TRANSACTIONS OF THE ROYAL, SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1999) 93,85590 85

Topical treatment with hexadecylphosphocholine (Miltex@) efficiently reduces parasite burden in experimental cutaneous leishmaniasis

Ruprecht Schmidt-Ott’, Thomas Klenneti, Peter Overath’ and Toni Aebischer’ ‘Max Planck-Institut fiir Biologie, Corrensstrape 38, 72076 Tiibingen, Germany; ‘ASTA Medica AG, Frankfurt/Main, Germany

Abstract Ether-lipids and alkylphosphocholines have been found to have anti-leishmanial activity. Oral treatment with hexadecylphosphocholine (HePC) efficiently reduces parasite burden in murine visceral leishma- niasis. Drugs for the treatment of cutaneous leishmaniasis are most commonly administered parenterally, whereas efficient drugs for topical treatment are not in current use. Here we investigate the efficacy of topical treatment with HePC in mice infected with Leishmania mexicana or L. major, causative agents of cutaneous leishmaniasis in the New and Old World, respectively. BALB/c, CBA/J and C57BL/6 inbred mice do not control infection with L. mexicana because they do not mount an efficient Thl-type anti- parasitic lymphocyte response. In contrast, C57BL/6 mice are resistant to an infection with L. major, developing only transient lesions that heal spontaneously owing to an efficient Thl response. BALB/c, CBA/J and C57BL/6 mice were infected subcutaneously with L. mexicana amastigotes, causing nodular lesions after 5 months. Topical treatment with HePC (Miltex@) was highly effective in reducing parasite burden and healed established lesions. The treatment did not induce a Thl response in L. mexicana- infected susceptible mice and most of the mice relapsed. In resistant C57BL/6 mice infected subcutaneously with 2 X lo6 L. major promastigotes at the tail base, nodular lesions developed after 2 weeks. Topical treatment with Miltex reduced the parasite load and the mice healed their lesions much faster than the untreated infected controls. The clinical application of Miltex for treatment of cutaneous leishmaniasis may be highly efficient because humans, similarly to resistant mice, in general do not relapse after healing. Clinical trials should be straightforward considering that Miltex is an approved drug for the treatment of breast cancer metastases.

Keywords: Letihmaniu mexicana, LRic;hmania major, experimental infections, mice, hexadecylphosphocholine, topical treatment

Introduction Cutaneous leishmaniasis, a disease resulting from

infection with species of the genus Leishmania including L. mexicana and L. major, represents a health problem in endemic regions such as the Middle East (KLAUS et al., 1994) and-Central America (GREVELIN~~ & LERNER~ 1996). In 1992. the worldwide incidence was estimated to be at least 300000 cases (ASHFORD et al., 1992). Clinical manifestations in humans are focal lesions that may persist for months to years. Drugs currently in use for the treatment of cutaneous leishmaniasis such as sodium stibogluconate, amphotericin B, and pentami- dine are most commonly administered parenterally using a repeated high dose regimen to ensure a sufficient concentration of drug at the site of the lesion. Topical therapy of cutaneous lesions, preferably as an ointment or lotion, would be more suitable, reducing the risk of systemic side-effects. Moreover, topical treatment would be more feasible in a rural setting and should increase compliance by allowing patients to treat themselves. However, at present such a treatment is not in current use. Efficacy of topical treatment with paromomycin has been assessed in patients with cutaneous leishmaniasis, but the results were not satisfactory (BEN SAL.AH et al., 1995; KNOBLOCH & DEMAR, 1997; NEVA et al., 1997; SOTO et al., 1998). In addition, increasing resistance to the conventional treatment of cutaneous leishmaniasis with antimonials has been reported in several regions of the world (BERMAN, 1997). Therefore, the development of new drugs against cutaneous leishmaniasis, preferen- tiallv with tonical efficacv, is hiahlv desirable.

Several ether-lipids and alkybhbsphocholines includ- ing hexadecylphosphocholine (HePC) have been shown to be active against L. donovaninromastiaotes (CROFT et al., 1987) aswell as intracellular amastigotes (CROFT. et al.. 1996‘1 in vitro. HePC is also active against other Le;shmania species in vitro (btANIER4, 1992x Oral treat- ment of L. donovani- or L. infanturn-infected mice with

Address for correspondence: Ruprecht Schmidt-Ott, Physiolo- gisches Institut, UniversitHt Tiibingen, Gmelinstrasse 5, 72076 Tiibingen, Germany; e-mail ruprecht.schmidt-ott@tuebingen.mpg.de

HePC was superior to sodium stibogluconate in redu- cing parasite load in liver and spleen<KUHLENCORD et al.. 1992; KAUFMANN-Ko~ etal., 1996; LE FICHOUX et al., 1998). Preliminary results of a clinical trial in India suggest that HePC may be an effective oral therapy for human visceral leishmaniasis (ASTA Medica AG, un- published data). The mechanism of its leishmanicidal activity is unknown, but its activity against promastigotes in culture is indicative of a direct toxic effect on the parasites. A perturbation of the parasite ether lipid metabolism and the elvcosvlnhosnhatidvlinositol (GPI) anchor biosynthesis-as weli as ihe membrane signal transduction was postulated by LUX et al. (1996). Also, HePC was shown to increase the interferon-gamma (IFN-y) production by human peripheral bloodmono- nuclear cells ~HOCHHUTH et al.. 1992) and. thus. could have immunomodulatorv act&-v. ‘B&y activates macrophages, stimulating their capacity to eliminate intracellular pathogens (GREEN et al., 1991) and po- tentiate their secretion of interleukin-12 (IL-12), a promoter of Thl-type differentiation of CD4+ T-cells (ROMANI et al., 1997). Resistant inbred mouse strains controlling L. mexicana or L. major infections exhibit a protective Thl cell response whereas susceptible strains do not (HEINZEL et al., 1991; AFONSO & SCOTT, 1993; SATOSKAR et al., 1995; GUEVARA-MENDOZA et al., 1997). Susceptible mouse strains with an established L. major infection can be induced to mount a protective Thl response: combined leishmanicidal and immuno- modulatory agents induce cure and longlasting immunity to reinfection. This has been shown for com- binations of sodium stibogluconate with either anti-IL-4 antibodies LABORS & FARRELL, 1994) or with IL-12 (LABORS er al., 1995). Thus, by enhancing IFN-y nroduction HePC might be beneficial in cutaneous ieishmaniasis by activating macrophages, increasing their leishmanicidal potential (MURRAY et al., 1983; TITUS et al., 1984) and by supporting a protective Thl cell response.

In this study, we investigated, first, the efficacy of HePC for the topical therapy of cutaneous leishmaniasis in susceptible mice infected with L. mexicana and resist- ant mice infected with L. major and, second, whether this

RUPRECHT SCHMIDT-OTT ETAL.

treatment promotes a disease-controlling Thl response in susceptible mice.

Material and Methods Parasites

L. mexicana amastigotes (strain MNYC/BZ/62/ M379) were isolated from dorsal lesions of CBA/J mice. Lesion tissue was homogenized and taken up in phos- phate-buffered saline (PBS) containing 140-mM NaCl, 3-mM KCl, 1.5-mM KH#Od and 21-mM NazHPOd. Amastigotes were separated by differential centrifuga- tion, washed, and resuspended in PBS at 2 X 10’ amastigotes/mL. For lymphocyte stimulation, axe- nit amastigotes, grown as described by BATES & TETLEY (1993), were washed twice in PBS and exposed to 46°C for 10 min. Therebv, more than 90% ofthe narasites lose their ability to transform into promastigotei in vitro.

L. major promastigotes (MRHO/IR/76/vaccine strain) were grown in semi-defined medium 79 (BRUN & SCHONEN~ERGER, 1979) supplemented wi& 10% inactivated fetal calf serum CFCS). Promastinotes from stationary-growth phase cult&es here used fo; infection of mice.

Miltex ” treatment One millilitre (about 40 drops) of Miltex”’ (ASTA

Medica AG, Frankfurt/Main. Gerrnanvj contains 60 me of Hel?C, 3i 6 mg of 3~propo&propyl~~eglycol, 158 rng of 3-hexyloxypropyleneglycol and 158 mg 3-nonyloxy- propyleneglycol in distilled water.

Female BALB/c, CBA/J and C57BL/6 mice were purchased from Charles River Breeding Laboratories (Sulzfeld, Germanv) and kept in a conventional animal facility in this institute. All mice were aged 8- 12 weeks.

BALB/c, CBA/T and C57BL/6 mice were infected subcuta;edusly a;“the tail base’ with lo7 virulent L. mexicana amastigotes. C57BL/6 mice were infected subcutaneously at the tail base with 2 X lo6 L. major promastigotes. Five months later (L. mexicana-infected mice) or 3 weeks later (L. major-infected mice), treat- ment of the established lesions was initiated. One drop of Miltex (about 1.5 mg HePC) was applied daily with a small brush directly on to the lesions for 5 days a week, during 5 weeks (L. mexicana-infected CBA/J and C57BL/6 mice) or 2 weeks (L. mexicana-infected BALB/c mice and L. major-infected C57BL/6 mice). The parasite burden of draining lymph nodes (inguinal and lower periaortic) and spleen was assessed in groups of at least 3 mice per strain at the onset of therapy and 3 weeks after the end of treatment (L. mexicana-infected mice) or immediately after the end of treatment (L. major-infected mice). In L. mexicana-infected mice the lymphocytes of the draining lymph nodes were typed and the functional phenotype of CD4+ lymph node cells was assessed.

Estimation of parasite burden Single-cell suspensions of draining lymph nodes and

spleen were prepared. The organs were cut into small pieces, taken up in ice-cold 15-mM HEPES-buffered balanced salt soiution containing 150-mM NaCl, 4-mM KC1 and 2.2 mM KH?POa. DH 7.2, sunnlemented with 5-mM EDTA and 2% F6.$-(BSS/EDyA), and passed through a fine wire mesh. Cells were taken up in semi- defined medium 79 and counted. Cell suspensions were serially diluted in quadruplicate in half-log&ithmic steps over 8 wells in 100 UL of semi-defined medium 79 in 96- well flat-bottom plates (Greiner, Niirtingen, Germany) and incubated at 27°C. After 4-5 days, cultures were inspected for the presence of flagellated parasites. The total number of parasites per organ was estimated from the highest dilution at which a minimum of 1 out of 4 replicate wells contained parasites (TITUS et al., 1985). The presence of persistent parasites at the original lesion site was determined as follows: scar tissue homogenate was taken up in 1 mL of semi-defined medium 79 and a

5OO+L sample was inspected for the presence of flagel- lated parasites after 4 days of culture at 27°C.

Typing of lymph node cells Cells from the draining lymph nodes of individual

mice were labelled with FITC- or PE-conjugated mono- clonal antibodies (mAbs) FUvi4-5, YTS169.4 and RA3- 6B2 (Pharmingen, Hamburg, Germany) which bind to CD4+ cells, CD8+ cells or B-cells, respectively, and with propidium iodide to exclude dead cells. The relative proportions of CD4+, CD8+ and B-cells were deter- mined by fluorimetry on a FACScan (Becton Dickinson FACS Systems).

Stimulation of CD4’ lymph node cells Single-cell suspensions of draining lymph nodes, pre-

pared as described above, from 3 mice ner arouu were pooled. Lymph node cells were incubate2 scmulta- neouslv with the mAbs 53-6.7. RA3-6B2. Ml 170 and RB6-8.C5 which bind to CDg+ cells, B-cells,’ macro- phages, or granulocytes, respectively, and the CD4+ cells were then negatively selected by magnetic cell sorting according to the manufacturer’s instructions (MAC& Milteni sotec, Bergisch-Gladbach, Germany), ieaving a population enriched in CD4+ T-cells (77-97%). The p&fied CD4+ cells were cultured in ‘96-well round- bottom plates in 200 & modified Dulbecco’s minimal essential medium (Dh4EM) (Gibco. Eggenstein, Ger- many) supplemented with .lo% F6S, -z-mM LLgluta- mine, 100 U

4 mL penicillin, 100 pg/mL streptomycin

and 5 X lo- -M 2-mercaptoethanol in a humidified atmosphere of 5% COz in air. The cell samples were stimulated in duplicate with heat-killed L. mexicana culture-amastigot& (final concentration 1 O6 amasti- gotes/mL), freeze-thaw lysates of L. mexicana pro- mastigotes (final concentration equivalent to 1 O6 promastigotes/mL) or concanavalin A (Con-A) (Phar- macia, Freiburg i. Br., Germany (final concentration 2.5 B/mL) in the presence of 10 2. u-radiated (2500 rad) syngeneic spleen cells from healthy donor mice as antigen-presenting cells (APC) at various effector cell/APC ratios (l:l, 1:3, 1:lO). Supernatants were harvested 24 h later for lymphokine assays. The lympho- kines IL-4 and IFN-y were assayed with the specific indicator cell lines CT4.S and WEHI 279 as described previously (MORRIS et al., 1992). CD4+ T-cell pro- liferation was assessed by pulsing overnight with 1 PCi ‘H-thymidine/well and measuring cellular uptake of ‘H-thymidine by liquid scintillation counting on Day 3.

Results Parasite reduction

Subcutaneous infection at the tail base of BALB/c, CBA/T and C57BLi6 mice with lo7 L. mexicana amas- tigotdsled to large &dular lesions after 5 months. At the onset of treatment mean parasite numbers in draining lymph nodes in BALB/c, CBA/J and C57BL/6 mice were 7.3 X 105, lo5 and 2.1 X 104, respectively, and 7900, 650 and 1200 in spleens (Table I). In all mice, visible and palpable nodules had disappeared at the end of treatment. In BALB/c and C57BL/6 mice treatment reduced the parasite burden in draining lymph nodes and spleen below the detection limit (< 100 parasites per organ) whereas in CBA/J mice the parasite burden decreased about lOOO-fold in lymph nodes and about 5-fold in the spleen (Table 1). Persistent parasites could be detected in the scar tissue of 3 of 6 BALB/c mice and 3 of 3 CBA/J mice but in none of 5 C57BL/6 mice (detection limit: 2 parasites per homogenized scar).

In C57BL/6 mice subcutaneous infection at the tail base with 2 X lo6 L. major promastigotes led to small nodular lesions after 3 weeks. At the onset of treatment the mean parasite number in draining lymph nodes was 9000 (Table 2‘1. Five weeks after infection nalnable lesions‘could nb longer be detected in Milted-triated mice whereas they were still palpable in untreated mice.

HEXADECYLPHOSPHOCHOLME TREATMENT OF EXPERIMENTAL. CUTANEOUS LEISHMANIASIS 87

Table 1. Estimation of parasite number in L. ma- icana-infected mice

Pre-treatmenta Post-treatmentb

Draining Draining Mouse lymph lymph strain nodesC Spleen nodes= Spleen

BALB/c 410 000 22 000 <loo <loo 1 100 000 42 000 <loo <loo

700 000 550 <loo <loo

CBA/J 130 000 650 100 <lOO 70 000 700 100 100

Cl00 <loo

C57BL/6 24 000 950 <loo <loo 52 000 1150 <IO0 <IO0

8000 1600 <loo <loo

Mice were infected subcutaneously with lo7 L. mexicana amastigotes at the tail base. Five months post infection estab- lished lesions were treated for 5 weeks (CBA/J and C57BL/6 mice) or 2 weeks (BALB/c) by topical application of MiltexB. The total parasite number in draining lymph nodes and spleen in individual mice was estimated by culturing single-cell suspen- sions in serial dilution. aAt onset of treatment, i.e. 5 months post infection. b3 weeks after end of treatment. CInguinal and lower peri-aortic lymph nodes.

Table 2. Estimation of parasite number in drain- ing lymph node9 of L. major-infected mice

Pre-treatmentb Not treatedC Post-treatment’

26 800 59 200 30 770 2000 15 780 3600 50

7700 53 000 10

C57BL/6 mice were infected subcutaneously with 2 X lo6 L. major promastigotes at the tail base. Three weeks post infection established lesions were treated for 2 weeks by topical applica- tion of MiltexR. The total parasite number in draining lymph nodes of individual mice was estimated by culturing single-cell suspensions in serial dilution. ‘Inguinal and lower peri-aortic lymph nodes. b3 weeks post infection. c5 weeks post infection.

The mean parasite number in draining lymph nodes in untreated mice was more than 1 OOO-fold higher than in Miltex-treated mice (Table 2). No parasites could be detected in the spleen in any mouse (detection limit: 100 parasites per spleen).

Cellular composition of draining lymph nodes L. mexicana infection was associated with alteration of

lymph node cellular composition in all 3 mouse strains investigated (Fig. 1). Five months after infection, there was a strong increase in the nodular B-cell number in all mouse strains, which is consistent with previous results showing polyclonal B-cell proliferation in cutaneous leishmaniasis (SOLBACH et al., 1987; LOHOFF et al., 1988). CD4+ and CD8+ T-cell numbers remained unchanged with the exception of a 3-fold increase in CBA/J mice. After Miltex treatment cell counts were reduced to the values found in healthy mice, with the exception of T-cell numbers in BALB/c mice that were lower than in healthy control mice.

Reactivity of CD4+ lymph node cells Five months after infection with L. mexicana, CD4+

cells from the draining lymph nodes were isolated and their proliferation in response to heat-killed amastigotes or freeze-thaw lysate of promastigotes was investigated. Following stimulation with heat-killed amastigotes, cells

25

T BALBlc

CDB+ B -Cells

25 C57BU6

“0 20

?r

CC%+ B-cells

l

CD4+ CD8+ B-cells

Fig. 1. Cellular content in draining lymph nodes. Mice were subcutaneously infected with 1 O7 L. mexicana amastigotes at the tail base. Five months later established lesions were treated as described in Table 1. Single-cell suspensions of draining lymph nodes of 3 mice per strain were analysed as described in Material and Methods. Total cell number/l06 f standard deviation are shown; white bars: syngeneic healthy control mice; blackbars: pre-treatment, i.e. 5 months post infection; greybars: post-treatment, i.e. 3 weeks after end of treatment. * Significantly different from syngeneic healthy control mice (f-test, P < 0.05). + Significantly different from pre-treatment values (c-test, P < 0.05).

88 RUPRECHT SCHMIDT-OTT ETAL.

from BALB/c and C57BL/6 mice incorporated 12 and 17 times more 3H-thvmidine into their DNA than unstimulated cells, whereas cells fi-om CBA/J mice did not respond. The proliferation of CD4+ lymph node cells of healthy BALB/c, CBA/J or C57BL/6 mice following stimulation with heat-killed L. mexicana amastigotes did not differ from unstimulated cells (Fig. 2). Stimulation of CD4+ cells with killed Leishmania amastigotes orfreeze- thaw lysate of promastigotes in the presence of syngeneic spleen cells from healthy mice as APC did not induce detectable production of either IL-4 (threshold of detec- tion 1 U/mL) or IFN-y (threshold of detection 0.1 U/mL) in cultures from healthy, infected, or treated mice. However, lymphokine production could be de- tected in cells from healthy, infected, or treated mice stimulated with Con-A (Table 3), showing high levels of IL-4 and undetectable or low levels of EN-y. After Miltex treatment there was no reduction in amastigote- specific T-cell proliferation (Fig. 2) or change in the lymphokine production profile of CD4+ cells (Table 3). CD4+ lymph node cells of all mouse strains produced no detectable IL-4 or EN-y following Leishmania-specific stimulation.

0 Healthy control

BALBlc CBA/J C57BU6

Fig. 2. Leishmania-specific proliferation of purified CD4+ cells from draining lymph nodes before and after Miltex@ treatment. Mice were subcutaneously infected with 10’ L. mexicana amastigotes at the tail base. Five months later established lesions were treated as described in Table 1. Single-cell suspensions from inguinal and lower peri-aortic lymph nodes of 3 mice per mouse strain and time point were pooled and CD4+ cells negatively selected by MACS. Cells (105) were co-incubated with lo5 irradiated (2500 rad) naive spleen cells and stimulated with heat-treated L. mexicana axenic amastigotes (106/mL). Cells were pulsed overnight with 1 uCi 3H-tbymidine and ‘H incorporation was measured by liquid scintillation counting on Day 3. The specific stimulation index (SI) is shown. The background c.p.m. values (syngeneic healthy mice/mice before treatment/mice after treatment) of cells from BALB/c, CBA/J and C57BL/6 mice were 400/1000/950, 2000/10300/2000 and 2000/8800/1500, respectively.

Discussion With increasing resistance to conventional anti-

leishmanial drugs (BERMAN, 1996), the treatment of cutaneous leishmaniasis has become a major concern for health authorities in endemic countries and in travel medicine.

Lesions in cutaneous leishmaniasis are in general focal. To reduce the risk of systemic drug-toxicity, treatment should therefore be topical, preferably as an ointment or lotion to be applied by the patient. However, efficient drugs for topical treatment are not in current use. CROFT

Table 3. Concanavalin A-induced lymphokine production of purified CD4+ cells fkom draining lymph nodes of L. mexicana-infected mice

Infected mice

Mouse strain

Healthy Pre- Post- mice treatmenta treatmentb

IL-4 IFN-v IL-4 IIN+ IL-4 IIN+

BALB/c 18.1 <O.l 48.6 3.2 433-5 3.1

%i,6 16.2 CO.1 34.1 <O.l 13.2 2.8

53 <O.I 12.7 <O.l 50.4 1.9

Mice were infected subcutaneously with 10’ L. mexicana amastigotes at the tail base. Five months post infection estab- lished lesions were treated by topical application of Miltex@ as described in Table 1. CD4+ cells from draining lymph nodes were stimulated with 25 ~.lg/rnL concanavalin A as described in Material and Methods. Cytokines were measured in 24-h supematants. IL-4 (U/mL) was assayed with the specific lymphokine responsive cell line CT4S. IFN-y (U/mL) was assayed with the specific lymphokine responsive cell line WBHI 279. aAt onset of treatment, i.e. 5 months post infection. b3 weeks after end of treatment.

et al. (1987) reported the anti-leishmanial activity of ether lipids and their derivatives, showing best results for the alkylphosphocholine HePC. HePC was originally developed as an anti-tumour agent. The Dhatmaco- kinetics (KW-I-ING et al., 1992y and toxic&y (ASTA Medica, unpublished data) of HePC have been exten- sively studied, and epicutaneous application of HePC has been well tolerated and devoid of systemic side- effects in humans (DUMMER et al., 1993; BURK et al., 1994). A micellar formula for the topical treatment of skin metastasis in patients with breast cancer (Miltex) (UNGER et al., 1992) was aDDrOved bv the German federal health authority in 199iand in se;eral European countries since. Here, we investigated the efficiency of this formula for topical treatment of established lesions in L. mexicana or L. major-infected mice.

BALB/c, CBA/J and C57BL/6 mice infected with L. mexicana developed progressively growing lesions. Sus- ceptibility of these mouse strains, as estimated by the kinetics of lesion growth, correlated with the narasite burden in draininglymph nodes (BALB/c > CeA/J > C57BL/6). In all L. mexicana-infected mice visible and palpable lesions disappeared after the second week of treatment. In BALB/c mice treatment was then stopped, but was continued for a total of 5 weeks in CBA/T and C57BL/6 mice. In the scars from 6 of 14 mice (3 BALB/c and 3 CBA/J mice) parasites could be detected 3 weeks after the end of treatment. Parasite numbers in draining lymph nodes and spleen, distant from the site of drug application, were very efficiently reduced. Systemic uptake of HePC in mice reaching leishmanicidal con- centration in body fluids could be responsible for the svstemic reduction of narasite burden; however. detect- able plasma levels after epicutaneous application of Miltex were not found in humans fASTA Medica AG. unpublished data). Alternatively,‘ burden reduction might be due to reduced dissemination from the lesion site and a failure of parasites to thrive under suboptimal temperature conditions for growth in internal organs (BI~GEL et al., 1983). Resisfent inbred mouse s&ins controlling L. major infections or IL-4 knock-out mice controlling L. m&icana infections exhibit a protective Thl cell response whereas susceptible strains do not (HEINZEL et al., 1991; AFONSO & SCOTT, 1993; SA- TOSKAR et al., 1995; GLJEVARA-MENDOZA et al., 1997). To investigate the possibility that the combined leishma- nicidal and TIN-y-inducing activities of HePC would induce protective immunity in susceptible mice, we determined the functional phenotype of CD4+ cells from

HEXADECYLPHOSPHOCHOLINE TREATMENT OF EXPERIMENTAL CUTANEOUS LEISHMANIASIS 89

draining lymph nodes of L. mexicana-infected suscepti- ble mice of different genetic background before and after treatment with He%. Before treatment, we detected substantial proliferation of CD4+ cells in BALB/c and C57BL/6 mice, indicating the presence of Leishmania- snecific CD4+ cells. Sumrisinelv. these cells did not groduce detectable amounts ofIE4 or EN-y, indicating a differential suppression of specific T-cell reactivity affecting only lymphokine production but not prolifera- tion. These findings extend the data of h?ONSO & SCOTT (1993) who have shown suppressed lymphokine production of lymph node cells from susceptible C57BL/6 mice chronically infected with L. mexicana amazonensis. Con-A induced strong IL-4 production and low or undetectable amounts of IFN-y in healthy mice as well as in L. mexicana-infected mice. This indicates that, independent of leishmanial infection, CD4+ cells from lymph nodes draining cutaneous sites express mainly the IL-4-producing Th2 phenotype. A possible explanation for this observation might be a preferential Th2 induc- tion in peripheral lymph nodes draining cutaneous sites. Alternatively, Th2 cells might home preferentially to lvmnh nodes drainina cutaneous sites. This could be inferred from findings-showing that lymphocyte homing to peripheral lymph nodes is mediated by I..-selectin (WARNOCK et al., 1998) and that the L-selectin-positive subpopulation of memory CD4+ cells contain Th%-like cytokine-producing cells (KANEGANE et al., 1996). There was no substantial change in rhe Leishmania- antigen (heat-killed amastigotes or promastigote freeze-thaw lvsate) or Con-A-induced lvmnhokine nro- duction after -Miltex treatment, in particular no @N-y production could be detected. We conclude therefore that topical treatment of HePC does not promote a protective Thl-type response in susceptible mice. In our study, 9 of 12 L. mexicana-infected mice relapsed 6 months after the end of treatment, presenting lesions at the initial site or other parts of the body. Thus, HePC- treated suscentible mice are not able to suunress the pathogenicity-of persistant parasites. L. maj&infected resistant C57BL/6 mice treated with Miltex for 2 weeks had healed their lesions without leaving visible scars while untreated mice still presented palpable lesions. Onlv few uarasites could be detected in draining 1vmDh nodes of heated mice, whereas draining lymph nodes from untreated mice harboured high numbers of para- sites. In this case, mice did not relapse with or without HePC treatment.

In immunocompetent humans with cured cutaneous leishmaniasis, parasites are thought to persist, without causing clinicai relapses (BERHE it al., 1995; ~~ELGADO et al.. 1996) as is the case in L. maiorinfected and cured C57&L/6 mice (AEBISCHER et al.; 1993). Therefore, by rapidly reducing the parasite burden the topical treat- ment of cutaneous leishmaniasis in humans may shorten the duration of disease without subsequent relapses. Because Miltex is an approved drug for topical treatment of breast cancer metastases, application of this drug for treatment of cutaneous leishmaniasis should be straight- forward.

Acknowledgements We thank Professor Dr Hansjijrg Eibl for invaluable advice,

Dorothee Harbecke for excellent technical assistance, and Dr Frank Weise for reviewing the manuscript.

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Received 3 September 1998; accepted for publication 25 September 1998

The Parasites of Homo sapiens: an Annotated Checklist of the Protozoa, Helminths and Arthropods for which We are Home. R. W. Ashford & W. Crewe. Liverpool: Liverpool School of Tropical Medicine, 1998. vii+728pp. Price klO.00 plus E2.50 mailing charge in UK, E4.00 elsewhere*. ISBN o-9508756-9-4.

About as interesting to read as the London (or Liver- pool) telephone directory, this book is none the less a very valuable check-list, as nearly comprehensive as the authors could make it, of the parasites of humans (the authors use the term ‘man’, stating in bold type that this is done ‘without gender implication’). There is a brief introduction, definitely more readable than a telephone directory, which includes, among other things, an in- clusive definition of ‘parasite’ as ‘a eukaryote organ- ism for which another organism can be described as its habitat’-as good a definition as any. The authors stress that the term should be used for individual organisms only, not for species, since some (e.g., Fasciola hepatica) are parasitic as adults but not as one or more of the larval stages.

The body of the text is the check-list proper. This

*This book can be ordered directly from Professor R. W. Ashford, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK (e-mail ljb@liverpool.ac.uk).

consists of brief listings of each species, including syn- onym(s) (where appropriate), status (i.e., prevalence), distribution, habitat, host(s), transmission, zoonotic status (i.e., relative importance of human and non- human hosts in maintaining populations), and one or a few key (not usually primary) references-just over 100 in all. Brief notes clarifying or amplifying the listings have been added in a few cases.

The 399 parasite species and 3 subspecies (the authors’ figures) listed are in taxonomic order, fi-om Protozoa to Arthropoda, taking in Trematoda, Cestoda, Nematoda and Acanthocephala on the way. Ticks and trombiculid mites have been expressly excluded. Oneunfortunate slip, pointed out to me by the senior author, is that Leishmania amazonensis and L. mexicana have been placed in the wrong subgenus ( Viannia instead ofleishmania) . I did not spot any other error, though there are many organisms listed about which I know very little, or less.

The book concludes with a short summary (2 pages) in which the parasites are tabulated according to abun- dance and zoonotic status. Most (328) of the 402 parasites depend on animals other than humans for their maintenance, 37 are shared with other hosts, and 37 appear to be restricted to human hosts.

There is a taxonomic index of 6 pages. John Baker

c/o Royal Society of Tropical Medicine and Hygiene Manson House 26 Portland Place London WlN 4EY, UK