Kerima Maasho

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

Live Leishmania promastigotes can directly activate primary human natural killer cells to produce interferon‐gamma

Natural killer (NK) cells have been implicated in the natural protection and healing of leishmaniasis by their ability to secrete the macrophage activating cytokine interferon (IFN)γ. Previous studies have demonstrated that early production of interleukin (IL)‐12 triggers IFNγ secretion by NK cells. Here we report that live Leishmania promastigotes (the form that is injected by the vector) can directly induce human peripheral blood NK cells from healthy donors to IFNγ secretion in the absence of IL‐12 and professional antigen presenting cells. Killing of promastigotes abolishes this property. This novel mechanism of activation of the innate immune response may be relevant for establishment of infection and thus also the design of vaccines against leishmaniasis.

A Leishmania Homologue of Receptors for Activated C-Kinase (LACK) Induces Both Interferon-γ and Interleukin-10 in Natural Killer Cells of Healthy Blood Donors

Natural killer (NK) cells from individuals unexposed to Leishmania organisms proliferate with high interferon (IFN)-gamma secretion in response to crude Leishmania antigen preparations. In an attempt to identify the molecules that induce blood cells to proliferate and to secrete cytokines, we tested the effect of a 36-kDa Leishmania homologue of receptors for activated C-kinase (LACK) on peripheral blood mononuclear cells from unexposed individuals. Mainly CD8(+) and NK cells proliferated in response to LACK. At both the mRNA and soluble protein level, the main sources for LACK-induced IFN-gamma and interleukin (IL)-10 were T and NK cells. Furthermore, in the presence of anti-major histocompatibility complex (MHC) class II antibody, there was inhibition of LACK responses in both CD4(+) and CD16/56(+) cells, with a marked decrease in IFN-gamma but with an increase in IL-10 production. We conclude that the response to LACK is part of the response to Leishmania organisms in unexposed donors described elsewhere. That this NK-dominated response is MHC class II sensitive, whether through a direct or indirect effect, is discussed.

Natural killer cells in cross-regulation of IL-12 by IL-10 in Leishmania antigen-stimulated blood donor cells

We have previously shown that natural killer (NK) cells play a role in protection against leishmaniasis. Furthermore, we have shown that NK cells in mononuclear cells derived from unexposed donors are induced to proliferate in vitro in response to leishmanial antigens. Since interleukin (IL)‐12, a strong inducer of NK cells, acts on the early events in NK cells and T‐cells, and is considered as an adjuvant for use in a potential antileishmaniasis antigen, we wished to investigate how this cytokine influences the in vitro Leishmania induced proliferative and cytokine response in healthy donors. We demonstrate that in an innate response to Leishmania antigen involving NK cells, a critical level of IL‐12 is required to induce interferon (IFN)‐γ secretion below which, IL‐10 is released in amounts which apparently inhibit IFN‐γ secretion and cellular proliferation. However, at higher IL‐12 levels, there is simultaneous secretion of IFN‐γ and IL‐10 as well as proliferation of cells. In a similar vein, exogenous IL‐10 in turn inhibited IFN‐γ secretion as well as proliferation when used at low/medium concentrations, but at high concentrations this effect was abolished and replaced by the simultaneous detection of IFN‐γ, IL‐10 and proliferation. The contribution of NK cells in cross regulation of these two very important immuneregulatory cytokines and the effect of exogenous IL‐12 in a Leishmania driven response are discussed.

Induction and abrogation of LACK reactive cells in the evolution of human leishmaniasis

Peripheral blood mononuclear cells (PBMC) from cutaneous leishmaniasis patients with ongoing Leishmania aethiopica infection and individuals cured/under treatment from L. infantum or L. donovani infection were stimulated in vitro with LACK, the Leishmania homologue of receptors for activated C kinase. The LACK protein is conserved in related leishmanial species and is expressed both in the promastigote and amastigote stages of Leishmania. Our results show that LACK induced marked NK and some CD8+ cell proliferation in PBMC from cutaneous leishmaniasis patients with active disease. These responses were coupled with high levels of IFN‐γ and IL‐10 production. At the concentration tested, the proliferative responses to freeze‐thawed Leishmania antigen (Ft‐Leish) were higher, while the levels of IFN‐γ were consistently lower than that of LACK. Although cells from individuals cured of leishmaniasis could respond to whole Leishmania lysate by proliferation and IFN‐γ production, there was no evident response to LACK. Ethiopian controls tested at the same time also showed LACK induced proliferation with IFN‐γ and IL‐10 responses. Thus LACK reactivity in terms of proliferation and cytokine induction were present in cells from some healthy donors and most of the patients with active lesions, while this response was absent in individuals cured of L. infantum or L. donovani leishmaniasis. Since cure from leishmaniasis often results in life‐long protection, and active but not cured patients showed in vitro responses to LACK stimulation, questions arose as to how this highly immunodominant molecule functions during human leishmanisasis. Some possible mechanisms are discussed.

Leishmanial amastigote antigen P-2 induces major histocompatibility complex class II-dependent natural killer-cell reactivity in cells from healthy donors

Innate mechanisms involving natural killer cells have been implied to play an important role in immunity against Leishmania infection. Previous studies have evaluated responses to three purified amastigote antigens, P‐2, P‐4 and P‐8, of Leishmania pifanoi. The P‐4 and P‐8 antigens have been demonstrated to induce protection in mouse models, as well as to induce cellular responses in American cutaneous leishmaniasis patients. Cells from Leishmania aethiopica‐infected leishmaniasis patients preferentially responded to P‐8 and, to a lesser extent, to the cysteine proteinase, P‐2. In this study, it is shown that cells from healthy donors, including cells from truly naïve donors (cord blood), could be stimulated to proliferation and cytokine production by P‐2. The main proliferating cell types in healthy adult donors were CD16/56+ and the CD8+ cells. Blocking of major histocompatibility complex (MHC) class II with α‐MHC class II antibodies markedly inhibited proliferation and interferon‐γ (IFN‐γ) production, whereas interleukin‐10 production was not affected. Experimental evidence indicates that CD4+ cells were not necessary for the proliferative and IFN‐γ responses; however, an adherent cell population was required. Furthermore, CD16/56+ cells expressing MHC class II were expanded following P‐2 stimulation. The responses to P‐2 show a striking similarity to responses induced by the vaccine candidate Leishmania homologue of receptors for activated C‐kinase (LACK) in healthy donors. The responses described here may not be desirable when aiming at inducing protective immune responses with a vaccine, and the implications of these results for the development of vaccines against leishmaniasis are discussed.

Ivermectin‐induced immunopotentiation in onchocerciasis: recognition of selected antigens following a single dose of ivermectin

Onchocerciasis is associated with blindness and gross skin changes, believed to be a consequence of the immune response to antigens released from the offspring of the female worm of Onchocerca volvulus, the microfilariae (mf). An effective microfilaricidal drug is now available which quickly reduces the mf burden without affecting the adult worm. There exist foci in onchocerciasis endemic areas where some of the patients have many mf in their skin but relatively few clinical symptoms. This state of hyposensitivity is believed to be due to immunosuppression. The aim of this study was to address the question of the basis of, and the effect of ivermectin treatment on this immunosuppression. Female adult worms of O. volvulus were used as whole or fractionated antigens to stimulate peripheral blood mononuclear cells. Microfilariae are found in the reproduction tract of the female worms, and thus an antigen preparation of the female adult O. volvulus contains both exclusive adult antigens as well as antigens from microfilariae. Cells were obtained from onchocerciasis patients, individuals of similar socio‐economic status living in the same Ghanaian village, but who showed no parasitological or clinical evidence of onchocerciasis (exposed endemic controls), healthy Ghanaians living in areas where transmission of onchocerciasis does not seem to occur (non‐exposed endemic controls) and unexposed healthy Swedish donors. As a group, cells from onchocerciasis patients proliferated to a lesser degree than cells from the exposed endemic control and the non‐exposed endemic control groups to the whole worm antigen, whereas the phytohaemagglutinin (PHA) response was strongest in the patients. Proliferative responses of above 1000 ct/min to fractions of the worm extract were only evident in the cells from a few individuals in each of the various groups. However, 28 days following ivermectin treatment, cells from all onchocerciasis patients were able to mount significantly enhanced proliferation to a fraction of approximately 96 kD (fraction 3), while only four of nine of this group showed an increased response to the whole worm antigen. The proportional increase in the response to the whole organism in these individuals was of a much lower magnitude than the increased response to fraction 3. The O. volvulus antigen‐specific immunosuppression observed in these onchocerciasis patients appears to be due to suppressive antigens which have the capacity to mask the potential response to selected antigens of O. volvulus, and ivermectin treatment possibly modulates the immune response, allowing for stepwise recognition of such antigens. Since ivermectin treatment kills only the microfilariae and not the adult worm, the putative suppressive antigens would be expected to be from the microfilariae.

Evaluation of amastigote reactive cells in human cutaneous leishmaniasis caused by Leishmania aethiopica

Lymphoproliferative responses to three affinity chromatography purified amastigote antigens of Leishmania pifanoi, P‐2, P‐4 and P‐8, were evaluated in peripheral blood mononuclear cells (PBMC) from patients with Ethiopian cutaneous leishmaniasis. Antigen‐stimulated cells were analysed for the percentage of CD4+, CD8+ and CD16/56+ cells and the expressed levels of gamma interferon (IFNγ) and interleukin (IL)‐10 were determined in culture supernatants. The amastigote antigens induced cellular responses in leishmaniasis patients with heterologous Leishmania parasite infection. These responses were compared to those of freeze‐thawed L. aethiopica promastigote antigen stimulation. The membrane protein (P‐8), and to a lesser extent the megasomal/cytoplasmic cysteine proteinase(P‐2), induced proliferation with high levels of IFNγ and IL‐10 production in cells from patients with active L. aethiopica lesions. CD16/56+ NK cells were the main cell types induced to proliferate in response to P‐8 and P‐2 stimulation, followed by CD8+ cell populations. P‐4 had no such effect. This contrasts from previous studies of New World human leishmaniasis where P‐4 and P‐8 were stimulatory. The success of a particular molecule in the induction of a response with a protective phenotype may be dependent on the infecting Leishmania spp. To our knowledge, there are no studies that directly compare the New versus Old World cutaneous leishmaniasis in respect of NK cell and IL‐10 responses. Our studies indicate that some leishmanial molecules are recognized across the species, while others are apparently more species specific.