Shoshana Frankenburg

Epidemiology of cutaneous leishmaniasis

Leishmaniasis refers to an infection caused by protozoan parasites of the genus Leishmania. Of the three clinical expressions of the disease—cutaneous, visceral, and mucocutaneous—the cutaneous form (CL) is the most abundant. It is important to recognize that CL does not refer to a single disease. At least 12 species of the genus Leishmania are known to cause CL, and each parasite species has its own characteristic vectors and reservoirs. Bradley has pointed out that in no other parasitic genus infecting man is there such a “diversity of species and strains, variety of pathogenic manifestations, and ecologic richness of transmission patterns.”1 It is now clear that only by recognizing the complexity of this group of diseases—including the intricate patterns of interaction between hosts, reservoirs, and vectors—can progress be made in formulating effective methods of control.

A simplified microtechnique for measuring human lymphocyte proliferation after stimulation with mitogen and specific antigen

Cutaneous leishmaniasis is a parasitic disease characterized by a marked cell-mediated response. In vitro measurements of this response in humans have so far been used to a limited extent probably because of the relatively large amounts of blood demanded for conventional cell proliferation studies. The microtechnique here described enables lymphocyte proliferation to be performed with small amounts of blood (100 microliter) which can be obtained by finger prick, and do not require Ficoll separation prior to cultivation of the mononuclear cells. The blood was aspirated into sterile capillaries, introduced into tubes containing heparin, and directly distributed into wells. The response to Leishmania major, to tuberculin purified protein derivative (PPD) and to mitogen was compared to the response of mononuclear cells after Ficoll separation, and no marked difference was found between the two methods. Using the described method, individuals cured from a Leishmania major infection showed a high response to the specific antigen, as compared to normal controls. The potential use of this microtechnique for epidemiological studies is discussed.

Capture of Tumor Cell Membranes by Trogocytosis Facilitates Detection and Isolation of Tumor-Specific Functional CTLs

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

Changing patterns of cutaneous leishmaniasis in Israel and neighbouring territories

The most frequent form of cutaneous leishmaniasis (CL) in Israel and the neighbouring territories is due to Leishmania major, which is endemic mainly in the Jordan Valley and in the Rift Valley. CL due to L. tropica is much less common, and in the past only sporadic cases have been reported. In this study we present data obtained during the years 1988-1992 regarding CL in the area. Our clinic has diagnosed a total of 371 leishmaniasis cases, most of whom acquired the infection in the Jordan Valley, mainly during June and July. About one-third of the patients had single lesions, and one-third more than 5 lesions. We also describe an outbreak of leishmaniasis in Kfar Adumim, a village 15 km east of Jerusalem, where leishmaniasis was previously unknown. Parasites were characterized by the polymerase chain reaction and by immunostaining, and found to be both L. tropica and L. major. The localization of the homes of the affected people on a slope where hyraxes were abundant suggests that these animals might have been involved in the transmission of L. tropica in this area.

Action of leishmanial excreted factor (EF) on human lymphocyte blast transformation

Summary. The effect of Leishmania tropica major excreted factor (EF) on human immune and normal mononuclear peripheral blood cells has been studied. The response of lymphocytes to stimulation either specifically with leishmanial antigens or non‐specifically with phytohemagglutinin (PHA) in the presence of EF was tested by the uptake of 3[H]thymidine. The results showed that EF inhibits the response of cells from immune donors to leishmanial antigens and from normal donors to PHA or PPD. Adherent and non‐adherent cells were separated and the effect of EF on both populations was analysed. The results showed that EF inhibited blast transformation if both EF and antigen were presented to each of the separate populations. The inhibition of the adherent cells (mainly monocytes) was more marked than the inhibition of the nonadherent population (mainly lymphocytes).

Efficacious topical treatment for human cutaneous leishmaniasis with ethanolic lipid amphotericin B

Old World cutaneous leishmaniasis (CL) is a widespread and potentially disfiguring protozoa1 infection, caused by the infection of dermal macrophages with Leishmania parasites. Despite the various implemented measures to prevent the disease, there is a steady number of infected individuals who seek treatment. Systemic treatments available are very often unjustified owing to their toxicity (e.g., pentavalent antimonials) or to their variable efficacy (e.g., ketoconazole). Topical treatment, the obvious way to treat a localized skin disease, still poses a challenge. Paromomycin-containing preparations have proven effective in some cases (El-On et al., 1985), but not in others (KLAUS et al., 1994; BEN SALAH et al., 1995), and often cause substantial skin irritation. Amphotericin B (AmB), a potent antileishmanial antibiotic, has not been previously delivered topically to dermal lesions. We have recently reported preliminary results showing that a colloidal dispersion of AmB and cholesteryl sulphate, in the presence of 5% ethanol and in the absence of glucose, has significant therapeutic effect in the treatment of CL lesions. The presence of glucose, on the other hand, inhibited the therapeutic effect (VARDY et al., 1999). Here we present results obtained in 1999-2000 on the treatment of 17 patients with AmB, in a prospective placebo-controlled study. Each patient had at least 1 placebo-treated and 1 AmB-treated lesion.

Efficacious Topical Treatment for Murine Cutaneous Leishmaniasis with Ethanolic Formulations of Amphotericin B

ABSTRACT The goal of the present study was to evaluate the antileishmanial effects of topically applied lipid-based formulations containing amphotericin B (AmB) in CBA mice as a model for human cutaneous leishmaniasis. Such treatment, if efficacious, is expected to be superior to systemic treatments since, by acting in a localized manner, it will require lower, and therefore less toxic, drug dosages. Three preparations of AmB complexed to polar lipids were tested: Fungizone (mixed micelles composed of AmB and deoxycholate), Amphocil (AmB and cholesteryl sulfate complex), and ABPLC (AmB and phospholipid complex). All these formulations killed parasites in vitro with similar efficacies but were ineffective when they were applied topically. However, Amphocil and ABPLC, but not Fungizone, when dispersed in an aqueous solution containing 5 to 25% ethanol, induced a statistically significant improvement in lesion size from week 2 or 3 onward (a total of 15 mg of AmB per kg of body weight was applied over 3 weeks). AmB biodistribution measurements following topical application of Amphocil, determined by high-pressure liquid chromatography, showed that AmB was detectable in the skin but not in the internal organs. Application of at least 10 times more drug was necessary to obtain detectable levels of AmB in the internal organs. After application of therapeutic doses of ABPLC, very low levels of AmB were detected in the internal organs. These experiments show for the first time that AmB administered topically as a complex either with cholesteryl sulfate or with phospholipids and in the presence of ethanol can penetrate the skin and kill sensitive organisms in a localized manner by using very low total drug concentrations.

Lysis of murine macrophages infected with intracellular pathogens by interleukin 2-activated killer (LAK) cells in vitro

To determine if murine lymphocytes activated by interleukin 2 (IL-2) were cytotoxic against syngeneic elicited peritoneal macrophages (M phi) infected with intracellular pathogens, splenocytes that had been cultured with IL-2 for 5 or 10 days were studied in vitro. These cells, [lymphokine-activated killer (LAK) cells] showed significantly greater cytotoxicity against M phi infected with Leishmania major or Legionella pneumophila than against uninfected M phi. Preferential cytotoxicity against infected M phi was best shown using effector-to-target-cell ratios of 1:1-20:1; when ratios greater than or equal to 40:1 were employed, uninfected M phi were also lysed. The extent to which M phi that had been incubated with L. major were lysed depended upon the proportion of M phi containing intracellular organisms. After infection with L. major, the duration of incubation did not appear to influence the degree of lysis by LAK cells.

Effective immunization of mice against cutaneous leishmaniasis using an intrinsically adjuvanted synthetic lipopeptide vaccine

Two peptides representing predicted T-cell epitopes of gp63, a major surface glycoprotein of the parasite Leishmania major, were used in vaccines tested in a murine model of cutaneous leishmaniasis. Either subcutaneous or intraperitoneal immunization in saline with a peptide representing gp63 amino acids 467-482 (p467) significantly protected CBA mice against the development of severe cutaneous lesions only when the peptide was intrinsically adjuvanted by covalently adding a lauryl-cysteine moiety (LC-p467) to its amino terminus during synthesis. In marked contrast, administration of p467 alone, cysteinyl-p467 or gp63 protein in saline resulted in some disease exacerbation. Splenic cells of LC-p467 immunized mice stimulated in vitro with LC-p467 displayed strong proliferative responses and secretion of IL-2, IFN-tau and GM-CSF (but not IL-4 and IL-10) suggesting that immunization with the lipopeptide induced the TH1 type cytokine responses associated with cell-mediated immunity. The safety, efficacy, ease of production and standardization of such lipopeptide vaccines suggest that they have significant potential for the development of vaccines for humans against leishmaniasis or other parasitic or viral diseases that require cell-mediated immunity for protection.

Autologous Melanoma Vaccine Induces Antitumor and Self-Reactive Immune Responses That Affect Patient Survival and Depend on MHC Class II Expression on Vaccine Cells

Purpose: Autologous melanoma cells display a broad variety of tumor antigens and were used for treatment of American Joint Committee on Cancer stages III and IV melanoma as an adjuvant or active therapy. Survival data and immune response were evaluated in vaccinated patients. Experimental Design: Forty-seven patients received 2,4-dinitrophenyl–conjugated autologous melanoma vaccine as an adjuvant (23 patients) or therapy (24 patients). CD4 and CD8 T-cell response in blood sampled before vaccination and after five or eight vaccine doses was evaluated against melanoma cells and autologous peripheral blood mononuclear cells using IFNγ enzyme–linked immunospot. Serum levels of antilivin, an inhibitor of apoptosis, and anti-gp100 IgG were determined. Results: The immunologic effect of the vaccine differed between the two groups of patients. In the adjuvant group, there was a significant increase in CD8 melanoma-reactive T cells (P = 0.035) after vaccination and an increase in antimelanoma CD4 T cells correlating with improved overall survival (P = 0.04). In the therapeutic group, there was no objective tumor regression; antimelanoma T-cell reactivity increased by a small amount, stayed the same, or in some cases decreased. In all patients, a significant increase was noted in CD4 T-cell reactivity against autologous peripheral blood mononuclear cells (P = 0.02), which did not affect survival. Increased antilivin IgG was associated with improved survival. Expression of MHC class II on melanoma cells was vital for the immunogenicity of the vaccine. Conclusion: Autologous melanoma cell vaccine is capable of inducing effective antimelanoma CD4 T-cell activity associated with improved survival. Patients with active metastatic disease generally displayed reduced immune response and gained little from active immunization.