Association of renal allograft rejection with virus infections

CLINICAL STUDIES

Association of Renal Allograft Rejection with Virus Infections

CARLOS LOPEZ, Ph.D.*

RICHARD L. SIMMONS, M.D.t

S. MICHAEL MAUER, M.D.

JOHN S. NAJARIAN, M.D.t

ROBERT A. GOOD, M.D., Ph.D.*

With the technical assistance of

SHARON GENTRY

Minneapolis, Minnesota

From the Departments of Pathology, Surgery

and Pediatrics, University of Minnesota, Min-

neapolis, Minnesota 55455. This work was

supported by Grant No. AM 13083, Al-00798

and Al-08677 from the U.S. Public Health

Service, Contract NCI 71-2261 from the Na-

tional Cancer Institute, the National Founda-

tion-March of Dimes, and the John A. Hart-

ford Foundation. Requests for reprints should

be addressed to Dr. Richard L. Simmons,

Box 185 Mayo, University of Minnesota Hos-

pitals, Minneapolis, Minnesota 55455. Manu-

script accepted August 15, 1973. * Present address: Memorial Sloan-Ketter-

ing Cancer Center, 410 East 68th Street,

New York, New York 10021.

t John and Mary R. Markle Foundation

Scholars in Academic Medicine.

Sixty-one immunosuppressed renal transplant recipients were systematically screened for virus infections and the findings correlated with their clinical course. Only herpesvirus (cytomegalovirus, herpes simplex and herpes zoster) were consistently isolated. The onset of virus infections could usually be associated with clinical syndromes. Patients without virus infections were usually asymptomatic. The clinical syndrome associated with virus infection consisted of fever, leukopenia and renal allograft rejection. Renal biopsy, performed at the time serum creatinine levels were elevated, revealed classic rejection; most rejections were reversed by increasing the dose of steroids. Patients continued to excrete virus even after antibody response and clinical recovery. Virus infections do not appear to be incidental findings in transplant patients except after recovery when the virus persists in the immune patient. The clear-cut association between virus infection and rejection episodes suggests a pathogenic relationship. The two mechanisms which seem to best explain the relationship are (1) the virus infection acting as an adjuvant and triggering the rejection of the allograft or (2) the allograft rejection activating a latent virus infection.

lmmunosuppressed patients are known to be susceptible to a variety of opportunistic agents including viruses. A particularly

high incidence of cytomegalovirus (CMV) infections has been

reported in renal transplant recipients [l-4]. Occasionally, these infections have been associated with clinical manifesta-

tions such as “transplantation pneumonia,” [5,6] hepatitis

[4,7,8] and hkterophil-negative infectious mononucleosis

[1,7,8]. However, CMV has most often been isolated from

asymptomatic transplant recipients and has not been con-

sistently associated with specific clinical syndromes [1,2,9].

A consensus has developed that CMV is an incidental finding

of little note in most transplant patients. Simmons et al. [IO], first noted that rather mild febrile illnesses appeared to pre-

cede and accompany renal function deterioration consistent with a diagnosis of allograft rejection. They asked whether

infections might not trigger rejection. Others have also noted

the association between infection and rejection [11,12]. Our

study was designed to critically analyze the role of virus infections in transplant patients and to correlate the appearance of viral infections with the clinical picture.

280 March 1974 The American Journal of Medicine Volume 58

METHODS

The procedures for the selection of donors and recipi-

ents, technics of transplantation, standard immuno-

suppressive regimens and diagnosis and standard

treatment for rejection episodes have previously been

described 1131. Briefly, the immunosuppressive regi-

men included methylprednisolone on the day of and

the 2 days after transplantation, antilymphoblast glob-

ulin for 14 days after surgery, and decreasing amounts

of azathioprine and prednisone to maintenance doses.

Patients were given 2 to 21 day old leukocyte-poor

blood cells to bring their hematocrit level to 30 per

cent, before surgery and then when necessary there-

after. The study group consisted of 61 patients who

had received renal allografts at the University of Min-

nesota Hospital. Forty-eight of these patients were

studied after readmission to the hospital for fevers or

rejection episodes. Thirteen patients were chosen at

random before the transplant for serial study through

their course.

All viral studies were carried out at the Virus Labo-

ratory, Minnesota State Health Department. Fresh

urine, sputum and stool samples were collected for

viral isolation at weekly intervals for 2 to 6 weeks after

the patient was started on the study, and then once

every 2 to 4 weeks thereafter. The frequency of sam-

pling was. to a certain extent, dependent on the fre-

quency of the patients’ routine clinic visits to the hos-

pital. Bronchoscopic washings and tissue obtained at

pulmonary or renal biopsy or at autopsy were cultured

when collected. Urine specimens were centrifuged,

and the sediment was resuspended in 2 ml superna-

tant fluid. Ten per cent suspensions were prepared

from tissues and stool, and were then centrifuged to

eliminate coarse debris. These supernatants, sputums,

and urines were then treated with antibiotics and inoc-

ulated onto monolayers of primary human amnion,

monkey kidney, HeLa and human skin fibroblast ceils

for routine viral isolation [14,15]. Sputums and 10 per

cent lung suspensions were inoculated into embryo-

nated eggs when influenza virus was suspected. Inoc-

ulated human amnion, monkey kidney, and HeLa

monolayers and eggs were observed for cytopathic ef-

fect (CPE) for at least 1 week; the human skin fibro-

blast monolayers were observed for at least 3 weeks.

If CPE was observed, supernatant fluid and cells were

passed to new cultures or eggs, and if CPE was again

noted, they were studied for presence of a virus. Most

viruses were identified by neutralization of CPE with

type specific hyperimmune serums obtained from the

Center for Disease Control, Atlanta, Georgia. CMV

was identified by its typical CPE in human fibroblast

monolayers [15], and its presence was frequently con-

firmed by the indirect immunofluorescence (IIF) test

using convalescent human serums obtained from Cen-

ter for Disease Control [16]. For this test, infected skin

fibroblasts were grown on slides and stained by the

method of Hilgers et al [17]. An hemadsorption test

[14] with guinea pig red blood cells was used on mon-

key kidney cells 7 days after inoculation to detect vi-

ruses such as the parainfluenza viruses which do not

cause a distinctive CPE. The cultures and technics

employed will detect most of the co’mmonly isolatable

human viruses. Viruses which would be missed by

these technics include rhinoviruses, rubella virus, cer-

tain coxsackie viruses and parvoviruses.

Serum samples were collected at least as often as

samples for virus isolation and were used to determine

complement fixing (CF) antibody titers to CMV (strain

AD-169, Microbiological Association, Bethesda, Mary-

land), herpes simplex virus (HSV) and herpes zoster

virus (HZV) [15]. Since strain Ad-169 will not detect

all the various strains of CMV [18], a local isolate was

used in the IIF test to detect antibody response to

CMV in one serum negative by CF test with AD-169.

Skin fibroblasts, infected with the local isolate, were

grown on slides and used as antigens for the test. Se-

rums with a CF antibody titer to AD-169 also had an

IIF antibody titer to the local isolate. IIF antibody titers

of serum from eight patients were two to fourfold

higher than the CF antibody titers. The presence of

Australia antigen was determined by the gel diffusion

method [19].

Immediately after obtaining renal tissue. a sample

was snap frozen in isopentane precooled in liquid ni-

trogen. The tissue was sectioned at 4 to 6 p in a Lip-

shaw cryostat and processed for immunofluorescent

microscopy as previously described [20]. The tissue

was stained with fluorescein isothyocyanate conjugat-

ed monospecific antiserums against human immuno-

globulins G (IgG), M (IgM), A (ISA), beta-C CPIC), properdin, fibrinogen and albumin which were pre-

pared as previously reported [20,21]. Certain clinical and laboratory criteria were defined

in order to make clinical correlations with the virologic

data. For this study, a significant febrile episode was

defined as an oral temperature of 10A’F for 2 or more

days within a 7 day period. A few episodes of fever

during the first 2 weeks after the transplant were ex-

cluded since they were clearly associated with com-

mon surgical complications or allergy to antihuman

lymphoblast globulin [13]. Clinical rejection was de-

fined as an increase in serum creatinine level of 0.5

mg/lOO ml greater than the base line level and eleva-

tion usually to a value greater than 2.0 mg/lOO ml

even though a lesser elevation of serum creatinine

level may frequently prompt the empiric institution of

antirejection treatment (131. Virus infection was con-

sidered to be present when a fourfold or greater rise in

CF or IIF antibody titer occurred or when the virus

was isolated from the patient. Leukopenra was arbi-

trarily defined as a total peripheral leukocyte cell

count less than 5,000 cells/mm3 for 2 or more con-

secutive days.

RESULTS

Viral !solation and Seroconversion. Although the virologic methods used in this study are capable

of isolating many viruses, only members of the

herpesvirus group; namely, cytomegalovirus

RENAL ALLOGRAFT REJECTION AND VIRUS INFECTIONS-LOPEZ ET AL.

March 1974 The American Journal of Medicine Volume 56 281

RENAL ALLOGRAFT REJECTION AND VIRUS INFECTIONS-LOPEZ ET AL

61+ - Study Group

8 (13%) - No Virus 53 (87%) - At Least One Herpes

Infections

A // rction

2. Technical

Complications

6 .- Study

Controls

7 (11.4%)“” HZV

Infections

15 (24.6%)“” HSV 47 (77%)“” CMV y/ y ions

3 - Bacterial 15 - Infected 29 - Onset of

Complications Before Study Infection

Detected

l 1,335 sampies were collected and tested for virus isolation and serum antiviral antibody

l *lO patients had HSV and CMV infections, 2 had HZV and CMV infections, and 2 had HZV, HSV, and CMV infections

Figure 1. Herpesvirus infections in the study group of renal transplant recipients.

(CMV), herpes zoster virus (HZV) and herpes

simplex virus (HSV) were consistently isolated. A

poliovirus was isolated from each of two patients,

but these isolations could not be repeated 1 week

later, and clinical manifestations were not associ-

ated with the isolation. Several patients had hepa-

titis-associated antigen in their serum; but, as oth-

‘ers have observed [22,23], this is not usually associated with clinical symptoms or alterations in

liver function in transplant patients.

Figure 1 details the herpesvirus infections de-

tected in the study group. Of the 61 patients stud-

ied 53 (87 per cent) had at least one infection

with a herpesvirus, whereas the remaining 8 (13

per cent) had no evidence of a virus infection.

Since 48 of 61 patients were admitted to the study

because of clinical symptoms of various types, a

truer incidence of virus infections was determined

using the group of 13 patients selected at random

before the transplant. Eleven (85 per cent) of

these 13 had at least one infection with a her-

pesvirus; 10 (77 per cent) of 13 had a CMV infec-

tion, 3 (23 per cent) of 13 had a HSV infection and 1 (8 per cent) of 13 a HZV infection. The in-

cidence of herpesvirus infections in the small

group of patients chosen at random is surprisingly similar to that in the larger group of selected pa-

tients (Figure 1). Fourteen of the 61 study patients had infections with two or more herpesvi-

ruses; 10 patients had CMV and HSV infections, 2 had CMV and HZV infections and 2 had CMV,

HSV and HZV infections (Figure 1).

Diagnosis of CMV infections was based on the

isolation of virus in 39 of 47 patients (8 of 10 in

the 13 patients selected at random). The diagno-

sis was based on serologic evidence alone in 8 of

the 47 patients (2 of 10 in the 13 selected at ran-

dom). Thirty-four of the 39 patients in whom virus

was isolated showed seroconversion to CMV. The

five patients who did not produce antiviral anti-

body died of mixed infections and form a special

group which has been previously described [24].

CMV was isolated from the urine alone in 17 pa-

tients, from the sputum alone in 7 patients, from

the urine and sputum in 14 patients, and from kid-

ney biopsy tissue alone in 1 of the 39 patients in

whom virus was isolated. CMV was, therefore,

detected in the urine of 31 of the 39 patients in

whom the virus was isolated. Of the 31 patients

who showed viruria, 30 (97 per cent) continued to

excrete virus after the appearance of serum an-

tiviral antibody. Nine of these patients excreted

virus in their urine for at least 5 months after ser-

oconversion and 1 patient for 12 months. Clinical-Virologic Correlations. Only 8 of the 61

study patients showed no laboratory evidence of viral infections. Two of these eight patients ei-

ther had technical complications or were not stud-

ied long enough to be used as controls. Of the 6

remaining patients, 2 were among the 13 selected at random who were studied serially before and

after they received their transplant. Five of these six patients have remained free of detectable virus infections for 6 consecutive months after receiv-


ing the transplant and one has remained virus free

for 20 consecutive months. Only one of these six

virus-free patients had fever, leukopenia and clini-

cal rejection at any time.

Forty-seven patients ha&f laboratory evidence of

CMV infections. The clinical-virologic correlations

were obscured in three patients who had exten-

sive bacterial complications; these three patients

have, therefore, been excluded from the analysis.

In addition, the onset of viral infections could not

be determined in all patients since 15 of 47 with

CMV infections were found to have CF antibody

titers 2 16 when first studied; 12 of 15 had titers

2 64; and 12 of 15 patients whose CF antibody

titers were high initially were excreting CMV when

first studied. Although fourfold or greater in-

creases in CF antibody titers were detected, the

exact onset of seroconversion could not be deter-

mined since the titers were already rising when

first studied. Most patients with high antibody ti-

ters, who were excreting CMV, manifested viruria

many months after the onset of CMV infections

and were usually asymptomatic during this entire

time.

Twenty-nine of the 47 patients in whom CMV

infections developed were studied early enough

and thoroughly enough to accurately determine

the time of onset of their infections. Onset of virus

infections was defined as a positive culture after

several (at least three) attempts had been nega-

tive or a fourfold or greater increase in CF anti-

body titer. Negative cultures were not detected

prior to the positive cultures found in seven pa-

tients. In each of these patients seroconversion

alone was used to determine the onset of virus in-

fections. In four patients serum antibodies to CMV

never developed and the onset of infection in

these patients was determined by the first positive

culture after several attempts had been negative.

Of the 29 well studied patients, 23 had CF anti-

body titers of less than 8, 2 had titers of 8, 1 had

a titer of 16, and 3 had titers of 32 when first

started on the study. In 28 on these 29 well stud-

ied patients, clinical findings could be correlated

with the onset of virus infections, whereas in only

one patient the initial isolation of virus or serocon-

version was not associated with any clinical ill-

ness. Ten of these 29 patients were among the 13

patients selected at random.

Fever: Nineteen of the 29 patients (9 of the 10

selected at random) with laboratory evidence of

CMV infections had fevers (defined as an oral

temperature greater than 101°F for 2 days in any

7 day period), whereas only 1 patient without a virus infection had fever after receiving the trans-

plant. The average time after fever for serocon-

version in 15 of the 19 patients was 25 days (f standard error [SE] 3.3 days). The average time

after fever and before the first positive CMV cul-

ture in 15 of the 19 patients was 21 days (*SE 4

days). Only two patients had positive viral cultures

3 and 21 days before the febrile episode. Positive

cultures were collected from 3 to 11 days after

the onset of fever in 5 of the 15 patients from

whom virus was isolated. Each patient was his

own control, since 17 of the 19 patients were afe-

brile during the period after transplantation and

before the febrile episode later associated with

the virus infection.

Although CMV could sometimes be isolated at

the time of onset of fever or shortly thereafter, the

average time of viral isolation was only 4 days be-

fore seroconversion. Since a rise in CF antibody

titer cannot be detected until 14 to 28 days after

clinical infection [25,26], seroconversion appears

to be a better indicator of the onset of virus infec-

tion than isolation of the virus.

Leukopenia: CMV’infection was also shown to be

associated with episodes of leukopenia (defined

as a total peripheral leukocyte count less than 5,000/mm3 for 2 or more consecutive days). In

18 of the 29 patients (7 of the 10 patients select-

ed at random) with laboratory evidence of CMV

infections leukopenia developed; whereas only 1

of the patients without virus infections was leuko-

penic after receiving the transplant. Thus, of 19

patients with leukopenia, 18 had laboratory evi-

dence of CMV infection. Onset of leukopenia usu-

ally preceded seroconversion by an average of 13

days (&SE 3.3 days). In five patient’s a rise in CF

antibody titer preceded leukopenia by 4, 6, 7, 60

and 118 days. The average time from onset of

leukopenia to positive culture in 11 patients was

16 days (*SE 3.5 days). Leukopenia followed the

first positive culture in five patients by 2 to 12

days and in one patient by 132 days. Many of

these patients had several leukopenic episodes

after the onset of the virus infection and while

they continued to have high CF antibody titers and

to excrete virus. However, no other episodes of

leukopenia were detected in these patients be-

tween the date of the transplant and the onset of

CMV-associated leukopenia.

Clinical rejection: An association between CMV

infections and episodes of elevated serum creati-

nine levels was also evident. Twenty-one of the 29

patients (7 of the 10 patients selected at random)

with laboratory evidence of CMV infections had

rejection episodes, whereas only 1 of 6 control patients without virus infections showed signs of

rejection. Thus, of 22 patients with rejections, 21

had CMV infections. Only one episode of allograft

RENAL ALLOGRAFT REJECTION AND VIRUS INFECTIONS-LOPEZ ET AL,


RENAL ALLOGRAFT REJECTION AND VIRUS INFECTIONS-LOPEZ ET AL

(a) Fever

(b) Serum creotinine

Figure 2. Times after transplanta-

tion of onset of fever, elevated serum

creatinine level, leukopenia and de-

I

patients in whom fever during first 14

,

days after receiving the transplant was not included since this can be

0 20 40 60 80 100 120 caused by surgical trauma and anti-

DAYS POST TRANSPLANT lymphoblast globulins.

r ~-~ 1

(d) C yt;;;s whichever first) in group of 29 well

studied patients. Asterisk indicates

tectable cytomegalovirus (CMV) in-

fection (seroconversion or isolation,

4UG

lmurm 10’

Mg f day 2’13

‘Or: r, Fisure 3. Clinical course of oatient

w;h typical CMV-associated syndrome. Spiking fevers were followed

by elevated serum creatinine levels

KG I and leukopenia. Serum anti-CMV

Salumedro, 60 antibody was detected 3 weeks after FGdiotmn

‘ 4 ,

aolyslr I 4 the onset of ciinical signs, and virus

L j 1 ~1i1lr~I~~,:ll,rr.l,l~11:L~1l!I1I.Jl~~ was isolated shortly thereafter. CMV

; /I 8 z lil ‘(5 24 <‘4 5? ii ,1:’ 114 48 ‘,2 56 gr\ 64 6R 72 76 80 = cytomegalovirus; Cr = creatinine; L-qr PoIf - Transplant Bx = renal biopsy.


rejection occurred during the period after the

transplant and prior to the virus-associated rejec-

tion episode. The average time after rejection for

seroconversion in 16 of the 21 patients was 19

days (&SE 3.9 days). One patient had his first re-

jection episode 107 days after the onset of the

virus infection and while his CF antibody titer re-

mained elevated. CMV was isolated from 19 of

the 21 patients with rejections; the average time

after the onset of rejection for the first positive

culture was 26 days (z!= SE 4.6 days); in four pa-

tients virus was isolated during the first 3 days of

the rejection episode. In six patients virus was

isolated before the onset of rejection; the average

time before rejection in five patients was 9 days

(h SE 2.5 days), and the other patient excreted

CMV for 121 days before the rejection episode.

The clinical syndrome: Of the 29 patients with CMV infections, 4 had fevers without clinical re-

jections, 6 had clinical rejections without febrile

episodes, 15 had both fevers and clinical rejec-

tion, and only 4 had neither clinical illness associ-

ated with their virus infections. Whether the rejec-

tion episode occurred in conjunction with a febrile

episode or not, the fever was always closely cor-

related with laboratory evidence of virus infection.

Of the 15 patients who had both fevers and rejec-

tion in association with CMV infection, 13 (87 per

cent) had their initial fevers before or on the

same day as the rejection episodes.

The typical syndrome can be seen by examin-

ing the frequency distribution of times after trans-

plantation that fever, leukopenia, elevated serum

creatinine levels, and laboratory evidence of CMV

infection appeared (Figure 2). The incidence of

CMV infections was highest between 30 and 70

days after the transplant, i.e., shortly after the

peak incidence of fevers, leukopenia and renal

functional deterioration.

The clinical course of a typical patient with this

syndrome is shown in Figure 3. Spiking fevers

were followed by deterioration of renal function

and leukopenia. CMV infection was demonstrated

by a rise in CF antibody titer 3 weeks later, and

shortly thereafter virus was isolated from urine

and sputum. Biopsy of the donor kidney at the

time the serum creatinine level was elevated

revealed histologic changes consistent with clas-

sic rejection (Figure 4). Treatment with increased

doses of steroids and local radiation was under- taken, and fever, leukopenia and elevated serum

creatinine levels returned to normal. Biopsies of

the renal grafts of five other patients with concurrent infection and elevated serum creatinine levels

were all consistent with rejection, and immunoflu-

orescent studies revealed no evidence of virus-


Figure 4. Renal biopsy specimen from patient whose

course is shown in Figure 2. Specimen taken at time

serum creatinine level was elevated; it was perfectly

compatible with allograft rejection.

complex nephritis [27]. Culture of four of the five

biopsy specimens revealed no evidence of viral

infestation of the kidney itself.

Nineteen of the 21 patients who had elevated

serum creatinine levels in association with CMV

infections received similar antirejection therapy.

Two kidneys regained normal function without

treatment, and two kidneys were lost despite

treatment. These latter two kidneys demonstrated

changes perfectly compatible with rejection. Cul-

ture of the renal tissue revealed CMV but no evi-

dence of virus-complex nephritis [27].

Two patients were not treated for elevated

serum creatinine levels. Figure 5 shows the clini-

cal course of one of these patients with fever, leu-

kopenia and clinical rejection associated with a

CMV infection. When serum antiviral antibody

reached a high titer, serum creatinine and tem-

perature spontaneously returned to normal levels and remained there. No renal biopsy was per-

formed in these patients.

Other herpesvirus infections: Systemic infections with HSV and HZV could also be associated with



0 4 8 I2 16 20 24 28 32 K 40 44 48 52 56 fx 64 68 72 7% 80

Days Port-Tronrplont

Figure 5. Clinical course of patient

with typical CMV-associated syn-

drome who was not treated for reiec-

tion episode. CMV = cytomegalovi-

rus; Cr = creatinine; CF = comple-

ment fixing antibody.

fever, leukopenia and allograft rejection. Three

patients, two with HSV infections and one with

HZV infection, showed significant increases in CF

antibody titers to their respective viruses, and

virus was isolated from the urine of the two pa-

tients with HSV infections. As in the patients with

CMV infections, virus was isolated shortly after

the onset of fevers, leukopenia and allograft re-

jection. The infection had to be systemic, as evi-

denced by isolation of the virus from urine or a

marked increase in serum antibody in order to

demonstrate this association. Cold sores, appar-

ently due to HSV, without concomitant rise in

serum antibody could not be associated with

fever, leukopenia or allograft rejection.

in titer to CMV, but they attributed these virus in-

fections to the greater susceptibility of the patients during antirejection therapy.

COMMENTS

The role of CMV in clinical syndromes appears

difficult to evaluate since many transplant recipi-

ents are asymptomatic by the time the infection is

detected. The finding of CMV in asymptomatic pa-

tients studied at random may merely reflect the

continued excretion of virus for long periods of

time after an initial infection. If these patients are

studied only in this period, they will usually appear

to be asymptomatic, and viral infection will appear

to be of little clinical import. However, if these

same patients are studied at the time of onset of

virus infection, an association between virus in-

fection and spiking fevers, leukopenia and clinical

rejection can be established.

CMV infections have been shown to be a common Establishment of a precise etiologic association

finding in immunosuppressed renal transplant re- of the onset ‘of viral infection with rejection is

cipients [9]. Infection rates of from 70 to 90 per complicated by the characteristics of the virus in-

cent have been established by other surveys [1,2]. fection; virus can usually only be isolated from 5 Hill et al. [28] and Hedley-Whyte and Craighead to 10 weeks after the initial exposure [29]. This [6] were the first to implicate CMV in an etiologic can be readily seen by studying the time course of role in clinical disease of allograft recipients. They exogenous CMV infection in nonimmunosup- found cytomegalic inclusion cells in lung and pressed patients in whom a postperfusion mono- other tissues of patients who died of transplanta- nucleosis develops [25,26,29-321,. since the tion pneumonia. The etiologic relationship has exact time of exposure to the virus can be deter- since been confirmed by others [1,8]. In addition, mined. In these patients, fevers develop 2 to 4 Rifkind et al. [3] first noted rejection episodes in weeks after transfusion with fresh blood contain- two patients at the time they were showing a rise ing CMV. Serum antibody titers rise to detectable


levels about 10 to 20 days after the onset of fe-

vers. Positive cultures are usually detected shortly

after the onset of fever but sometimes not until

mudh later.

One cannot assume that CMV infections in im-

munosuppressed patients will follow the patterns

of infection in nonimmunosuppressed patients.

However, if the patterns are roughly comparable,

clinical infection should be present from 14 to 28

days before seroconversion. In our patients, onset

of fever, leukopenia and clinical rejection ap-

peared 25, 13 and 19 days before seroconversion

or at the time when clinical infection might be ex-

pected. The correlation of CMV infections with

these clinical manifestations, as well as the tem-

poral assocration between them, indicates that

they are probably true manifestations of the virus

infections.

In our study, the peak onset of fevers was 10 to

40 days after transplantation indicating that, if the

period of time between “exposure” to CMV and

clinical illness is the same in these patients as it

is in nonimmunosuppressed patients with postper-

fusion mononucleosis [25,26,29-321, our patients were “exposed” to infectious doses of CMV at

about the time, or shortly after, they received the

transplant. One explanation for the high incidence

of CMV infections immediately after the renal

transplant is that the profound immunosuppression

may activate a latent virus in these patients. An-

other possible interpretation of our findings is that

CMV is always present in the environment of

transplant patients and that the more intense im-

munosuppression regularly experienced by these

patients in the period immediately after receiving

the transplant is responsible for our observation

that initiation of the CMV infection dates to that

time. The many blood transfusions these patients

require might expose them to CMV [29]. Since

the incidence of CMV infections is lower in non-

immunosuppressed surgical patients who receive

blood transfusions than in immunosuppressed

renal transplant recipients [29], immunosuppres-

sion must play a role in the higher incidence of

virus infection. Recent studies by Huraux et al.

[33] moreover support the concept that reactiva-

tion of existing virus infections occurs in the im-

munosuppressed patient. Their studies showed

significant rises in antibody titer to several viruses

in two patients shortly after allotransplant. Since

these patients probably did not develop several new virus infections, all with viruses known to be

latent, these increases in serum antibody probably

indicated reactivation of preexisting latent in-

fections. Further supportive evidence comes from

Gardner’s [34] laboratory where immunosuppres-

sion of wild mice alone lead to a high incidence

(50 per cent) of mouse CMV infections.

There are several possible explanations for the

association of herpesvirus infections with deterio-

ration of renal function. First, the systemic virus

infection could lead to infestation of the grafted

kidney causing damage to the kidney accompa-

nied by moderate nonspecific deterioration of

renal function not related to allograft rejection.

This possibility seems unlikely since virus could

not be cultured from the renal biopsy specimens

obtained during the rejection episode, and biopsy

specimens taken at this time showed the typical

histologic characteristics of rejection.

A second possibility is that antigens which

cross react with donor histocompatibility antigens

are expressed during the herpesvirus infection,

and an immune response directed against these

viral-induced antigens might lead to rejection of

the kidney. Herpesviruses have been shown to in-

duce antigens in infected cells which cross react

with blood group substances [35], and a number

of bacteria have been shown to share antigenic

specificities with histocompatibility antigens

[36,37]. An analogous mechanism might occur for

viruses and histocompatibility antigens. However,

the fact that deterioration in renal function pre-

Ceded the increase in antiviral antibody titer

argues against this interpretation. Indeed, when

rejection episodes were untreated, the rise in an-

tiviral antibody heralded a decrease rather than an

increase in serum creatinine levels indicating that

antiviral antibody was associated with reversal

rather than initiation of rejection.

Still another possibility to explain the concur-

rence of allograft malfunction and viral infection is

the formation of circulating viral antigen-antibody

complexes that lead to immune complex nephritis

[27]. lmmunofluorescent studies of renal biopsy

specimens taken during rejection episodes dem-

onstrated no evidence of immune complex nephri-

tis.

Rifkind et al. [3] have suggested that severe

antirejection therapy probably makes the patient

more susceptible to infection, thereby explaining

the association between infection and rejection.

Evidence against this possibility was derived from

two patients in whom rejection episodes were not

treated but, nonetheless, in whom herpesvirus in-

fections were detected shortly thereafter. There are two possibilities which seem to best

explain the association between herpesvirus infec-

tions and rejection. The first possibility is that a

rejection episode might activate a latent virus in-




fection. Activation of a latent herpesvirus infection was demonstrated in rabbits [38] in conjunction with anaphylactic shock, or a second exposure to

the antigen. Graft versus host reactions have also

been shown to activate latent viruses [39-411.

Other studies have shown that lymphocytes,

transformed by mitogens or antigens, can be

more easily infected than can normal lympho-

cytes, and such cells appear to support viral mul-

tiplication much better than do nontransformed

cells [42]. It seems likely that lymphocyte trans-

formation is an integral part of the rejection pro-

cess; thus, recently transformed lymphocytes

would be available for virus infection. Therefore,

clinical or subclinical, rejection occurring soon

after transplantation might activate a latent CMV

infection.

The second possibility is that virus may act as a

nonspecific stimulus or adjuvant upsetting the

very delicate immunologic balance between donor

organ and host. The postulate presupposes that

there is a state of subclinical rejection present in

most allografted kidneys and that a state of rela-

tive immunologic adaptation of host to graft be-

comes established with time. Random biopsy

specimens of well tolerated allografts usually

show some evidence of rejection, however mini-

mal [43-451. In addition, humoral and cell-me-

diated immunity to functioning allografts have

been repeatedly demonstrated [43,46-491. Simi-

larly, there is much evidence to support the idea

that bacterial agents (i.e., endotoxin, BCG, infec-

tions) can nonspecifically stimulate immunologic processes [50]. Endotoxin, for example, can facil-

itate the rejection process itself [51]. Perhaps vi-

ruses, too, or virus antibody interactions can do

likewise. This possibility receives further support

from patients with postperfusion mononucleosis

caused by CMV. In these patients delayed hyper-

sensitivity to ampicillin and other immunologic ab-

normalities developed concurrent with their virus

infection [52,53]. Furthermore, if we can again

compare this clinical syndrome to postperfusion

mononucleosis, “exposure” to infectious doses of

virus precedes clinical rejection by about 2 to 4

weeks. Although one cannot say that infection

triggers rejection just because it precedes it,

these data lend added weight to this possibility.

The interrelationship between immunosuppres-

sion, infection and allograft rejection must cer-

tainly be complex. For example, the rejection pro-

cess probably begins long before clinical episodes

are diagnosed, and immunosuppression may con-

spire with rejection to activate the virus infection

soon after transplantation. Our data, because of

its epidemiologic nature, cannot determine wheth-

er infection activates allograft rejection or rejec-

tion activates a virus infection. Experiments are in

progress in this laboratory using a mouse CMV

model to determine which of these two possibili-

ties might best describe the situation in renal

transplant recipients. If, in fact, the virus infection

can trigger rejection episodes, then the develop-

ment of active antiviral drugs and/or the develop-

ment and use of vaccines for common herpesvi-

ruses might lessen the incidence of clinical re-

jection in transplant recipients.

ACKNOWLEDGMENT

The able technical assistance of Mr. Joseph Carey

and Ms. Charlene Edelman is gratefully acknowl-

edged.

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Association of renal allograft rejection with virus infections

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