle

stiffness.(1,2,3,4,5)

No etiological treatment is available with mainly conservative and supportive therapy. Easy-moderate intensity

adaptive physical education and sport activities could improve

the transitory muscle stiffness.(6)

Mexiletine response, a Na+ channel blocker, is effective in treating the myotonia. The best responses obtained

when the drug blocks voltage-dependent mutant channels, but

there is also a partial effect in more severe cases with the blockade of wild type channels. It seems that the Me7

derivative, that inhibits the Na+ channels in M2 gating mode, is

more effective than Mexiletine. Other membrane stabilizers are

also available.(7,8,9,10,11,12,13)

CASE PRESENTATION

The proband, a 17 years old female, born of a non-

consanguineous marriage, complained of difficulty in rising up

quickly from the bed in the morning describing a,,muscle

tension “ sensation when standing up from sitting position. As

an only child she had unremarkable antenatale and perinatal

history. She was born at 40 weeks of pregnancy with a weight of

3300g, 51 cm length, in cephalic presentation. She presented with normal motor and cognitive development. With a family

history of diabetes (maternal grandmother), thyroid nodules (the

mother) she was diagnosed at the age of 16 years with

autoimmune thyroiditis. The probable onset was in childhood at the age of 9-

10 years. She described a painless intermittent muscle tension

and restriction in executing rapid movements for short periods

of time (seconds/minutes) with poor localization in the lower limbs. The complains were limited to movement initiation after

prolonged rest –sleep with slight improvement with repeated

movements. No relation to intense physical activity, rest after

physical activity, fasting or carbohydrates or potassium rich food was noticed. No episodes of prolonged motor deficit,

myalgia, history of constipation/respiratory dysfunction or

recurrent infections were mentioned. The mother denied early

symptoms, in infancy or early childhood, as difficult eye opening during cry, sucking or mastication. Latterly she

presented with fatigue, moderate effort dyspnea and tachycardia,

mild decline in attention and memory skills, hypersomnia and

episodic speech impairment. First evaluated by the pediatrician at the age of 15

years she was diagnosed and repeated treated for tetany due to

low plasmatic calcium levels (8.32 mg/dl NV 8.5-11 mg/dl) and 25-OH vitamin D (12.66 ug/L NV 20-70 ug/L), with no clinical

improvement. The endocrinological investigations: high

antitireoglobuline antibodies (157 UI/ml NR <4.11 UI/ml) and

TSH plasma levels, with normal hormonal thyroid and pituitary



AMT, vol. 22, no. 3, 2017, p. 23

levels and ATPO levels concluded chronic autoimmune

thyroiditis with euthyroidia.

On careful clinical examination in our clinic the 17 years old patient presented with a mild athletic aspect, short

stature T=155 cm (perc 10-25 CDC), W= 46 kg (perc 3-10

CDC), BMI=19 (perc 10-25 CDC), no alopecia or skin lesions.

The clinical examination of both parents was normal. The first neurological examination, in summer,

revealed intermittent latency in tongue movement with normal

eye movement, no ptosis, lid lag, facial weakness, dysphonia or

dysphagia. The calf muscles, thighs and proximal muscles in the upper limbs were mildly hypertrophied. There was evidence of

provoked myotonia, on percussion of the thenar eminence,

emphasized by cold water immersion, mild active (hand grip)

myotonia. No tongue or eye orbicular myotonia was obtained. Extensive investigations were performed:

echocardiogram, cardiac ultrasound, creatinkinase levels, lipid

profile, imunogramme, blood glucose were all normal. The

electromyography revealed the presence of typical myotonic runs and a myogenic aspect (Figure 1).

Figure no. 1. Myotonic discharges on needle EMG

The cerebral MRI revealed a pituitary microadenoma

with normal sella turcica and no cortical atrophy or white matter

lesions( Figure 2).

Figure no. 2. T1-weighted sagital acquisition shows a small

pituitary adenoma

The ophthalmological examination excluded posterior

subcapsular cataract or other anomalies. The diagnoses was done based on the nonprogressive

painless myotonia, athletic appearance, percussion myotonia and

EMG confirmation of the myotonic discharges.

Although the disease was diagnosed by corroborating the anamnestic, clinical data and the EMG route “the gold

standard" for diagnosis remains the screening for CLCN1

mutation.

The disease has no etiological treatment, the therapy being conservative and supportive: thermal protection of

extremities is recommended the cold stressing the myotonic

phenomenon. Easy-moderate intensity adaptive physical

education and sport activities could improve the transitory

muscle stiffness. To reduce the myotonia and due to the lack of

Mexiletine another membrane stabilizer treatment with

Carbamazepine 10 mg/kg/day was initiated. Moderate

improvement of the myotonic phenomenon was obtained. Late prognosis does not imply severe disability due to

the lack of progression of the symptoms in the adulthood. Life

expectancy is not affected.

A genetic testing should be undergone because some mutations (Gly230Glu, T310 M) are correlated with aggravated

myotonia and weakness during pregnancy. Appropriate

professional orientation was recommended. Advise regarding

aggravating medication was given: β2-agonists: Fenoterol, Ritodrine, monocarboxylic amino acids and depolarizing muscle

relaxants.

DISCUSSIONS There were several difficulties in establishing a

diagnose.

The patient denied any exacerbation of the myotonia

by cold, hunger or stress exposure or ,,warm up phenomenon”- reduced myotonia after repeated activity, but the follow up

confirmed them as triggers. The absence of correlation with

carbohydrate and potassium rich food and episodic paralysis

excluded other chanellopathies- hyper K periodic paralysis, K sensitive myotonia.

The electromyography confirmed the myotonic

discharges, therefore excluding a possible pseudomyotonic

phenomenon associated to the autoimmune thyroiditis. Acquired myotonia due to drug toxicity was rapidly

excluded. Myotonia permanens, a Sodium Channel - α subunit

(SCN4A; Nav1.4) chanellopathy, was excluded due to the

intermittence of symptoms. The lack of exacerbation after potasium rich foods and the absence of delayed,,paradoxical “.

myotonia made a diagnoses of myotonia fluctuans improbable.

The recent complaints of fatigue, intolerance to

moderate effort, mild decline in attention and memory skills,

hypersomnia and episodic speech impairment, family history of

diabetes, together with the mildly myopathic EMG pattern,

could have been clues for a slow progressive myotonic

dystrophy. The absence of muscle wasting or other physiognomy features, eye, brain and other systems

involvement excluded Steinert myotonic dystrophy and

proximal myotonic myopathy (PROMM) .

The mild athletic allure, age at onset, lack of significant progression of the myotonia represented

pathognomonic criteria for congenital myotonia. In the absence

of suggestive family history and with a borderline phenotype on

clinical bases no assignation in the Becker or Thomson form could be done. The screening of the CLCN1A mutations

remains the only framing resource.

CONCLUSIONS

Myotonia does exist! Awareness of the myotonic

phenomenon could have hasten the diagnosis and treatment. It is

important to differentiate between tetany and other types of muscle stiffness like myotonia. Hypocalcaemia associated with

intermittent muscle stiffness can be a diagnostic pitfall.

Acknowledgment:

This work has been conducted in the Pediatric Clinic Hospital Sibiu, within the Research and Telemedicine Center in

Neurological Diseases in Children - CEFORATEN project (ID

928 SMIS-CSNR 13605) financed by ANCSI with the grant



AMT, vol. 22, no. 3, 2017, p. 24

number 432 / 21. 12. 2012 thru the Sectoral Operational

Programme “Increase of Economic Competitiveness”.

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http://emedicine.medscape.com/article/1171678-clinical



AMT, vol. 22, no. 3, 2017, p. 25

GUT MICROBIOTA AND FOOD ALLERGY

CORINA CAZAN

1

1“Lucian Blaga” University of Sibiu, Pediatric Clinic, Research and Telemedicine Center of Neurological Diseases in Children

Keywords: food

allergy, dysbiosis,

probiotics

Abstract: In the last decade, the role of the intestinal microbiota in the development of immune tolerance

to food is increasingly appreciated. An altered microbiota and an early life dysbiosis were suggested in

food allergy pathogenesis with the possibility to be involved in disease persistence. The factors that

induce dysbiosis involved in food allergy are caesarian, lack of natural feeding, antibiotics use and low fibers diet or high-fat diet. The data suggest that dysbiosis occurs before the onset of clinical

manifestations of food allergy. Modulation of microbiota by probiotics and prebiotics represents an

encouraging strategy for prevention and treatment of food allergy.

1Corresponding author: Corina Cazan, Str. Pompeiu Onofreiu, Nr. 2-4, Sibiu, Romania, E-mail: [email protected], Phone: +40745

998833


Food allergy is a real health problem, and the

prevalence increased dramatically over the last decade. Food

allergy affects 6% of children under five years of age and 3% of

adolescents. Increasing prevalence of food allergy and that of other atopic diseases suggests an important role for

environmental factors in early childhood.(1)

The mechanisms of food allergy seem to be promoted

by gut dysbiosis. Recent studies report a pathogenetic role of gut

microbiota in the development of food allergy. The interaction

between gut microbiota and immune or non-immune cells

supports oral tolerance. The maturation of microbiota is related

to Th1/Th2 ratio, with a shift in Th1 cell response. Alteration of gut microbiota or dysbiosis, affects

microbiota homeostasis producing a shift of the Th1/Th2

cytokinic production, with a predominance in favor of the Th2

response.(1) Several factors responsible for gut microbiota alteration and the onset of food allergy are caesarian, artificial

feeding, drugs use such as antibiotics or gastric acidity inhibitors

and low fibers diet or high-fat diet.(2) The data suggest that the

use of antibiotics induces an increase in the prevalence of food allergy. Several studies demonstrated that neonatal or maternal

antibiotic treatment reduced intestinal microbial diversity in

both faecal and ileal samples and improved food allergen

sensitization. Antibiotic use during pregnancy and the treatment in

the newborns increase the risk of cow’s milk allergy in infants

and children.(1) Recent data support the concept that intestinal

dysbiosis or some changes or alteration in intestinal microbiota composition during early life can influence the development of

food allergy and other allergic diseases.(1)

DNA-based gut microbiota studies suggest the

existence of placenta microbiome in relation to the innate Toll-like receptor. This data correlates the maternal exposures during

pregnancy and the increased risk of allergic diseases. Some

authors demonstrated that in vaginal birth the skin and intestinal tract are colonized by Lactobacillus species.(3) Caesarian-born

babies have a bacterial pattern of the skin and intestinal tract

composed of Staphylococcus and Streptococcus species

overlapping with the bacterial strains on the mother’s skin. The cesarian-born infants have a higher risk to develop the allergic

diseases compared with vaginally newborn.(3) Several studies

demonstrated that the neonates have a complex gut microbial

communities in the first week of life, with dynamic fluctuations

in bacterial microbiota from 1 to 3 years of age.(4) Bifidobacterium, Proteobacteria, Bacteroides, and much less

Firmicutes (including the Lactobacillus spp., from the vaginal

flora) massively colonize the neonatal gut in the early life. In

neonates weighing less than 1200 g, gut microbiome is

dominated by both Firmicutes and Tenericutes and with much

less dominance of Actinobacteria. The collective data improve

the concept that the infant may be first seeded in utero by the

placenta-associated microbiome by length of gestation.(4) Another study, suggests that infants diagnosed with atopic

dermatitis, IgE-mediated, present an increased level of faecal

Escherichia coli.(5) Current data reported risk factors for

allergic disease including early-life antimicrobial exposure, caesarian birth and formula feeding.(6) Other studies support the

association of formula-feeding without prebiotics and allergic

diseases. Formula supplemented with prebiotics provides

support and diversity for Bifidobacterium longum, a protective species, in allergic children.(7,8)

Both maternal and neonatal gut microbiota in the early

postnatal life are considered as risk factors involved in allergic

diseases. This concept has been demonstrated for infants diagnosed with allergic disease and decreased Bifidobacterium

in the gut.(3) Allergic sensitization in early-life involved

microbiota imbalance in the stage of immunological

development.(3) Kalliomäki and colleagues presented the patterns of early microbiota linked to dysbiosis with increased

pathogenic agents and decreased commensal species involved in

development and maintenance of immune homoeostasis.(3,5)

The commensal gut microbiota target different cellular components of the innate and adaptive immune compartments to

promote oral tolerance. One mechanism by which the

commensal microbiota influence the outcome of the allergic response is by modulating the innate lymphoid cells

(ILC).(9,10) The commensal microbiota also targets the

adaptive immune response to promote tolerance, in part by

direct action on T cells and B cells. Depletion of the commensal flora by antibiotic treatment is associated with the development



AMT, vol. 22, no. 3, 2017, p. 26

of T helper type 2 (Th2) cell-type allergic responses and higher

serum IgE levels.(11) Another tolerance promoting mechanism

is based on short chain fatty acids (SCFAs) production. SCFAs are end-products resulting from bacterial fermentation of dietary

fibers.(12). A high-fiber diet protects against allergic

inflammation by altering the composition of the microbiome by

increased Bacteroidetes and decreased Firmicutes abundances with a subsequent increase in circulating levels of SCFAs.(13)

Gut Microbiota in Allergic Disease.

Novel research approaches based on next-generation

sequencing systems have highlighted potential biomarkers species in allergic patients. Bacteroidaceae and Clostridium

species were suggested by Melli et al.(14,15) as relevant

candidates. It seems that Bacteroidaceae are responsible for

intestinal mucin secretion while Clostridium strains are related to the immune response.

Early exposures in non-breast feeding infants to

Clostridium species have been proposed for an increased risk of

associated allergic conditions.(16,17) Moreover, an elevated ratio of Firmicutes vs Bacteroidetes were described in food

allergic infants.(9,18) Prediction of food allergy at one year of

age has been computed in a study group of 166 infants (the

Canadian Healthy Infant Longitudinal Development-CHILD). The patients were profiled for gut microbiota

composition at three months of age. Data were retrieved from

next gene sequencing reports denoting a higher proportion of

Enterobacteriaceae versus Bacteroidaceae and pointing to a possible parameter of immaturity.(9,19). Bacteroidaceae species

reaches to dominate the intestinal flora with increasing

age.(9,20) Bacteroidetes deficiency was revealed in pregnant

mothers of eczematous atopic infant but without correlation of the same microbiota deficiency in infants (9,21).

Probiotics and Prebiotics in Allergic Diseases.

Probiotics are considered as living micro-organisms

modulating the health of the host if given in appropriate amounts. Probiotics have a potential role in gut microbiota and

immune responses regulation. In the last decade, considerable

interest and efforts were invested in preventing and treating the

allergic diseases. Strains of Bifidobacterium bifidum, lactis,

breve or longum, Lactobacillus reuteri, rhamnsosus GG,

acidophilus, casei, salivarius and paracasei were evaluated in the

management of allergic disease.(22)

Although many systematic reviews tackled the probiotic supplementation to prevent allergic disease, there was

insufficient facts and statistics in the proposed panel of WAO

(World Allergy Organization) to sustain this approach for the

high risk pairs mother-child in the cases of allergic family history(22,23).

Moreover, there was no caveat concerning the type or

dose of the probiotic strain for clinical applications.(9,23).

However we found some research papers with positive data in this respect. For example, a meta-analysis performed by

Cao et al. showed a 14% prevalence reduction in atopic

dermatitis, administering probiotics in pregnancy and early infancy.(24) Another meta-analysis of 25 probiotic trials

reported significant clinical improvement in children receiving

probiotics. A new study on 43 children reported that a four-week

use of Lactobacillus salivarius convincingly alleviated clinical symptoms while a randomized trial of 40 infants claimed that

four weeks Bifidobacterium bifidum treatment notably

improved symptoms compared with placebo.(25,26,27). Still, in

infants there isn’t enough evidence to support the benefit of probiotic use (9,28). Furthermore, in the same note with WAO,

recent guidelines proposed by European Academy of Allergy

and Clinical Immunology, couldn't point out any premise to

recommend the use of probiotics for food allergy and

anaphylaxis prevention.(29) It seems that probiotic association

to oral immunotherapy is showing notably results (30) Prebiotics have the ability to stimulate probiotic

strains in gut microbiota. Oligosaccharides in human milk

provide beneficial effects in maintaining the balance of

intestinal microbiota in infants. The synthetic oligosaccharides are added as a supplement to infant formulas to foster gut

microbiota development.(31)

CONCLUSIONS New intervention targets are needed to document

alteration or change of intestinal colonization and involvement

of the microbiota in the pathogenesis of allergic disease.

Acknowledgement: Part of this work has been conducted in the Pediatric



project (ID 928 SMIS-CSNR 13605) financed by ANCSI with the grant number 432 / 21. 12. 2012 thru the Sectorial Operational

Program “Increase of Economic Competitiveness”.

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11. Hill DA, Siracusa MC, Abt MC, et al. Commensal bacteria-derived signals regulate basophil hematopoiesis and

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12. Smith PM, Howitt MR, Panikov N, et al. The microbial

metabolites, short-chain fatty acids, regulate colonic Treg

cell homeostasis. Science 2013, 341:569– 573. 13. Trompette A, Gollwitzer ES, Yadava K, et al. Gut

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MB, Gut microbiota and allergic disease in children, Ann

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D, de Morais MB, Intestinal microbiota and allergic diseases: a systematic review, Allergol Immunopathol

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16. Almqvist C, Oberg AS. The association between caesarean

section and asthma or allergic disease continues to

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on clinical effects of probiotic Lactobacillus salivarius LS01 in children affected by atopic dermatitis. J Clin

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http://refhub.elsevier.com/S1081-1206(15)00684-5/sref26


















































































































AMT, vol. 22, no. 3, 2017, p. 28

MICROBIOTA AND THE IMMUNE SYSTEM, A COMPLEX

LIAISON

IOANA MĂTĂCUŢĂ-BOGDAN

1

1“Lucian Blaga“University of Sibiu, Pediatric Clinical Hospital of Sibiu, Research and Telemedicine Center in Neurological

Diseases in Children

Keywords: microbiota,

immune system, complex liaison

Abstract: Based on millennia of symbiotic relationship, gut microbiota and the human organism can be

viewed as a whole. Recent evidence support the fact that microbiota shapes the human organism at many levels: tissues, cells, immune system and connection between them. It appears natural that such a

long complex and dynamic interdependence in not yet fully understood. The relationship goes up to the

molecular level and one of the major implications is the intimate liaison between microbiota and

immune system with divergent effects on several aspects such as: regulatory and modulatory effects, shaping both local and systemic immune mechanisms, implication in immune disorders, cancerogenesis

and immune therapy.

1Corresponding author: Ioana Mătăcuţă-Bogdan, Str. Pompeiu Onofreiu, Nr. 2-4, Sibiu, Romania, E-mail: [email protected] Phone:

+40740 024274


INTRODUCTION

GUT MICROBIOTA

Over the years more and more evidence appear

concerning the complex relation between the microbiota and the

immune system. The human organism, the microbiota and the immune system interconect in ways far from being completly

descovered. Every study in the field brings more and more

arguments of the remarkable link between these components.

The gut microbiota and the human body can be considerred a meta-organism – community of biological entities

that comunicate between them. Microbiota reunites an enormous

number of bacteria, viruses, fungi and other microbial species.

(1, 2, 3) The mathematically calculated number of cells in the human body is 3,72 x1013.(4) Over 100 trillion cells compose

the gut microbiota, which outnumbers host’s cells, with a

complexity of 500-1000 species (2), 7000 strains (3) and over

600.000 genes (compared to the 22.000 genes of the human body). (4)

The microbiome represents a biomass of aproximately

2 kilograms.(5,6) Adding the enormous enzymatic capabilities

the posibilities of interaction with the host are unlimited and not only in terms of digestion, nutrients production, detoxification,

defense against pathogens, but also regulatory function over the

immune system.(1,7)

Relationship between the microbiota and the human host depends of many factors such as: genetic particularities of

the host, co-infection, nutritional conditions. Mutualistic,

parasitis and commensal interaction can be described based on

the benefits of both the host and the microbe. Therefore, mutualism implies benefits for both species involved, parasitism

implies benefits only for the micoorganisms obtaining nutrients

from the host and commensalism defines a relationship with benefits for the microorganism without harming the host.(1)

Inside the microbiota organisms form complex network of

microb-microb relationships.(8)

Recent studies support the evidence that human are not born as sterile as thought before, amniotic fluid, placenta,

meconium and umbilical cord being populated with microbes

probably implicated in immune-tolerance towards commensal bacteria.(8,9,10,11)

Bacterial colonization acquierd at birth depends on

various factors: genetic factors, maternal inflammatory

conditions (11), type of birth, gestational age, infant’s type of food, use of antibiotics.(7) Vaginally delivered infants are

colonized with bacterial species similar to mother’s vaginal and

faecal microbiota such as Lactobacillus spp., Prevotella spp. and

Sneathia spp. For caesarean-section born infants the first contact is with bacterial species found on the skin Staphylococcus spp.,

Corynebacterium spp. and Propioni bacterium.(7,8)

Preterm infants have a dominant colonization with

Clostridium difficile (7), breast-milk fed infants have microbiota preferentially colonized by Bifidobacterium while formula-fed

infants have gut colonization with Staphylococcus, E.coli,

Bacteroides, Lactobacilli.(1,7)

Both diversity and stability of the microbiota acquired at birth are low, manifests high instability and suffers

modifications during the first year of life, becoming stable in

several years (5,8,12,13) and gains an incredible resistance in

front of aggression, phenomenon known as “colonization resistance”.(1,8)

The composition of gut microbiota has interpersonal

variations and can be modified by a number of factors such as:

dietary factors, vaccination, hygiene practices, use of antibiotics and xenobiotics.(7,8,11)

Gut microbiota is a major palyer in immune

homeostasis, having a profound impact on host both innate and

adaptative, local and systemic immunity.(1,7,8,12) The relationship between the host’s immunity and microbiota is

bidirectional – the immune system controls the microbiota and

the microbiota has the capacity to calbrate and modulate the immune system.(1,14)

INTERACTIONS BETWEEN THE IMMUNE SYSTEM

AND MICROBIOTA

Digestive tract is a lymphoid organ, the lymphoid structures are reunited in GALT (gut-associated lymphoid

tissue) and MALT (mucosal-associated lymphoid tissue) and



AMT, vol. 22, no. 3, 2017, p. 29

have tree major locations: Peyer’s patch, lamina propria and

intraepitelial follicules.

All levels of innate immunity are implicated in a relationship with microbiota, on both level of development and

function. The influence of microbiota over the immunity can be

demonstrated by using germ-free models where animals are

reared in a sterile enviroment, without being exposed to microbes.(7,15)

The development of lymphoid structures is orchestrated by

the gut microbiota.(15)

- Antigen presenting cells of the Peyer’s patches and microbiota are interconnected, so antigen presenting cells

manifest immune tolerance to normal gut microbiota, and

microbiota has regulatory effects on these cells

development Under the same conditions of stimulation these cells produce higher levels of IL-10, compared to

antigen presenting cells from the spleen.(7)

- Macrophages from the microbiota proximity have a

modified phenotype, responsable of „inflammation anergy”.(7)

- M cells are intestinal epitelial cell, specialized for the

phagocytosis and transcytosis with the ability to induce

specific immune responses in the Peyer’s patches.(16) - A particular class of natural killer cells (IL-22+

, NKp46+) is

selected over the influence of microbiota. (7)

- Intestinal Epitelial Cells production of antimicrobial factors

are regulated by microbiota.(7) Microbiota and acquired immunity:

- All subtypes of T CD4 cells (Th1, Th2, Th17, T reg) have

development and activities regulated by microbiota which

has an important role in the development of T cells, certain bacterial species induce development of a certain T cell

subtype. IL-17 and IL-22 produced by Th 17 are the main

effectors that contribute to the maintenance of the

intestinal homeostasis.(1,7) - The number and cytotoxicity of T CD8+ of the intestine are

adjusted permanently by interaction to microbiota.(7)

- B cells located in Peyer’s patches need permanent stimulation

from the gut microbiota in order to maintain a normal number

and IgA production. In germ-free models was demonstrated the

reduction of the number, cellularity and IgA production in the

studied animals. Studying the same models it has been showed

that plasma levels of Ig A and Ig G are low, plasmatic Ig M levels are normal, while Ig E levels are high both in the intestine

and in plasma.(1,7) A relationship between the microbiota

composition and the cells producing Ig A was discovered, so a

reduction of the nmber of Ig A producing cells was noted when the animal was appendectomized and colonized, demonstating

the liaison between the microbiota and the development of the

immune system. (3)

IMPLICATIONS OF GUT MICROBIOTA IN IMMUNE

DISEASES

Because of the influences of gut microbiota over both

local and systemic immune responses, it has been linked to a number of immune and autoimmune, gastro-intestinal and

extraintestinal diseases.(7,15) The mechanisms involved are

complex and not fully understood.

The most studied entities are Crohn disease and ulcerative colitis, reffered to as inflamatory bowel disease and

linked to the microbiota. Etiology of inflamatory bowel disease

is very complex and reunites genetic factors, immunological

factors concerning the host, environmental factors, infections, stress and microbiota.(1,3,17,18,19) Hygiene hypothesis appears

to be implicated - recent studies show that improved health

measures determine intestinal immunological distrurbances

inducing autoimmunity.(5)

More and more evidence suggest that dysbiosis is not

only present but specific to this disease. Some studies show that normal microbiota contributes to the inflammation present in

inflamatory bowel disese, while other studies indicate

inflammation itself as the cause of dysbiosis.(1,7)

Another attractive idea is that commensal bacteria with high inflammatory abilities can contribute to the disease by

stimulatind both the innate and aquired immune responses to

different antigens and even commensal bacteria, which can be

demonstrated by the high levels of antibody against antigens from the microbiota sources.(1,20,21,22, 23)

Based on of the Crohn disease phenotypes it was

possible to classify the microbiota, recent studies showing that

in ileal and colonic forms of the disease the microbiota is distinct.(14,24) This ascertainment permitted the conclusion that

different mechanisms are involved for different clinical

manifestations.(14,25)

Dysbiosis was recently related to rheumatoid arthritis, especialy in patients with an under six months duration of the

disease. The liason between gut microbiome and arthritis in

reffered as the „gut-joint axis’ hypothesis”.(14) In this disorder

microbiota can play a both inhibitory and augmentatory role based on the imunological target: Th 17 lymphocites or T reg.(7)

In a murine model it was demostrated that the reduction of Th

17 response associates with a reduction of disease activity. (1)

Ankylosing spondylitis has, also been linked to the gut microbiota, based on the observation that one tenth of the people

with ankylosin spondylitis develop in time one form or another

of inflamatory bowel disease, up tu 70% of of the persons

diagnosed with spondylitis have gut inflamation. Dysbiosis in the cecal microbiome could be demonstrated in these cases, and

even more, a association with HLA-B27 and a increasead Th17

population in the mucosa.(7,26)

Dysbiosis appears to be involved as a causative factor in systemic lupus eritematosus, as shown in recent studies using

murine models.(14)

Psoriasis and the psoriatic arthritis were associated

with modifications of gut microbiome similar to those observed

in Intestinal bowel disease and with high levels of secretory

IgA.(14,27)

Type 1 diabetes was linked to the reduction of Tregs

in the intestine.(7) Incidence of type 1 diabetes is higher for nonobese diabetic mouse in germ-free models, while

colonization with SFB (segmented filamentous bacteria) offers

protection to the disease.(1)

CANCER AND THE LINK TO MICROBIOTA The International Agency for Cancer Research

(IACR) has indicated that 10 microorganisms are carcinogenic

to humans, the mechanisms involved concerning either the host

or the microorganism. They are responsible for almost one fifth of the malignancies that appear in humans.(28)

Intestinal cancer and colo-rectal cancer are the result

of gastrointestinal inflammation due to the immune stimulatory effect of commensals and the infiltation of intestinal tumors with

immune cells.(1,5,29,30,31,32,33)

Liver cancer was linked to microbiota by more

mechanisms: microbiota can induce DNA damage by specific bile acids (1, 34), the breakdown of intestinal barrier can induce

hepato-carconima (35), and endotoxemia can promote it’s

progression.(36)

OTHER ASSOCIATIONS WITH MICROBIOME Other associations between microbiota and

pathological states could be established. Obesity and metabolic

syndrome (5,37), circulatory disease (5,38), drug metabolism



AMT, vol. 22, no. 3, 2017, p. 30

and toxicity (L- DOPA, sulfasalazine, digoxin, simvastatin)

(5,39,40), post surgical recovery and autism (5,41) were related

to the gut microbiome.

CONCLUSIONS

All the implications of gut microbiota in the complex

aspects of immune system are, most certain far from being completely discovered. More and more connections and

interconnections appear permanently due to the new and

advanced technologies used in studying them. It seems logical

that an ecosystem millennia old offers surprising connections. The immune system is still under a continuous process of

discovery, so new liaisons, implications and revelations are

possible at any time.

Microbiota is implicated in different aspects of the immune system and the immune system controls and

orchestrates the microbiota.

Dysbiosis has been linked to many pathological states,

immune and autoimmune disorders, allergy, cancerogenesis, metabolic syndrome, multiple sclerosis and rheumatological

diseases.

Acknowledgement:

Part of this work has been conducted in the Pediatric Clinical Hospital Sibiu, within the Research and Telemedicine


project (ID 928 SMIS-CSNR 13605) financed by ANCSI with the

grant number 432 / 21. 12. 2012 thru the Sectoral Operational Programme “Increase of Economic Competitiveness”.

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AMT, vol. 22, no. 3, 2017, p. 32

RISK FACTORS FOR BRONCHIOLITIS IN INFANTS AND THE

ROLE OF FECAL MICROBIOTA PROFILES

OLGA CÎRSTEA

1, OXANA TURCU

2, NINEL REVENCO

3

1,2,3„Nicolae Testemițanu” State University of Medicine and Pharmacy, Chișinău, Republic of Moldova

Keywords: infant,

bronchiolitis,

microbiota, lung health

Abstract: The significant burden of lower respiratory tract disease in infancy determines emerging

studies aimed to find new therapeutic and preventive measures. One of the most frequent and potentially

severe lower respiratory tract diseases is bronchiolitis that affect mainly infants under six months of age,

but leading also to long-lived structural alterations of airways and immune cells mediated susceptibility toward allergic sensitization and asthma. Experimental models showed promising results in

demonstrating the important role of the host microbiota in influencing the function and development of

the child immune system. Another authors showed interesting correlations between maternal diet during

pregnancy and immune system function in infant, including protective factors against severe bronchiolitis. In conclusion, revealing risk factors and protective immune mechanisms at an early age

may lead to new opportunities for targeted interventions in children and decrease the morbidity from

lower respiratory tract diseases in early years.

1Corresponding author: Olga Cîrstea, Str. Grenoble, Nr. 149, MD-2060, Chișinău, Republic of Moldova, E-mail: [email protected], Phone:

+37369038523


INTRODUCTION

Globally, acute lower respiratory infection is the

leading cause of childhood mortality and the highest

hospitalization rates among infants, especially between 30 days

and 60 days of age, is due to bronchiolitis.(1,2) Bronchiolitis is a

frequent lower respiratory tract disease of infancy, being mainly

a viral disorder. Clinical manifestations are determined by

morphological changes in the airways, specifically by inflammatory changes and swelling of the mucosa, and mucus

hypersecretion in bronchioles. Evolution of the diseases may

vary from moderate severity symptoms to progressive

respiratory distress caused by obstruction of lower airways. Factors associated with an increased risk of progression to

severe disease or mortality include congenital heart disease,

bronchopulmonary dysplasia, congenital anomalies of the

airways, maternal smoking, immunocompromised host.(3) Viruses that cause bronchiolitis include human rhinovirus,

influenza, parainfluenza viruses, adenovirus, human

metapneumovirus, but the most common is respiratory syncytial

virus (RSV).(4) The vast majority of children (about 90%) gets infected with RSV in the first years of life, and amongst them up

to 40% will develop lower respiratory tract infection.(5)

Respiratory syncytial virus is also found to be one of the most

important risk factors for childhood asthma onset. Therefore, one the most intriguing research area nowadays is to find

underlying immunoregulatory and antiviral pathways that may

have a protective role and potentially may decrease the

susceptibility to both RSV-bronchiolitis and asthma.(6,7,8) A new area of interest is the gut–lung axis, in which

the fecal microbiota participates regulatory in the immunologic

responses to environmental challenges in the lungs, including infectious agents, allergens or endogenous antigens. Until

recently, in the absence of an active infectious process such as

pneumonia or bronchiolitis, much of the respiratory tract was

assumed to be sterile. New emerging data from research tend to demonstrate different physiologic and immune cross-linked

mechanisms between different organs and fecal microbiota.(9)

Hasegawa et al. (10) in a recently published study have revealed

specific changes in the nasopharyngeal microbiota in infants

with RSV-bronchiolitis. However, it is not clear yet whether

changes in the composition of the gut and/or lung microbiota

predisposes to RSV-bronchiolitis or is simply a consequence of

disease.

Immediately after birth, the newborn is colonized by a diversity of environmentally associated microbiota such as

microbes derived from the mother’s vagina and feces during

vaginal delivery. In the first hours of life the breast milk further

diversifies the neonatal microbiota by providing milk oligosaccharides and other microbial metabolites such as short-

chain fatty acids (SCFAs).(11,12) Interestingly, in infants born

by Cesarean section, the gut microbiota profile correlates mostly

with bacterial populations of the mother’s skin, this finding seen also in formula-fed infants.(11,12,13,14) Short-chain fatty acids

serve as a significant source of energy in the gastrointestinal

tract for both the local microbiota and intestinal epithelial cells,

and also have a variety of regulatory immune functions.(15) Interestingly, the supplementation of infant formulas with

prebiotics or probiotics does not fully reproduce the effect of

breast milk on the development of the gut microbiota in the

newborn.(16) Nevertheless, there are rapid changes of the gut microbiota in response to environmental factors that may affect

immune homeostasis.(17)

Experimental studies on mice demonstrated the key

role for the host microbiota in influencing the function and development of the immune system.(18,19) Emerging studies

suggest that microbial metabolites such as SCFAs, acetic acid,

butyric acid, and propanoic acid play an important role in regulating host physiology and immunity: by the mechanism of

histone deacetylases inhibition they stimulate the formation of a

tolerogenic, anti-inflammatory cell phenotype; also, peripheral

blood mononuclear cells and/or neutrophils being exposed to acetate, propionate, or butyrate consecutively suppress NF-κB



AMT, vol. 22, no. 3, 2017, p. 33

and downregulate the production of the pro-inflammatory

cytokine TNF-α.(20,21,22)

Hasegawa et al.(23) studied fecal microbiota profiles in 40 infants with bronchiolitis in a case-control study, who

were compared with 115 healthy children of the same age.

Authors identified four distinct types of bacteria that prevail,

such as Escherichia in 30% of children, Bifidobacterium in 21% of children, Enterobacter associated with Veillonella species

(22%), and in 28% microbiota consisted of Bacteroides species.

Interestingly, Bacteroides species was the type of bacteria

associated with bronchiolitis, as authors concluded based on the results of the multivariate analysis. Moreover, an inverse

correlation was found for Escherichia and Bifidobacterium

species.(23) This is an important insight into the future

therapeutic approaches aimed to modulate gut microbiota in order to decrease the rate of acute respiratory infections in

children.

Conversely, Sjögren et al.(24) reported an association

in early infancy of high quantity of Bacteroides fragilis in the gut with reduced levels of proinflammatory mediators in the

blood.

Another plausible hypothesis is that of the reverse

causation, meaning that viral infection influence the gut microbiota and lead to intensive growth of Bacteroides species

in the intestine.(25) Moreover, other research groups reported an

association of these species of gut bacteria higher morbidity in

early infancy.(26) The underlying mechanism of this correlation remains to be elucidated, but these observations bring more

evidence to support the hypothesis that bronchiolitis in infants is

associated mostly with deficit of gut microbiota rather than

microbial diversity.(27) Consequently, a deeper understanding of the diversity

of factors that determine susceptibility to bronchiolitis at an

early age may lead to new opportunities for targeted

intervention, including primary prevention. Existing up to date evidence highlights the positive influence of the microbiome on

the maturation of the immune system of the child. Accordingly,

clinical and preclinical studies results may indicate alternative

approaches aiming to reduce the risk and severity of viral

bronchiolitis in infants by modifying the infant microbiota.

Nevertheless, it is important to highlight the role of the

breast milk. Besides being a primary source of nutrition for the

child it also contains different immune factors that affect the immune response. Studies showed that in breast-fed infants the

pH of the intestinal contents is acidic (pH=5.0), whereas in

formula-fed infants the pH is neutral (pH=7.1).(28)

Oligosaccharides from the milk represent the major factor that maintains the growth of Bifidobacteria and consequently they

may influence gastrointestinal and circulating SCFAs levels.(29)

This way, milk oligosaccharides presumably have two roles –

one is to act as prebiotics by supporting selectively the growth of commensals that are more competitive comparing with

potentially pathogen bacteria, and secondary milk

oligosaccharides have direct anti-pathogenic and pro-tolerogenic roles in gut mucosa by acting as glycan receptor decoys for

microbial adhesion factors and preventing pathogen

attachment.(29)

Moreover, in several epidemiological studies researchers tried to link the probability of a severe RSV

bronchiolitis in infants who are not breast-fed, although this

finding is not universal.(30,31,32,33) All these studies

demonstrate that breast milk is one of the most important environmental determinants of bacterial colonization and may

significantly influence the immune homeostasis and postnatal

development of gut microbiota and lung health.

In addition to these mechanisms, an important

research area is the composition of the maternal intestinal

microbiota in relationship with the infant intestinal microbiota and lung health.(34) It was studied the association of maternal

transient colonization during gestation and immune responses in

the offspring. For this purpose, pregnant germ-free mice were

inoculated during late gestation with a bioengineered strain of Escherichia coli (HA107) that was cleared from the intestine

within 72 hours.(35) Noticeably, results of the study showed that

colonization with E. coli during last gestational months was

associated with a better intestinal immune system in the child, manifested by increased levels of group 3 innate lymphoid cells

and F4/80+ CD11c+ mononuclear phagocytes, the latter

potentially may inhibit the inflammatory processes in the

intestine, as animal studies showed. Moreover, experimental models showed that maternal gut microbiota during the

pregnancy period influence positively the child’s systemic

immune response. This hypothesis was confirmed by the

improved immune response within the child spleen following intraperitoneal administration of lipopolysaccharide.(36)

However, other studies failed to find an association

between SCFAs and the numbers of mononuclear cells and the

group 3 innate lymphoid cells in the offspring’s intestine.(37) Interestingly, Ferolla et al.(38) found an interesting correlation

between maternal dietary preferences and child lung health in a

prospective birth cohort study that included more the fifty

thousand children. The highest number of children amongst those who had a respiratory infection (1293 patients) were

diagnosed with RSV infection (>60%); human RV was found in

22% of cases and influenza in 4% of children. One intriguing

result of the study was that the a more severe form of bronchiolitis developed children with RSV who were born from

mothers whose diet was rich in carbohydrates and poor in fruits

and vegetables. The causative relation is not known but one

plausible hypothesis is that this diet may alter maternal gut microbiota and consequently influence negatively fetal systemic

immune response.(38)

CONCLUSIONS Various studies aimed to reveal the role of gut

microbiota on lung health and protection against bronchiolitis in

children. However, this pathogenetic mechanism continues to be

a subject of ongoing and future research because molecular pathways and networks between the gut microbiota and lung

mucosal immunity may become a potential therapeutic target in

the complex treatment of bronchiolitis in early life.

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AMT, vol. 22, no. 3, 2017, p. 35

GUT MICROBIOTA AND IRRITABLE BOWEL SYNDROME

CORINA CAZAN

1

1“Lucian Blaga” University of Sibiu, Pediatric Clinic, Research and Telemedicine Center of Neurological Diseases in Children

Keywords: gut

microbiota, dysbiosis,

irritable bowel

syndrome

Abstract: Irritable bowel syndrome is defined as chronic or recurrent abdominal pain in the absence of

a demonstrable pathology. The mechanisms that explain the role of microbiota in the development of IBS

include altered composition of the microbiota, mucosal immune activation, gut inflammation, small

intestinal bacterial overgrowth, dysbiosis, increased intestinal permeability and impaired mucosal barrier function. Modulation of the intestinal microbiota through dietary changes and use of probiotics,

prebiotics and antibiotics has been proposed for IBS treatment strategy.

1Corresponding author: Corina Cazan, Str. Pompeiu Onofreiu, Nr. 2-4, Sibiu, Romania, E-mail: [email protected], Phone: +40745

998833


Chronic abdominal pain has an increased prevalence affecting up to 25 % of schoolchildren and teenagers. Using

conventional diagnostic methods, the majority of children with

chronic abdominal pain are diagnosed as abdominal pain-related

functional gastrointestinal disorder classified as functional dyspepsia, functional abdominal pain and irritable bowel

syndrome (IBS) and that is the most prevalent, affecting

approximately 60 % of children. IBS is phenotypically

subtyped, using the Rome III criteria, into IBS with constipation

(IBS-C), IBS with diarrhea (IBS-D), IBS mixed (IBS-M) and

IBS un-subtyped (IBS-U). In irritable bowel syndrome are

several factors that play an essential role in the symptom

expression including: visceral hyperalgesia, intestinal hyperpermeability, dysmotility, altered barrier function, gut

microbiome composition, low-grade immune activation, food

intolerance, colonic bacterial fermentation, gut inflammation,

genetics, environmental factors and psychosocial distress.(1,2) In the last decade, advances registered and reported in the

pathophysiology of irritable gut syndrome have revealed that the

alterations in intestinal microbiota are significantly implicated in

pathogenesis.(3) Dysbiosis as an alteration of the gut microbial balance

occurs as a cause in functional disorders and thus in irritable

bowel symptoms. Quantitative imbalance in the composition of

small gut microbiota defines intestinal bacterial overgrowth (SIBO). Quantitative changes cause diarrhea, abdominal pain,

bloating, excessive gas, malabsorption and severe motor

dysfunction.(5) Changes and alteration at the colonic microbiota

cause excessive gas production and excessive short chain fatty acids. Methane gas, carbon dioxide and hydrogen gas have as

clinical expression symptoms like abdominal pain and

abdominal distension. The increase of acetate butyrate and

propionate short chain fatty acids has as a consequence alteration electrolyte and water transport through the process of

acidification and deconjugation of bile acids. The carbohydrate

diet causes excessive hydrogen gas predictive for IBS-D and excess of methane gas production predictive for IBS-C.(4,5) The

gastrointestinal tract is first colonized in the intrauterine period

through the placental barrier transfer. Beneficial factors that

influence gut colonization are maternal microbiota, genetics and environmental factors, geographical origin, diet before and

during pregnancy and breast feeding. At birth, the infant intestinal tract is colonized by aerobic species and gradually

becomes anaerobic over a period of days. Recent data report that

microbiota composition has over 35,000 species and 70%

colonized the colon. The small intestine is colonized mainly by aerobic bacteria, and the large intestine is colonized by Gram-

negative and strictly by anaerobic bacteria.(4)

Dysbiosis as an alteration of the gut microbial balance

occurs as a cause in functional disorders and thus in irritable

bowel symptoms. Quantitative imbalance in the composition of

small gut microbiota defines intestinal bacterial overgrowth

(SIBO). Quantitative changes cause diarrhea, abdominal pain,

bloating, excessive gas, malabsorption and severe motor dysfunction.(5) Changes and alteration at the colonic microbiota

cause excessive gas production and excessive short chain fatty

acids. Methane gas, carbon dioxide and hydrogen gas have as

clinical expression symptoms like abdominal pain and abdominal distension. The increase of acetate butyrate and

propionate short chain fatty acids has as a consequence

alteration electrolyte and water transport through the process of

acidification and deconjugation of bile acids. The carbohydrate diet causes excessive hydrogen gas predictive for IBS-D and

excess of methane gas production predictive for IBS-C.(4,5)

Pathophysiology.

1. Dysbiosis. Current research suggests that gut microbiota ecosystem changes or dysbiosis have potentially

significant effect in the pathogenesis of gastrointestinal tract

disfunction and also in irritable bowel syndrome.(6,7) Alteration

in gut microbiota facilitates the attachment of pathogenic bacteria to the wall of the enteric tract, as a concept supported

by recent studies in IBS. The normal composition of the

microbiota includes Firmicutes, Bacteroidetes and especially

Bifidobacteria. Recent survey reports significant alteration of the normal intestinal microbiota composition in patients

diagnosed with irritable bowel syndrome.(4) Another study

reported significant changes in the ratio between the main species of intestinal microbiota composition in these patients.

The data reported maintain that the ratio of Firmicutes to

Bacteroidetes increases twice while Bifidobacterium decreases

quantitatively.(8) The level of inflammatory cytokines IL6 and IL8 increases significantly in relation to an excess of



AMT, vol. 22, no. 3, 2017, p. 36

Enterobacteriaceae in irritable syndrome.(9) In several studies

culture, based analysis the composition of microbiota suggests

an important imbalance for Lactobacillus and excess in lumen versus enteral mucosa.(9)

2. Postinfectious IBS (PI-IBS).

Postinfectious IBS occurs following an acute episode

of enteral infection sustained by abdominal pain, abdominal discomfort fever, vomiting and then persistent diarrhea

concerning psychological factors including anxiety and

depression. Recent studies estimate prevalence in PI-IBS at 3-

30% of subjects with persistent diarrhea after an acute gastroenteritic episode. The most common infectious agents

involved in the development of PI-IBS are Salmonella, Shigella,

Campylobacter, Staphylococcus, Clostridium difficile and

perfringens or viruses.(4) Several reports are indicating that patients diagnosed

with traveller's diarrhea develop over time post infectious

irritable bowel syndrome with a prevalence of 7-14%.The

current concept regarding the pathogenic mechanisms for postinfectious irritable gut syndrome suggests that this entity is

correlated with a lot of factors such as: 1.alteration of gut

motility, 2.increased intestinal permeability, 3.dysbiosis after the

use of antibiotics, 4. an increased numbers of enterochromaffin cells, 5.genetic predisposition, 6. determinant psychological

factors, 7. persistent of the inflammatory process , 8. continuous

activation of the immune system.(4,9) Increased gut

permeability and persistent of inflammatory process induce activation of the immune system characterized by an increasing

number of T lymphocytes, macrophages, mast cells and an

increased expression of inflammatory mediators and cytokines

such as IL2, IL6 and IL10. The results suggest an exposure to pathogenic agents that disrupt the barrier function, alter the

neuromuscular integrity and support the inflammatory process

by releasing inflammatory cytokines and mediators such as

IL1α, IL1β, IFNγ which sustains irritable bowel syndrome symptoms. Dysbiosis or alteration of the echogenic microbiota

occurs in post infectious irritable intestinal syndrome after using

antibiotics and resulting in suppression of antimicrobial

peptides.(4,9) Bacterial fermentation of undigested nutrients

induces a bacteriostatic effect by releasing short chain fatty

acids which decrease intraluminal pH.(4,9)

3. Excessive bacterial growth in the small intestine.

The composition of microbiota and quantitative representation differ significantly in the duodenum, jejunum,

proximal or terminal ileum and in the cecum compared to the

colon.(10) Intestinal microbiota colony counts balance is

maintained through mechanisms that include the presence of stomach acid, normal gastrointestinal motility, enzymes,

mucosal immunity and the integrity of the ileocecal valve.

Characteristic for small intestinal bacterial overgrowth (SIBO) is

the presence of more than 105 colony forming units/ml in the jejunum with symptoms and signs included bloating, diarrhea,

flatulence and malabsorption of nutrients. Symptoms and

nutrient deficiencies are induced by excess intraluminal small intestinal bacteria results from the fermentation of nutrients

producing gas, alteration of intestinal motility, alteration of the

immune response, hypochlorhydria, bile salt deconjugation

resulting in the suppression of the bacteriostatic effect of pancreatic enzymes and bile acids.(4,10) Excess intestinal

bacteria determine fat malabsorption causing steatorrhea or

secretory diarrhea. SIBO is associated with malabsorption and

irritable bowel syndrome. Lactulose and glucose breath tests are noninvasive diagnostic tests. Use of lactulose causes excess gas

hydrogen and methane in the proximal intestine which is

eliminated by the respiratory tract and detected by the breath

test. The bacteriological examination, the culture of the jejunal

aspirated sample is accepted and recognized as a standard

diagnosis in dysbiosis.(4,10) 4. Altered Immune Response. The current data support the

hypothesis that irritable bowel syndrome may be a condition of

low-grade inflammation and abnormal immune response.

Microbiota has a crucial role in the modulation of the immune response. Commensal bacteria including Bacteroidetes and

Firmicutes interact with intestinal epithelial cells and modulate

immune responses. Several studies demonstrate that Th1 and

Th2 have a different role in the mediation of immune response and confirm that Th1 and Th2 cells function interact to maintain

a balanced immune response. The available data suggest

increased levels of pro-inflammatory cytokines including IL-1β,

TNF-α, IL-6 and IL-8 in patients with IBS.(4)

Therapeutic strategies in IBS.

Probiotics are living micro bacteria with beneficial

effects on gut microbiota if given in appropriate quantity.

Recent studies reported that probiotics use is associated with better outcomes in irritable bowel syndrome. The benefits and

directions of action, in the context of dysbiosis and

inflammatory changes, include suppression of pathogens and

their adhesion effect, improvement of epithelial barrier function, colonic motility and immune response. Bifidobacterium infantis

an intensively studied strain in IBS, reduces the level of pro-

inflammatory mediators such as TNF-alpha, IL-6 and IL-12.

Lactobacillus has a similar effect on the IL-12 and IL-10 cytokine balance.(4,11) The essential characteristics of

probiotics are: stability in gastrointestinal tract without side

effects and genetic stability.(4,12) Other studies, which use the

probiotics added to milk for eight weeks, report a significant improvement in abdominal pain, discomfort, distension and

bowel movement.(4,12)

Prebiotics are undigested carbohydrates which are

designed to stimulate the growth and activity of beneficial bacteria, mostly Bifidobacterium and Lactobacillus. The

fermentation of prebiotics released short chain fatty acids as a

nutritional support for enterocytes.(13) Prebiotics include in

composition galactooligosaccharides, fructooligosaccharides

and inulin.(4,14)

Antibiotics. Rifaximin, derived from rifamycin, with

minimal systemic absorption and intraluminal high level, is

recommended in dysbiosis and small intestinal bacterial overgrowth.(4,15) The probiotic administered in combination

potentiates the effect of the antibiotic to improve the

symptoms.(4,16,17)

CONCLUSIONS In the last decade, advances in gastrointestinal

pathophysiology indicate the role of gut microbiota and

dysbiosis as relevant factors in IBS symptoms. Improved understanding of the microbiota especially in IBS leads to future

therapeutic strategies targeting the modulation of gut microbiota

composition. Acknowledgement:

Part of this work has been conducted in the Pediatric


Center in Neurological Diseases in Children - CEFORATEN project (ID 928 SMIS-CSNR 13605) financed by ANCSI with the

grant number 432 / 21. 12. 2012 thru the Sectorial Operational

Program “Increase of Economic Competitiveness”

REFERENCES 1. Chumpitazi Bruno P, Shulman Robert J., Underlying

molecular and cellular mechanisms in childhood irritable



AMT, vol. 22, no. 3, 2017, p. 37

bowel syndrome, Molecular and Cellular Pediatrics 2016.

2. Rajilić-Stojanović M, Jonkers DM, Salonen A, Hanevik

K, Intestinal Microbiota And Diet in IBS: Causes, Consequences, or Epiphenomena?, Am J Gastroenterol

2015.

3. Bhattarai Y, Pedrogo DAM, Kashyap PC, Irritable bowel

syndrome: a gut microbiota-related disorder?, American Journal of Physiology - Gastrointestinal and Liver

Physiology, Vol. 312 no. 1, 2017, G52-G62.

4. Ghoshal UC, Shukla R, Ghoshal U,

Gwee KA,

Siew C.

Ng, Quigley EM, The Gut Microbiota and Irritable Bowel Syndrome: Friend or Foe?, International Journal of

Inflammation, Article ID 151085, 2012.

5. Bures J, Cyrany J, Kohoutova D. et al., Small intestinal

bacterial overgrowth syndrome, World Journal of Gastroenterology, vol. 16, no. 24, 2010, pp. 2978–2990.

6. Bennet SMP, Öhman L, Simrén M, Gut Microbiota as

Potential Orchestrators of Irritable Bowel Syndrome, Gut

and Liver, Vol. 9, No. 3, 2015, pp. 318-331. 7. Shankar V, Reo NV, Paliy O, Simultaneous fecal microbial

and metabolite profiling enables accurate classification of

pediatric irritable bowel syndrome, Microbiome, 2015.

8. Ponnusamy K, Choi JN, Kim J, Lee SY, Lee CH, Microbial community and metabolomic comparison of irritable bowel

syndrome faeces, Journal of Medical Microbiology, vol.

60, no. 6, 2011, pp. 817–827.

9. Saulnier DM, Riehle K, Mistretta TA et al., Gastrointestinal microbiome signatures of pediatric patients

with irritable bowel syndrome, Gastroenterology, vol. 141,

no. 5, pp. 2011, 1782–1791.

10. Chandra S, Dutta U, Noor MT et al., Endoscopic jejunal biopsy culture: a simple and effective method to study

jejunal microflora, Indian Journal of Gastroenterology,

2011, pp. 1–5.

11. Lee BJ, Bak YT, Irritable bowel syndrome, gut microbiota and probiotics, Journal of Neurogastroenterology and

Motility, vol. 17, no. 3, 2011, pp. 252–266.

12. O’Mahony L, Mccarthy J, Kelly P et al., Lactobacillus and

Bifidobacterium in irritable bowel syndrome: symptom

responses and relationship to cytokine profiles,

Gastroenterology, vol. 128, no. 3, 2005, pp. 541–551.

13. Preidis GA and Versalovic J, Targeting the human

microbiome with antibiotics, probiotics, and prebiotics: gastroenterology enters the metagenomics era,

Gastroenterology, vol. 136, no. 6, 2009, pp. 2015–2031.

14. Silk DBA, Davis A, Vulevic J, Tzortzis G, Gibson GR, The

effects of a trans-galactooligosaccharide prebiotic on faecal microbiota and symptoms in irritable bowel syndrome,

Alimentary Pharmacology and Therapeutics, vol. 29, no. 5,

2009, pp. 508–518.

15. Pimentel M, Lembo A, Chey WD et al., Rifaximin therapy for patients with irritable bowel syndrome without

constipation, New England Journal of Medicine, vol. 364,

no. 1, 2011, pp. 22–32. 16. Prakash S, Rodes L, Coussa-Charley M et al., Gut

microbiota: next frontier in understanding human health

and development of biotherapeutics, Biologics, vol. 5,

2011, pp. 71–86. 17. Distrutti E, Monaldi L, Ricci P, Fiorucci S, Gut microbiota

role in irritable bowel syndrome, World J Gastroenterol.

Feb 21; 22(7): 2016, 2219–2241.

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https://www.ncbi.nlm.nih.gov/pubmed/?term=Jonkers%20DM%5BAuthor%5D&cauthor=true&cauthor_uid=25623659

https://www.ncbi.nlm.nih.gov/pubmed/?term=Salonen%20A%5BAuthor%5D&cauthor=true&cauthor_uid=25623659

https://www.ncbi.nlm.nih.gov/pubmed/?term=Hanevik%20K%5BAuthor%5D&cauthor=true&cauthor_uid=25623659

https://www.ncbi.nlm.nih.gov/pubmed/?term=Shankar%20V%5BAuthor%5D&cauthor=true&cauthor_uid=26653757

https://www.ncbi.nlm.nih.gov/pubmed/?term=Reo%20NV%5BAuthor%5D&cauthor=true&cauthor_uid=26653757

https://www.ncbi.nlm.nih.gov/pubmed/?term=Paliy%20O%5BAuthor%5D&cauthor=true&cauthor_uid=26653757

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675077/

https://www.ncbi.nlm.nih.gov/pubmed/?term=Distrutti%20E%5BAuthor%5D&cauthor=true&cauthor_uid=26900286

https://www.ncbi.nlm.nih.gov/pubmed/?term=Monaldi%20L%5BAuthor%5D&cauthor=true&cauthor_uid=26900286

https://www.ncbi.nlm.nih.gov/pubmed/?term=Ricci%20P%5BAuthor%5D&cauthor=true&cauthor_uid=26900286

https://www.ncbi.nlm.nih.gov/pubmed/?term=Fiorucci%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26900286

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734998/



AMT, vol. 22, no. 3, 2017, p. 38

THE IMPACT OF ANTIBIOTICOTHERAPY ON GUT

MICROBIOTA IN CHILDREN

OXANA TURCU

1, OLGA CIRSTEA

2, ALA HOLBAN

3, NINEL REVENCO

4

1,2,3,4„Nicolae Testemițanu” State University of Medicine and Pharmacy, Chișinău, Republic of Moldova

Keywords: antibioticotherapy,

microbiota, antibiotic

resistance, children

Abstract: Development of the intestinal microbiota in young age children is characterized by important

changes in microbial abundance, composition, and diversity (2). Most drug-based therapies interfere

with infants’ gut microbiota, but the most significant changes result after administration of antimicrobial

agents. The changes induced by antibiotherapy in gut microbiota may persist long periods of time, months and even years after the end of treatment (15, 16). The role of microbiota in host’s metabolism

and disease susceptibility it is well recognized. Repeated courses of antibiotics may have a significant

role in the pathogenesis of allergic, autoimmune and metabolic disorders later in life.

1Corresponding author: Oxana Turcu, Str. Ştefan cel Mare, Nr. 165, Chișinău, Republic of Moldova, E-mail: [email protected], Phone:

+37369358765


INTRODUCTION

The human microbiota is a selection of cells, genes, and

bacteria metabolites, eukaryotes, viruses that can inhabit in

humans, and is defined as a key element of our body function. It has a major role in nutrition, metabolic reactions, the host’s

innate and adaptive immune responses and body

development.(1)

Development of the intestinal microbiota in young age

children is characterized by important changes in microbial

abundance, composition, and diversity. These modifications are

influenced by a multitude of factors, such as mode of delivery,

family diet, diseases, and therapies used. Over the humans grows up, the microbiota is modified in such of structured,

means sequences of foreseeable species, similar across human

populations.(2) The link between microbiome and eukaryotes,

also viruses, is understandable as an ecosystem partnership, although less is known about their function.(3,4)

Although is generally recognized that the first contact of

newborns with microbes occurs after birth delivery, in the

presence of an abnormal amniotic liquid, recently it was found that the placenta and the meconium also contain microbes.(5)

These findings show that the first contact with microbes occurs

during intrauterine life of children, although the effects of this

interaction are still studied. In the light of these findings, it’s increasing the importance of any early external influence on

microbiota development such as antenatal chemoprophylaxis

with penicillin in pregnant women carrying group B

Streptococcus. The mode of delivery is another important determinant

of gut microbiota composition in infants. Lactobacillus,

Prevotella, and Sneathia species from vaginal microflora will

colonize infants’ digestive tract during vaginal delivery, while the intestine of the infants delivered by C-section is generally

colonized by Staphylococcus, Corynebacterium, and

Propionibacterium species from mother’s skin.(6) Pre- and postnatal administration of antibiotics during C-sections

delivery also can irreversibly disrupt the microbiome natural

process of maturation. Infants delivered by C-section present an

instable homeostasis of their gut microbiome resulting in an increased susceptibility to methicillin-resistant Staphylococcus

aureus.(7) Several studies confirmed that C-section delivery can

be associated with long-term health problems in late childhood

or adulthood, especially immunological disorders such as

asthma and type 1 diabetes.(8,9) Important changes of the gut microbiota composition

with increased concentration of Proteobacteria are noted in

premature infants, especially in those treated with broad-

spectrum antibacterial agents form the first days of life.(10)

Latest studies showed that extended courses of antibiotic

therapy in premature newborns increase the risk of late-onset

sepsis and necrotizing enterocolitis with high rate of mortality.

That is why the latest researches are focused on the relationship between modified composition of the intestinal ecosystem and

these illnesses in infants.(11)

Most drug-based therapies interfere with infants’ gut

microbiota, but the most significant changes result after administration of antimicrobial agents. Antibiotic therapy affects

both the target pathogen and commensal inhabitants of the

intestine, resulting in marked reduction of bifidobacteria

populations and multiplication of enterobacteria and enterococci with selection of drug-resistant microbes.(12,13) This is one of

the major causes of the changes in the normal evolution of

intestinal microbiota with reduced microbial fecal diversity and

its composition. The way and degree of severity of microbiota alteration depends on several factors as the spectrum of the

antibacterial agent, the route of administration, the dosage and

the length of treatment, number of the courses, and the

pharmacokinetic and pharmacodynamic properties of the drug. Broad-spectrum antibiotics are active against anaerobic

bacteria that dominate intestinal microbiota and can have

substantial negative consequences on the functional stability of

the microbiota.(14) Microbiota may return to a similar composition of the

original one after the end of treatment, but initial condition is

often not fully recovered and the changes induced by antibiotherapy may persist long periods of time, months and

even years. Extended terms of intestine ecosystem recovery can

be explained by important shift of bacterial population and by

short intervals between repeated courses of antibacterial drugs.(15,16)

http://mic.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.040618-0#R44



AMT, vol. 22, no. 3, 2017, p. 39

Previous assumptions that fever previous to antibiotic

treatment may alter the composition of gut microbiota in

children, have not been confirmed. Both fever and infections cannot explain all those changes in the intestinal flora which

persist long after treatment with antibiotics, although previously

it was thought that antibiotics have a short term impact on the

human microbiome.(17) Low gut bacterial diversity with important low

concentration of Bifidobacterium has been identified previously

to necrotizing enterocolitis onset. Preterm newborns exposed to

broad-spectrum antibiotics and infants whose mothers received antibiotic treatment before delivery presented high risk for

necrotizing enterocolitis development due to C. difficile

overgrowth.(18)

Chron’s disease was one of the first autoimmune disease for which was established a strong correlation between

repeated antibiotic courses in children younger than 5 and an

increase of Gram-negative bacteria like Porfiromonadaceae in

gut microbiota.(19,20) There are evidences to support that antibiotics may play a significant role in the pathogenesis of

irritable bowel syndrome, especially by reduction in gut

microbiota diversity.(21)

Numerous researchers have found a correlation between allergic disorders and Bifidobacteria

deficiency with Escherichia coli abundance in gut microbiota

after antibiotic intake during early childhood.(22,23,24) The

most recent studies confirmed the link of autoimmune and atopic diseases to gut microbiota dysbiosis, caused by the intake

of antibiotics during early life, which is a critical period for

development of the immune system and immunological

tolerance. Is sustained that intestinal microbiota contain activation factors for innate immunity, similar to

inflammation.(24)

Increased frequency of atopic diseases by maternal

intake of antibiotics during pregnancy was a topic of several discussions being linked to the broad-spectrum and the dosage

but this connection couldn’t be sustained in other two large

prospective case-control studies.(23,24)

Gut microbiota is an important factor in the control of

human body metabolism, particularly of energy homeostasis and

adiposity. Several metabolic disorders have been linked with gut

microbiota dysbiosis.(25,26) Antibiotic use it was recognized as

an aggravating factor in obese child and associated metabolic syndrome.(26) Latest studies confirmed a strong association

between macrolide antibiotics use in early life and increased

BMI, obesity or metabolic diseases in children or adults due to

abundance of Bifidobacterium and Bacteroides in the intestine, which normalized within 24 months.

The response of gut microbiota to amoxicillin use is

mild to moderate being characterized by short-term decrease in

aerobic Gram-positive cocci but the main disadvantage is the enrichment of gut microbiota with resistant enterobacteria.

Recently, human gut microbiota has been established as a

significant reservoir of antibiotic resistances not only in adults, but infants and children too.(27,28,29,30)

Antibiotic resistance lately it became a major public

health threats, mostly because of unnecessary overuse of

antibiotics. The development of resistance to harmful environment factors is a normal evolutionary process for

bacteria, but this process can go faster due to incorrect use of

antibacterial drugs. New researches found resistant strains of

bacteria not only in adults and children who received several courses of antibiotics, but in isolated human populations who

have never used antibiotics.(31,32)

Children with nutritional disorders present different

way of gut microbiota maturation and in result the response to

the influence of environmental factors is altered.

Undernutrition is characterized by a delay in the evolution of the microbiome. This immature microbiome is

associated with susceptibility to digestive infections, has

negative impact on nutritional correction measures and can

prolong the malnourished state. Curiously, Trehan I. et al. in their study showed that a short course of broad-spectrum

antibiotic (amoxicillin or cefdinir) can improve nutritional

treatment results and can reduce mortality rate between children

with acute malnutrition. These results are sustained by an important meta-analysis focused on the antibiotics impact on

children’s growth in low and middle income countries that

increased their weight after antibiotic therapy. On the other

hand, in developed countries obesity has become an important public health problem. A high-calorie diet and early antibiotic

exposure change gut ecosystem to abundance of Bacteroidetes

that have a higher capacity of energy harvesting and increase the

risk of obesity in older children.(32) Intestinal microbiota develops and evolves quickly

during infancy, on a predictable way, until it achieves a

physiological homeostasis and bacteria should remain stable for

life. The role of microbiota in host’s metabolism and disease susceptibility it is well recognized. Latest studies are focused on

the link between intestinal microbiota changes in early infancy

and metabolic or other diseases later in life.

Lately are discussed several opportunities of restoring gut microbiota, like toxin inhibitors for C.difficile and E.coli,

use of probiotics, phage therapy or transplant of fecal

microbiota. Probiotics are the most used strategy to maintain

and improve the microbiota components after antibiotic treatment. Probiotics are living microorganisms with health

consequences in humans, only if are properly used. (33) Some

opinions suggest that probiotics could have the capability of

antibiotic resistances transfer to intestine bacteria. Bacteria that normally inhabit human intestine can share resistance genes

among themselves. This type of transfer turns out to be a vast

problem when these safe commensal bacteria are converted to

pathogen pattern. In result intestine is reside with antibiotic

resistance genes, among both pathogen and commensal bacteria.

Once acquired, resistance genes become a relatively stable part

of gut intestine genome (34).

The beneficial impact of antibiotic drugs is delicate to enhance. Nevertheless, excessively widespread use of antibiotics

has created many threats for the present and future of human

population. The most important challenge is constant increasing

resistance of bacterial pathogens to antibiotics. In light of this facts, and given that bacterial infections

remain an important major public health problem, new studies

are required to develop strategies to abrupt or reduce negative

impact of antibiotics when their administration is needed.

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2. Yatsunenko T., Rey F., Manary M., et al. Human gut

microbiome viewed across age and geography. Nature. 2012; 486: 222-7.

3. Lim E., Zhou Y., Zhao G., et al. Early life dynamics of the

human gut virome and bacterial microbiome in infants. Nat

Med. 2015; 21: 1228-34. 4. Parfrey L., Walters W., Knight R. Microbial eukaryotes in

the human microbiome: ecology, evolution, and future

directions. Front Microbiol. 2011; 2:153.



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5. Aagaard K., Ma J., Antony K., et al. The placenta harbors a

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6. Dominguez-Bello M., Costello E., Contreras M., et al. Delivery mode shapes the acquisition and structure of the

initial microbiota across multiple body habitats in

newborns. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 11971-

5. 7. Centers for Disease Control and Prevention (CDC)

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8. Roduit C., Scholtens S., de Jongste J., et al. Asthma at 8

years of age in children born by caesarean

section. Thorax. 2009; 64: 107-13. 9. Bonifacio E., Warncke K., Winkler C., et al. Cesarean

section and interferon-induced helicase gene

polymorphisms combine to increase childhood type 1

diabetes risk. Diabetes 2011; 60: 3300-6. 10. La Rosa P., Warner B., Zhou Y., et al. Patterned

progression of bacterial populations in the premature infant

gut. Proc Natl Acad Sci U S A. 2014; 111: 12522-7.

11. Kuppala V., Meinzen-Derr J., Morrow A., Schibler K. Prolonged initial empirical antibiotic treatment is

associated with adverse outcomes in premature infants. J

Pediatr. 2011; 159: 720-5.

12. Tanaka S., Kobayashi T., Songjinda P., et al. Influence of antibiotic exposure in the early postnatal period on the

development of intestinal microbiota. FEMS Immunol Med

Microbiol. 2009; 56: 80-7.

13. Greenwood C., Morrow A., Lagomarcino A., et al. Early empiric antibiotic use in preterm infants is associated with

lower bacterial diversity and higher relative abundance of

Enterobacter. J Pediatr. 2014; 165: 23-9.

14. Sullivan A., Edlund C., Nord C. Effect of antimicrobial agents on the ecological balance of human

microflora. Lancet Infect Dis 2001; 1: 101-114.

15. Jernberg C., Lofmark S., Edlund C., Jansson, J. Long-term

ecological impacts of antibiotic administration on the

human intestinal microbiota. ISME J. 2007; 1: 56-66.

16. Jernberg C., Löfmark S., Edlund C., et al. Long-term

impacts of antibiotic exposure on the human intestinal

microbiota Microbiology 2010, 156 (11): 3216-23. 17. Penders J. et al. Factors influencing the composition of the

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18. Deshpande G., Rao S., Patole S., Bulsara M. Updated meta-analysis of probiotics for preventing necrotizing

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19. Hildebrand H., Malmborg P., Askling J., et al. Early-life exposures associated with antibiotic use and risk of

subsequent Crohn’s disease. Scand. J. Gastroenterol. 2008;

43: 961-966. 20. Manichanh C., Rigottier-Gois L., Bonnaud E., et al.

Reduced diversity of faecal microbiota in Crohn’s disease

revealed by a metagenomic approach. Gut. 2006; 55: 205-

211. 21. Yamini D., Pimentel M. Irritable bowel syndrome and

small intestinal bacterial overgrowth. J. Clin.

Gastroenterol. 2010; 44: 672-5.

22. Bisgaard H., Li N., Bonnelykke K., et al. Reduced diversity of the intestinal microbiota during infancy is associated

with increased risk of allergic disease at school age. J.

Allergy Clin. Immunol. 2011; 128: e641-e645.

23. Abrahamsson T., Jakobsson H., Andersson A., et al. Low

diversity of the gut microbiota in infants with atopic

eczema. J. Allergy Clin. Immunol. 2012; 434-440. 24. Risnes K., Belanger K., Murk W., Bracken M. Antibiotic

exposure by 6 months and asthma and allergy at 6 years:

findings in a cohort of 1,401 US children. Am. J.

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as a link between obesity and metabolic syndrome. J. Nutr.

Metab. 2012: 476380.

26. Francino M., Moya A. Effects of antibiotic use on the microbiota of the gut and associated alterations of

immunity and metabolism. EMJ Gastroenterol. 2013; 1: 74-

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27. Pehrsson E., Forsberg K., Gibson M., et al. Novel resistance functions uncovered using functional

metagenomic investigations of resistance reservoirs. Front.

Microbiol. 2013; 4:145.

28. Gueimonde M., Salminen S., Isolauri E. Presence of specific antibiotic resistance genes in infant faecal

microbiota. FEMS Immunol. Med. Microbiol. 2006; 48:

21-25.

29. Zhang L., Kinkelaar D., Huang Y., et al. Acquired antibiotic resistance: are we born with it? Appl. Environ.

Microbiol. 2011; 77: 7134-41.

30. Alicea-Serrano A., Contreras M., Magris M., et al.

Tetracycline resistance genes acquired at birth. Arch. Microbiol. 2013; 195: 447-451

31. Clemente J., Pehrsson E., Blaser M., et al. The microbiome

of uncontacted Amerindians. Sci. Adv. 2015; 348:

e1500183. 32. Langdon A. et al. The effects of antibiotics on the

microbiome throughout development and alternative

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Scientific Association for Probiotics and Prebiotics

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34. Dzidic S. et al. Antibiotic resistance in bacteria. Food

Technol. Biotechnol. 2008; 46 (1) 11-21.

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http://mic.microbiologyresearch.org/search?value1=Sonja+L%C3%B6fmark&option1=author&noRedirect=true

http://mic.microbiologyresearch.org/search?value1=Charlotta+Edlund&option1=author&noRedirect=true



AMT, vol. 22, no. 3, 2017, p. 41

LEFT INGUINAL HERNIA AND CRYPTORCHIDISM.

PARTICULARITIES OF TREATMENT AND EVOLUTION.

CASE REPORT

CRISTIAN-ŞTEFAN BERGHEA-NEAMŢU1

1“Lucian Blaga” University of Sibiu, Surgical Clinic-Pediatric Clinic Hospital, Sibiu

Keywords: inguinal

hernia in infants,

recurrent dehiscence,

severe anemia and hypoproteinemia

Abstract: Inguinal hernia in children can be associated with anemia and/or hypoproteinemia. These

kind of conditions can cause a bad evolution, with multiple surgical interventions (recurrent dehiscence,

infected surgical wound, dehydration). It is possible to be present a vicious circle: recurrent dehiscence

is explained by hypoproteinemia, but multiple surgical interventions can lead to hypoproteinemia, together with severe anemia.

1Corresponding author: Cristian-Ştefan Berghea-Neamţu, Str. Gh. Baritiu, Nr. 1-3, Sibiu, Romania, E-mail: [email protected] Phone:

+40722641331


INTRODUCTION

Inguinal hernia in children can be associated with anemia and/or hypoproteinemia, with its complications and

evolution particularities. There are some appropriate therapeutic

solutions, successfully applied in surgical practice.

PURPOSE

The purpose is to highlight the whole mechanisms

implied in evolution particularities of an inguinal hernia in

neonates.

MATERIALS AND METHODS

We present an infant, male, 21 days old, admitted in

Pediatric Surgical Clinic Sibiu at the end of December 2016 for specialty diagnostic and therapeutic advice in the context of the

present of a pseudotumor formation, with an elastic consistence,

and subjacent placed of the left inguinal arcade, which seems to

be contiguous, with the left scrotum swelling. The infants comes from two members family, the

mother, 17 years old, schoolgirl, and the father, 21 years old,

healthy, without family medical history, except paternal

grandmother, with type 1 Diabetes Mellitus. Is the first born in this family, from a physiological pregnancy, 2910 g the weight

at birth, 10 Apgar score, good postnatal evolution and almost

good weight gain, breastfeeding, exclusively; poor educational

and lifestyle conditions, means 6 persons are living in only 2 rooms; mother smoking during the pregnancy. The admittance is

required via Emergency room for a condition with acute onset,

in the same day, by the presence of the pseudotumor formation

in the left inguinal area, and ipsilateral scrotum.

RESULTS

The clinical exam in the first moment of admission

emphasize good general condition, no fever, 3500 g actual weight, subcutaneous cells tissue reduced at the level of thorax

and abdomen, the same left inguinal and ipsilateral scrotum

pseudotumor formation, the left scrotum difficult to examine; no other organs or system pathology. Clinical and anamnestic data

allowed the exclusion of the other conditions of the left inguinal

site, such as hydrocele, spermatocele, femoral hernia, spermatic

cord hernia, with the step diagnostic of Left inguinal and scrotal hernia, and suspicion of cryptorchidism.

The laboratory data were normal, and ultrasound ones

confirm the step diagnostic.

Given the possibility of an evolution towards strangulation, with alteration of intestinal viability, the surgical

treatment was considered to be immediate done, the surgical

intervention was a large one, with a medium anesthetic risk.

Under general anesthesia the surgical hernia cure was decided; also, the mesentery and left orchidopexy.

Was performed a limited transverse incision in the site

of left inguinal fold; was permeate at the left inguinal canal, and

with its wall opens was highlighted an edematous hernia sac, also endured, friable, the scrotum with intestinal content no taxis

reducible. With the sac open, the intestinal content appears

cyanotic and edematous along a 15 cm, with a proximal area of

stenosis, 0.5 cm length and permeable. Was also cured the mesentery, with local warm serum

application; good intestinal evolution after 10 minutes, and than

its content was reapplied in the peritoneal cavity. Was dissected

the edges of the spermatic cord sac; the sac close down and the Barker procedure too. Was performed a transversal incision on

the left scrotum, with the left orchidopexy. After surgery

procedures, the digital rectal examination had revealed a normal

feces. The patient was monitored in intensive care unit about 6 days, with pain management indication; no peritoneal abnormal

signs. Was discharged in good condition, but after a month the

patient had presented fever, bilious vomits, secondary acute

dehydration. The patient laboratory data showed leukocytosis, positive inflammatory markers, anemia, high blood urea, high

blood amylase.

Figure no. 1. The patient abdominal radiography (see,

multiple gases in the superior part of abdomen), with the

permission



AMT, vol. 22, no. 3, 2017, p. 42

Based on laboratory and radiology data, the final

diagnostic was: Intestinal Occlusion; Secondary Acute

Dehydration; Anemia. After nasogastric aspiration and intravenous

hydration, the patient was operated by exploratory laparotomy

on the upper site of umbilical region, with the median incision;

were highlighted a marked intestinal distension and a complete stenosis, 30 cm from ileocecal valve. Was practicing the stenosis

region resection, with T-T, double layer, anastomosis.

Figure no. 2. The patient at the first surgical reintervention

(with the permission)

After the first surgical intervention, a secretion and

repeated dehiscence of surgical wound are observed. It was

necessary a second surgical reintervention with blood

replacement in the same time (because of severe anemia). The bacteriologic exam of the wound secretion had revealed the

presence of Enterobacter cloacae; the labs exam revealed severe

anemia, severe hypoproteinemia.

Figure no. 3. The patient before the second surgical

reintervention (with the permission)

The second surgical reintervention consists in

resection of the affected area (near anastomosis).

Figure no. 4. The patient after the second surgical

reintervention (with the permission)

The post surgical monitoring has focus to the

treatment of anemia and hypoproteinemia, antibiotics for

infected surgical wound. The evolution was favorable, and the outcome was made in good condition.

Figure no. 5. The patient at the discharged moment (with

permission)

CONCLUSIONS The recurrence of dehiscence surgical wounds was

explained by hypoproteinemia, mainly, also by the surgical reintervention, but it did not associated with the dehiscence of T-T, double layer, anastomosis.

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