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Topical chemotherapy of cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) is one of the most important causes of chronic ulcerative skin lesions. The disease is endemic in many parts of the world, presenting a range of clinical forms - acute, chronic, recurrent and diffuse(1). Several species of Leishmania are involved, including L. major, L. tropica and L. aethiopica in the Old World, and several members of the L. braziliensis and L. mexicana complexes in the New World. Some forms of the disease produce only mild, self-limited lesions, while at the other extreme are the destructive mucocutaneous forms caused by L. braziliensis and L. panamensis(1-7). In all cases, chemotherapy tends to be difficult - often requiring prolonged parenteral administration of toxic drugs such as pentavalent antimonials or amphotericin B. Such drugs are also expensive and relatively inefficient in the sense that much of the active ingredient is excreted by the patient before reaching its target. Consequently, there is renewed interest in the development of active formulations suitable for topical application directly onto the lesions.

Journal Article↗

T-cell subpopulations, expression of interleukin-2 receptor, and production of interleukin-2 and gamma interferon in human American cutaneous leishmaniasis.

Leukocyte subpopulations, the expression of the interleukin-2 (IL-2) receptor, and the production of IL-2 and gamma interferon (IFN-gamma) were studied in the peripheral blood mononuclear cells of American cutaneous leishmaniasis patients that had been stimulated in vitro with either leishmanial antigen or mitogen (phytohemagglutinin M). The 75 patients examined were classified as having either the localized (LCL; 66 patients), mucocutaneous (MCL; 5 patients), or the rare diffuse (DCL; 4 patients) form of the disease. Patients with DCL, who are characterized by their defective cell-mediated immune response to leishmanial antigen, failed to express the IL-2 receptor and did not produce IFN-gamma when exposed to the antigen but did so when stimulated by phytohemagglutinin M. Both LCL and MCL patients showed strong proliferative responses to leishmanial antigen; these were by far the greatest in MCL patients. Both groups had significantly increased IL-2 receptor expression and IFN-gamma production after exposure to either antigen or mitogen, and these were highest in the MCL patients. Concerning the leukocyte subpopulations evaluated (CD2, CD4, CD8, CD20, MO2), the most significant findings were a decrease of both CD4+ cells and the CD4/CD8 ratio in MCL patients compared with the other groups. Considering IL-2 production, in response to phytohemagglutinin M both MCL and LCL patients showed amounts of IL-2 comparable to those of the controls. Our results help explain the anergy of T cells from DCL patients to leishmanial antigen, which could lead to a defective production of IFN-gamma and possibly contribute to their incapacity to kill the Leishmania parasite. Concerning MCL patients, the significantly increased expression of IL-2 receptor, decreased expression of the CD4 (helper-inducer of suppression) phenotype, and elevated IFV-gamma production might partially explains the state of hypersensitivity and mucosal damage exhibited by these patients.

Adult↗

Histologic characterization of experimental cutaneous leishmaniasis in mice infected with Leishmania braziliensis in the presence or absence of sand fly vector salivary gland lysate.

Leishmania braziliensis is the causative agent of human cutaneous leishmaniasis in parts of the New World. In the murine model of infection, L. braziliensis does not produce severe or lasting cutaneous lesions in either BALB/c or C3H mice. However, when the parasites are injected into BALB/c mice with salivary gland lysate of the sand fly vector for the parasite, infection is significantly enhanced, as measured by lesion size, parasite burden, and the outcome of infection. Histologic examination of these cutaneous lesions showed that initially, nodular and diffuse dermal infiltrates of neutrophils, eosinophils, and histiocytes occurred in all mice. Over time, the saliva-free lesions progressed to small organized granulomas of epithelioid macrophages that contained few parasites, with eventual resolution of inflammation and mild dermal fibrosis. The saliva-associated lesions progressed to extensive, poorly organized accumulations of heavily parasitized epithelioid macrophages, with persistent neutrophils and eosinophils, and minimal fibroplasia. These results indicate that sand fly salivary gland lysate markedly modifies the inflammatory response to infection with L. braziliensis.

Animals↗

[Metacyclics of Leishmania mexicana in the hypopharynx of Lutzomyia youngi experimentally infected].

Although it has been postulated that the natural transmission of leishmaniasis occurs by regurgitation of the parasites from contaminated proboscis of phlebotomine vector recent experimental results seems to oppose this thesis. Wild-caught and laboratory-reared females of Lutzomyia youngi, vector of Leishmania mexicana in the Venezuelan Andes, were infected on tarsal lesion of hamsters, inoculated 6 weeks previously s.c. with 2 x 10(4) amastigotes of L. mexicana s.l., isolated from Venezuelan case of diffuse leishmaniasis. The insects were kept at 23 degrees C and 80% R.H., on a 50% (v:v) solution of "unrefined" cane sugar. The parasites developed through 3 stages: i) differentiation of the amastigotes and exponential growth in the number of promastigotes, ending between 60 and 108 hrs, with a massive fecal elimination of large promastigotes; ii) a stationary phase in growth with flagellar adhesion to the cuticle of the stomodeum and synthesis of a gel that formed a plug between 60 and 120 hrs; iii) differentiation of metacyclics, which invade the hypopharyngeal duct in 7% of the insects, from the 5th day post-infection, and most frequently between the 6th and 9th day. The metacyclics measured 4.2/microns in body length, 1.07/microns in maximum with and the flagellum was 8.8/microns long. The parasites swam freely in the saliva of the hypopharyngeal duct (lumen 6/microns in diameter), from the apex to union with the salivary duct, without invading the latter. Similar results has been obtained in the same vector experimentally infected with other two strains of L. mexicana isolated respectively from wild Lu. flaviscutellata and from another case of diffuse leishmaniasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Atypical multifocal cutaneous leishmaniasis in an immunocompetent patient treated by liposomal amphotericin B.

Multifocal cutaneous leishmaniasis (MCL) is an extremely rare disease in South Europe, and it mainly affects immunosuppressed patients. We report a case of MCL in an immunocompetent patient affected by type II diabetes mellitus that clinically presented with three large ulcers on the legs with a non-linear distribution and several months later with an erythematous-crusty lesion on the left cheek. Diagnosis of leishmaniasis due to Leishmania infantum was formulated by PCR analysis. Given the diffuse and wide lesions, the unresponsiveness to previous local and systemic treatments, a parenteral i.v. therapy with liposomal amphotericin B at a dosage of 3mg/kg/day for 5 days was started and then repeated on the 14th and 21st days, leading to a clear improvement in the clinical picture. The different clinical expression and the evolution of leishmaniasis depend on both the parasite subtype and the host's immunity status. L. infantum manifests with an atypical clinical feature more frequently than other species. The differential diagnosis for multiple ulcers must include several skin diseases, such as cutaneous TBC, bacterial ulcers, traumatic ulcers, deep mycoses, and sarcoidosis. However, an MCL should always be considered in subjects coming from endemic areas. In our case, the multifocality, the size of the lesions and the unresponsiveness to other treatment indicate a short course treatment with liposomal B amphotericin that proved to be a suitable alternative to traditional drugs used in MCL.

Aminoglycosides↗

Topical buparvaquone formulations for the treatment of cutaneous leishmaniasis.

As the part of a study to develop buparvaquone (BPQ) formulations for the treatment of cutaneous leishmaniasis, the topical delivery of BPQ and one of its prodrugs from a range of formulations was evaluated. In previous studies, BPQ and its prodrugs were shown to be potent antileishmanials in-vitro, with ED50 values in the nanomolar range. 3-Phosphono-oxymethyl-buparvaquone (3-POM-BPQ) was the most potent antileishmanial and was chosen, together with the parent drug, for further investigation. The ability of the parent and prodrug formulations to cross human and murine skin was tested in-vitro using the Franz diffusion cells. Formulations intended for topical application containing either BPQ or 3-POM-BPQ were developed using excipients that were either acceptable for topical use (GRAS or FDA inactive ingredients) or currently going through the regulatory process. BPQ was shown to penetrate both human epidermal membranes and full thickness BALB/c skin from a range of formulations (gels, emulsions). Similarly, 3-POM-BPQ penetrated full-thickness BALB/c skin from several gel formulations. In-vitro binding studies showed that BPQ bound melanin in a dose-dependent manner and preferably bound to delipidized skin over untreated BALB/c skin (on a weight to weight basis). The results confirm that BPQ and its prodrug 3-POM-BPQ can penetrate the skin from several formulations, making them potentially interesting candidates for further investigation of topical formulations using in-vivo models of cutaneous leishmaniasis.

Administration, Cutaneous↗

Leishmania major: the suitability of East African nonhuman primates as animal models for cutaneous leishmaniasis.

The susceptibility of four species of East African nonhuman primates to experimental infection with Leishmania major was investigated. Four Syke's monkeys (Cercopithecus mitis), two vervet monkeys (Cercopithecus aethiops), two baboons (Papio cynocephalus), and two brown bushbabies (Galago garnettii) were each inoculated intradermally on the left eyelid, left ear, and nose with 0.1 ml of medium containing 1 x 10(7) promastigotes of a characterized L. major strain. All the nonhuman primates except the bushbabies developed erythema and conspicuous nodules on the eyelids and ears by 3 weeks PI. The nodules increased rapidly in size and ulceration was evident on the eyelids and ears by 49 days PI in the vervets, Syke's, and baboons. The aspirates were positive in culture or smears at 35, 49, 63, and 77 days PI. No parasites were observed in cultures or smears at 92, 105, 128, 147, and 161 days PI. The lesions in these animals began resolving by 84 days PI and were completely healed by 112 days PI. The exception was one baboon in which lesion healing did not start until around 147 days and was completely healed by 182 days PI. Cultures from the liver failed to demonstrate visceralization of the parasite in any of the animals throughout the 68 weeks of the experiment. Challenge with the same strain of L. major 6 months PI, corresponding to about 3 months after self cure, failed to produce infection in any of these experimental hosts. All the nonhuman primates except the bushbaby when challenged with the same strain of L. major at 12 months PI developed lesions and were positive for parasites at 14 and 28 days PI. Positive cultures were obtained from the eyelid and ear of one vervet up to 42 days PI. However, the lesion sizes in all these animals were smaller than in the initial infection and did not ulcerate. The nodules disappeared within 6 to 8 weeks as compared to 16 weeks in the initial infection. The histopathological appearance of the lesions varied from diffuse infiltration of plasma cells and lymphocytes which increased progressively to granulomata with epitheloid cells. This study shows that the vervets, Syke's, and the baboons are equally susceptible to L. major infection, while bushbabies are refractory. The vervets, Syke's, and baboons demonstrate a self-healing phenomenon within about 3 months which is comparable to that observed in humans infected with L. major. These three species of nonhuman primates are therefore considered as suitable models for drug or vaccine trials against human zoonotic cutaneous leishmaniasis.

Animals↗

[Histopathology of mucocutaneous leishmaniasis caused by Leishmania (Vianna) braziliensis].

A histopathological study of mucocutaneous leishmaniasis due to Leishmania (Viannia) braziliensis was carried out on 28 cutaneous and 114 mucosal biopsies, taken from Bolivian and Peruvian patients. This study showed similar histopathological findings in cutaneous and mucosal lesions. The cutaneous biopsies showed a strong epidermal hyperplasia occasionnally budding in the dermis. In the ulcerative area, the epidermis was totally necrosed and replaced by a fibrino-leucocytic edge. In the dermis, histio-lympho-plasmocytic infiltration was constantly found. The histiocytes often gathered in follicles sometimes with diffuse fibrosis. The parasites were encountered in 28.6 p. 100 of the biopsies. Whatever the mucosa concerned (i.e. nasal, palatal or lingual), the mucosal lesion was not different from the cutaneous lesion. The malpighian epithelium is either absent or the seat of a pseudo-epitheliomatous hyperplasia. Major histio-lympho-plasmocytic infiltration was found and extended through the depth of the lamina propria. Suppurative and fibrinoid necroses coexisted superficially and sometimes penetrated in depth. The parasites were found in about 30 p. 100 of the cases.

Adult↗

Lipid and lipopolysaccharide-like antigens of Leishmania promastigotes.

Extraction of whole promastigotes of Leishmania tropica major and L. donovani with a mixture of hexane and isopropanol (3:2) yielded three fractions containing immunological activity: lipids, where the activity was determined by radioimmunoassay; a lipopolysaccharide-like (LPS-like), water-soluble precipitate, where activity was determined both by radioimmunoassay and double gel diffusion, and the phenol: water extract of the lipid-free promastigotes, where activity was followed by double gel diffusion. The use of a solid state, lipid-based radioimmunoassay for detection of leishmanial antigens provided a sensitive measure of their activity with a considerable degree of species and serotype specificity. We found antibodies to leishmanial lipids in sera from immunized rabbits, convalescent mice, and human patients with confirmed cases of cutaneous leishmaniasis or kala azar. There was very little activity in normal human or animal sera. Analysis by SDS-polyacrylamide gel electrophoresis of fractions from promastigotes surface-labeled with galactose oxidase and sodium borotritiate and preliminary immunochemical characterization of the LPS-like antigen showed that it contained galactose, but otherwise differed immunologically and chemically from excreted factor (EF), the best characterized leishmanial antigen.

Animals↗

Influence of lysates of the salivary glands of Lutzomyia longipalpis on the development of a Leishmania-major-like parasite in the skin of the golden hamster.

Twelve years ago, some mice inoculated with Leishmania major were found to develop larger lesions, containing more amastigotes, if the inoculum used to infect them contained a lysate of salivary glands from Lutzomyia longipalpis than if no lysate was included. In the present study, outbred golden hamsters (Mesocricetus auratus) were each inoculated in a footpad with 10(4), 10(5), 10(6) or 10(7) stationary-phase promastigotes of a Leishmania-major-like parasite (MHOM/BR/71/BH49). Some of the inocula used each contained a lysate of the salivary glands from a laboratory-reared, female Lu. longipalpis. Only the hamsters inoculated with 10(7) promastigotes each developed macroscopic cutaneous lesions (all 10 co-inoculated with lysate but only two of the 10 co-inoculated with diluent). Each of the lesions developed into cutaneous nodule affecting the dermis and underlying subcutaneous tissue of the inoculated footpad, with, histologically, an intensive, diffuse and productive, inflammatory reaction. There were no apparent differences between the lesions of hamsters infected with inocula containing salivary-gland lysate and those seen in the animals infected with lysate-free inocula. Future studies will follow the histological changes at the sites of Lu. longipalpis bites.

Analysis of Variance↗

In vitro skin permeation and retention of paromomycin from liposomes for topical treatment of the cutaneous leishmaniasis.

Paromomycin (PA), a very hydrophilic antibiotic, has been tested as an alternative topical treatment against cutaneous leishmaniasis (CL). Although this treatment has shown promising results, it has not been successful in accelerating the recovery in most cases. This could be attributed to the low skin penetration of PA. Liposomal formulations usually provide sustained and enhanced drug levels in skin. The aim of this study was to prepare liposomal formulations containing PA and to investigate their potential as topical delivery systems of this antileishmanial. Large multilamellar vesicles (MLVs) were prepared by conventional solvent evaporation method. Large unilamellar vesicles (LUVs) were prepared by reverse-phase evaporation method. The lipids used were soybean phosphatidylcholine (PC) and PC:cholesterol (CH) (molar ratio 1:1). The skin permeation experiments across stripped and normal hairless mice skin were performed in modified Franz diffusion cells. The PA entrapment in LUV liposomes (20.4 +/- 2.2%) was higher than that observed for MLV liposomes (7.5 +/- 0.9%). Drug entrapment was 41.9 +/- 6.2% and 27.2 +/- 2.4% for PC and PC:CH LUV, respectively. The skin permeation was 1.55 +/- 0.31%, 1.29 +/- 0.40%, 0.20 +/- 0.08%, and 0.50 +/- 0.19% for PC LUV, PC:CH LUV, empty LUV +/- PA and aqueous solution, respectively. Controlled topical delivery, across stripped skin, was observed for PA entrapped in LUV liposomes.

Administration, Cutaneous↗

Cell-mediated immunity in experimental cutaneous Leishmaniasis.

Forms of cutaneous leishmaniasis are caused by Leishmania major, L. tropica, L. mexicana, L. amazonensis and L. panamensis. Like all leishmanial species, these are obligate intracellular parasites of the mononuclear phagocyte system, with a restricted range of vertebrate hosts including humans, dogs, rodents and arboreal animals. The disease evolves chronically, usually with slow healing, but can sometimes become nonhealing, diffuse disseminating or relapsing. The parasite exists within the macrophages of the vertebrate host in the amastigote form. These transform into extracellular flagellated promastigotes in the gut of the sandfly vectors. The promastigotes can then be injected into new vertebrate hosts as the insects feed. Promastigotes, and to a lesser extent amastigotes, can now be grown in tissue culture. This, together with the use of inbred mouse strains that are susceptible to most of the Leishmania species which are pathogenic for man, has facilitated great advances in our understanding of the immunological control of leishmaniasis. However, as Eddy Liew points out, there are still many unanswered questions.

Journal Article↗

Purification of two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis.

Two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis were purified using species-specific monoclonal antibodies and characterized. The molecular weights of the proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were approximately 70,000 and approximately 72,000, respectively. The 70 kDa protein, which appears as a diffuse band on silver staining, was resolved into a doublet by Western blotting with monoclonal antibody. Though of similar molecular weight and amino acid composition, the two proteins were shown to be distinct by peptide mapping and Western blotting of the purified material. The two proteins are recognized specifically by human visceral leishmaniasis serum and not by serum from cutaneous leishmaniasis or Chagas' disease. These proteins will be useful in developing a direct serodiagnostic assay for visceral leishmaniasis.

Animals↗

The pathology of cutaneous leishmaniasis in the Sudan: a comparison with that in other geographical areas.

The pathology of cutaneous leishmaniasis (CL) caused by Leishmania major zymodeme LON 1 in the Sudan was compared with that caused by L. major zymodeme LON 4 in Saudi Arabia and with that already described for L. tropica infections in Iran and for localized CL in the New World. The lesions were classified according to Ridley's five histological types. Most of the lesions in the Sudan and Saudi Arabia were of types B and C, characterized, respectively, by diffuse macrophage necrosis and focalized necrosis. B was the most common type in Nicaragua and Guyana whereas responses of types A (in which there are heavily parasitized macrophages without necrosis) and D (reactive tuberculoid) were the most frequent in Iran. The type-E response, which is similar to D but with virtual absence of plasma cells, was uncommon in all areas. The type-D reaction is a chronic relapsing disease when associated with L. tropica but not when associated with L. major. The major differences in the pathology of CL in different geographical areas most probably relate to differences in the Leishmania species involved. Minor differences, however, not only occur between patients from the same area but may also occur, with time, in the same patient. Detailed comparison between areas is therefore difficult; lesions on one patient may heal asynchronously and show different histological types at any point in time and rebiopsy from the same lesion during healing reveals changes from one histological type to another.

Animals↗

Experimental infection of Mystromys albicaudatus with Leishmania braziliensis: pathology.

Thirty-four Mystromys albicaudatus were injected intradermally with Leishmania braziliensis promastigotes and examined and killed during a 12-week period. All animals developed single cutaneous lesions at the sites of inoculation that began as a papular thickening in the dermis and progressed to a 2.0 cm crateriform ulcer. The histopathology of these lesions was characterized by granuloma formation and diffuse infiltrates of lymphocytes and plasma cells. Parasites were most frequently observed in vacuolated macrophages immediately underlying the necrotic debris of the ulcer. Histiocytes in which amastigotes were not identified were noted in aggregates at the margins of the inflammation. Russell's bodies were observed at 6 weeks post-infection, and discrete lymphocytic infiltrates bordered the inflammation at 12 weeks post-infection. It is suggested that M. albicaudatus is an excellent model of American cutaneous leishmaniasis.

Animals↗

Mechanisms of pathogenesis: differences amongst Leishmania species.

One of the features of the genus Leishmania is the diversity of tropism/disease resulting from infection. With notable exceptions, the form (visceral, cutaneous, diffuse cutaneous, mucocutaneous) and severity of disease is a function of the infecting Leishmania species together with host genetics and consequent inflammatory and immune responses. It has become evident from genetic and immunological studies using the murine model that the various members of the genus Leishmania differ in aspects of their 'approach' to the host immune system. We are just beginning to appreciate the complexities of these interactions, which have import for the development of a vaccine against leishmaniasis. In this paper, what is currently understood concerning the mechanisms of leishmanial pathogenesis (based upon studies employing the murine model) is briefly summarized.

Animals↗

Cutaneous leishmaniasis of the New World: diagnostic immunopathology and antigen pathways in skin and mucosa.

Non-specific chronic inflammation and/or granulomatous reaction are the main histopathological manifestations of cutaneous and mucocutaneous leishmaniasis of the New World. Plasma cell infiltration associated with collagen and vascular changes are data suggestive but not diagnostic of the disease. Specific diagnosis is only possible through demonstration of the parasite in the tissue examined. It is noteworthy that the parasites are usually scanty and difficult to demonstrate in the lesions. Biopsies from 40 patients with cutaneous or mucocutaneous leishmaniasis were examined using the immunofluorescence and immunoperoxidase techniques in order to demonstrate the parasite and/or antigen in the tissues. Nineteen biopsies showed non-specific chronic inflammation and 21 a granulomatous reaction. Parasites were found in 20% of the routine biopsies. The positivity through indirect immunofluorescence was 88.46% in frozen sections of fresh material and 89.28% in paraffin embedded tissue. The antigen positivity with the immunoperoxidase technique was 64.51%. Antigen was detected as amastigotes and also as diffuse material in the macrophage cytoplasm and adsorbed in the epithelial basement membrane and vessel walls. There was no difference in the positivity of antigen according to the type of inflammatory reaction.

Animals↗