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More than 40 agencies that fund health research capacity strengthening in low- and middle-income countries (LMICs) participate in the ESSENCE Health Research initiative, which has established a mechanism for reviewing and coordinating their funding. Taken together, the expected outcomes of implementation of the review mechanism are increases in the efficiency and equity in health research capacity strengthening activities with decreased duplication of efforts. The overall goal is increased support of research on national health priorities as well as improved pandemic preparedness in LMICs, and, eventually, fewer countries with very limited research capacity.

Why and when is it appropriate and relevant to use research funds for social innovation to support both conventional scholarly researchers and non-researchers working in collaboration?

Leishmania aethiopica is the main causative species for cutaneous leishmaniasis (CL) in Ethiopia. Despite its considerable burden, L. aethiopica has been one of the most neglected Leishmania species. In this review, published evidence on L. aethiopica history, geography, vector, reservoir, epidemiology, parasitology, and immunology is discussed and knowledge gaps are outlined. L. aethiopica endemic regions are limited to the highland areas, although nationwide studies on CL prevalence are lacking. Phlebotomus pedifer and P. longipes are the sandfly vectors and hyraxes are considered to be the main reservoir, but the role of other sandfly species and other potential reservoirs requires further investigation. Where and how transmission occurs exactly are also still unknown. Most CL patients in Ethiopia are children and young adults. Lesions are most commonly on the face, in contrast to CL caused by other Leishmania species which may more frequently affect other body parts. CL lesions caused by L. aethiopica seem atypical and more severe in their presentation as compared to other Leishmania species. Mucocutaneous leishmaniasis and diffuse cutaneous leishmaniasis are relatively common, and healing of lesions caused by L. aethiopica seems to take longer than that of other species. A thorough documentation of the natural evolution of L. aethiopica as well as in depth studies into the immunological and parasitological characteristics that underpin the atypical and severe clinical presentation are needed. Better understanding of CL caused by this parasite species will contribute to interventions related to transmission, prevention, and treatment. Keywords: Cutaneous leishmaniasis, Ethiopia, Leishmania aethiopica

BACKGROUND: The incidence of tuberculosis (TB) in sub-Saharan Africa is one of the highest in the world. OBJECTIVE: To evaluate the prevalence of TB, the annual risk of tuberculous infection (ARTI) and associated risk factors in rural Ethiopia. METHODS: A tuberculin skin test was performed among 2743 individuals in a rural community of Ethiopia around Ginci town, west of Addis Ababa, to estimate the prevalence of tuberculin reactivity and to assess factors associated with tuberculous infection. RE SULTS: Among 2743 volunteer participants, test results were available for 2640, 691 (26.2%) of whom had an identifiable bacille Calmette-Guérin (BCG) scar; 221 (8.3%) reported household contact with a known TB case. The overall prevalence of TST reactions of ≥10 mm was 29.7%. The ARTI was estimated at 1.7%. Tuberculin reactivity varied with age, sex, income and history of household contact with a TB case. Presence of BCG scar was not related to tuberculin reactivity. CONCLUSIONS: Our findings indicate that despite an effective TB control programme, TB transmission rates are still high in rural Ethiopia. Provision of isoniazid prophylaxis in close contacts of active TB cases among the poorest population groups may reduce TB incidence.

Background: Concern over the impact of HIV/AIDS has spurred significant investment in research and training in African institutions. In 2013, eight of the largest funders of biomedical research launched World RePORT, an open-access, visual database of research grants intended to identify synergies and gaps in research funding. We reviewed data from World RePORT for lessons learned and an assessment of the value of the database in developing new priorities for health research. Methods: Partners provided data on both direct grants awarded to African researchers and through collaborations with African institutions. We queried 2017 World RePORT Africa data to analyze the landscape and examined 2012-17 data to visualize funding trends in research on Ebola, sickle cell disease, diabetes, and cancer. Findings: In 2017, there were 3,794 research activities at 1,013 institutions across 44 African countries. Three countries-South Africa, Kenya, and Uganda-accounted for over half the records. Fifty-seven institutions receiving ten or more grants accounted for 54% of the total awards, but three-quarters of African institutions had only one or two grants. HIV/AIDS (49%), tuberculosis (16%), and malaria (10%) were the most common infectious disease topics and one third of grants were awarded for several categories of noncommunicable diseases (NCDs): cancer (14%), mental health (7%), and diabetes (3%). Marked increases in research on Ebola were evident after the 2014 outbreak, and funding for sickle cell disease, cancer and diabetes rose more slowly over time. Interpretation: World RePORT maps a landscape of biomedical research investments. In Africa HIV/AIDS remains the largest focus, but funding has grown in other disciplines as well. African institutions have become centers of excellence with each hosting more than 25 international awards from multiple funders. Plans are underway to expand World RePORT to become a more comprehensive body of information on research investments. Funding: World RePORT is supported by contributions of data and/or funding from its partners. Declaration of Interest: No authors declare any competing interests. Ethical Approval: Not required.

Immunosuppression contributes significantly to the caseload of visceral leishmaniasis (VL). HIV coinfection, solid organ transplantation, malnutrition, and helminth infections are the most important immunosuppression-related factors. This review briefly describes the challenges of these associations. East Africa and the Indian subcontinent are the places where HIV imposes the highest burden in VL. In the highlands of Northern Ethiopia, migrant rural workers are at a greater risk of coinfection and malnutrition, while in India, HIV reduces the sustainability of a successful elimination programme. As shown from a longitudinal cohort in Madrid, VL is an additional threat to solid organ transplantation. The association with malnutrition is more complex since it can be both a cause and a consequence of VL. Different regimes for therapy and secondary prevention are discussed as well as the role of nutrients on the prophylaxis of VL in poverty-stricken endemic areas. The authors received no specific funding for this work. Sí

Chronic asymptomatic worm infection, often in combination with tuberculosis (TB), is common in low-income countries. Indeed, a life without worm infestation, as is now the case in most high-income countries, is a recent condition for humankind. Worms and Mycobacterium tuberculosis give rise to different immune response patterns (Th2 vs. Th1 driven), and we have studied whether chronic worm infection affects the susceptibility to and control of TB in the low income country of Ethiopia. Our results, as well of those in the general literature, are inconclusive, although we have some rather strong data in support of adult deworming in relation to vaccination with bacillus Calmette-Guerin (BCG) against TB. In addition, we discuss briefly the putative relationship between chronic worm infestation and autoimmunity/allergy.

Bilateral research cooperation between Sweden (Sida/SAREC) and Uganda has supported major advances in institutional research capacity strengthening at Makerere University (2000–2013). This case study illustrates how a department within Makerere’s Faculty of Medicine (Department of Pharmacy and Therapeutics) has contributed to transforming the post-graduate educational process with formation of a productive research team comprised of faculty and students to advance a research agenda targeting strategic priorities of national significance. We explain the evolution of research as an element of national development and development assistance plus the evolution of doctoral studies and strategic research worldwide, in Africa, and at Uganda’s oldest national university, Makerere, where Sweden has contributed the largest amount of external funding 2000–2012. We conclude with lessons and recommendations about Ph.D. programme start up, recruitment, supervision, international collaboration, and research infrastructure and environment for building institutional research capacity.

SUMMARYPeripheral 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.

Summary Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.

Human cutaneous leishmaniasis (CL) has previously been reported in West Africa, but more recently, sporadic reports of CL have increased. Leishmania major has been identified from Mauritania, Senegal, Mali, and Burkina Faso. Three zymodemes (MON-26, MON-117, and MON-74, the most frequent) have been found. The geographic range of leishmaniasis is limited by the sand fly vector, its feeding preferences, and its capacity to support internal development of specific species of Leishmania. The risk of acquiring CL has been reported to increase considerably with human activity and epidemics of CL have been associated with deforestation, road construction, wars, or other activities where humans intrude the habitat of the vector. In the Ho Municipality in the Volta Region of Ghana, a localised outbreak of skin ulcers, possibly CL, was noted in 2003 without any such documented activity. This outbreak was consistent with CL as evidenced using various methods including parasite identification, albeit, in a small number of patients with ulcers. This paper reports the outbreak in Ghana. The report does not address a single planned study but rather a compilation of data from a number of ad-hoc investigations in response to the outbreak plus observations and findings made by the authors. It acknowledges that a number of the observations need to be further clarified. What is the detailed epidemiology of the disease? What sparked the epidemic? Can it happen again? What was the causative agent of the disease, L. major or some other Leishmania spp.? What were the main vectors and animal reservoirs?What are the consequences for surveillance of the disease and the prevention of its reoccurrence when the communities see a self-healing disease and may not think it is important? Keywords: cutaneous leishmaniasis; Ghana; Volta Region; West Africa; outbreak; follow-up (Published: 13 July 2011) Citation: Global Health Action 2011, 4 : 5527 - DOI: 10.3402/gha.v4i0.5527

Purpose of review Every year, over 8 million people develop tuberculosis and nearly 1.8 million die from it, despite extensive vaccination and drug treatment programmes. It is increasingly recognized that the diagnosis of tuberculosis, which relies heavily on century-old techniques, is one of the weakest links in the chain of tuberculosis control, hampering not just treatment but also the development of new drugs and vaccines. As a result, recent years have seen the initiation of large-scale studies aiming to identify biomarkers of Mycobacterium tuberculosis infection and disease. This review discusses initial results and future prospects for that work. Recent findings The key finding from recent work has been that no one factor seems able to explain the complex course of Mycobacterium tuberculosis infection. Multifactorial analyses have identified a variety of genes and proteins, mostly involved in bacterial persistence or host responses, that offer promise as biomarkers for different disease stages. Summary The challenge now is to validate the suggested biomarkers being described and then reduce them to clinical practice. If this can be done, it offers the possibility of greatly improved clinical management of tuberculosis, allowing segregation of patients and contacts into appropriate treatment regimens.

People who have recovered from leishmaniasis are believed to have long-lasting protection against subsequent infection. Understanding the immunological changes that are associated with protection from cure of and susceptibility to the disease are fundamental to both designing and evaluating vaccine candidates against the leishmaniases. In the quest for a vaccine against leishmaniasis, appropriate surrogate markers of immunity would be valuable and cost effective. Biomarkers would ease screening and selection of potentially efficient vaccine candidates. Moreover, biomarkers of disease may be used to monitor disease and aid therapeutic prognosis. This would be useful in the evaluation of both existing and new drugs, making invasive post-treatment evaluation redundant. Biomarkers may also be indicative of the severity of the disease and may be able to predict the outcome of an infection and indicate whether the patient will spontaneously recover, exhibit mild symptoms or if the disease is disseminating and will be severe. In this article we discuss the immunological changes associated with different forms of human leishmaniasis and the value of appropriate immunological biomarkers in finding an effective vaccine and an evaluation of therapies against leishmanial disease will be given.

SummaryNatural killer (NK) cells contribute to immunity as the first line of defence in numerous infections by early cytokine secretion and cytotoxicity. In Leishmania infection, NK cells contribute with interferon-γ and may assist in directing the immune response towards T helper type 1, which is essential for successful control of the parasites. Thus, NK cells may play an important role in both resistance and control of the infection. However, during Leishmania infection NK cells show signs of suppression. To explore the reason for this suppression, we exposed naive and interleukin (IL)-2 activated NK cells directly to promastigotes of Leishmania major in vitro. As a rapid consequence of contact between naive NK cells and promastigotes, expression of NK cell receptors show significant changes. We identify one of the major surface molecules of promastigotes, glycoprotein (gp) 63, as an important agent for these suppressive effects by using promastigotes of a gp63ko strain of L. major. Furthermore, proliferation of IL-2-activated purified NK cells is suppressed after exposure to the wild-type but not to gp63ko promastigotes. However, gp63ko L. major induced no NK cell proliferation when NK cells were co-cultured with peripheral blood mononuclear cells populations such as CD14+ monocytes or T cells.

The only vaccine available against tuberculosis (TB), BCG, so effective in experimental animal models, has been under scrutiny for a long time owing to its variable efficacy against pulmonary tuberculosis in adults. In this study, we evaluated whether anti-helminthic therapy prior to BCG vaccination could increase the immunogenicity of BCG vaccination in helminth infected population. We recruited volunteers with evidence of prior mycobacterial infection and who were asymptomatic carriers of helminths. The subjects were randomized to receive either anti-helminthic drugs or placebo. Three months later, BCG vaccination was administered to volunteers. Mycobacterial antigen-specific cytokine responses were assessed 2 months after vaccination. The results show that peripheral blood mononuclear cells obtained from the placebo group were found to have a lower frequency of IFN-γ (129 vs 191, p = 0.03) and IL-12 (149 vs 243, p = 0.013) producing cells per 2 × 105 PBMC (peripheral blood mononuclear cells) when stimulated in vitro with a mycobacterial antigen mixture (purified protein derivative (PPD)) compared to those from the dewormed group. On the other hand the placebo group had higher frequency of TGF-β producing cells in response to PPD (152 vs 81.3, p = 0.002) or the T cell mitogen concanavalin A (Con A) (210 vs 157, p = 0.03). However, no detectable IL-4 or IL-5 producing cells were observed when cells were stimulated with PPD. Comparable numbers of both cytokine producing cells were induced in both groups upon stimulation with concanavalin A (IL-4 217 vs 191, p = 0.08) and IL-5 (131 vs 103, p = 0.14). The data presented here demonstrate that chronic worm infection reduces the immunogenicity of BCG in humans and this was associated with increased TGF-β production but not with enhanced Th2 immune response.

Tuberculosis (TB) has once again become a major public health threat owing to the combined effects of deteriorating socioeconomic situations and the emergence of the HIV/AIDS pandemic. The only vaccine available against TB, although effective in reducing the burden of childhood TB, shows enormous variability in its efficacy against pulmonary TB, which is the most common form of the disease in adults. Most areas of high TB incidence and poor TB vaccine efficacy have a high prevalence of intestinal helminth infections. Such infections have been shown to cause a range of immunomodulation characterized by enhanced T helper 2-type cytokine profile, high immunoglobulin E levels and upregulated regulatory T-cell activity, as well as chronic immune activation. An altered background immune profile could have adverse effects on the outcome of subsequent infections and vaccinations. In support of this hypothesis, studies conducted in animals and humans living in worm-endemic areas have shown that helminths impair resistance against a number of infections of major public health importance, including TB, malaria and HIV/AIDS. Understanding such interactions could assist in the design of vaccines against these diseases.

SUMMARY Helminthes, infections widespread in the tropics, are known to elicit a wide range of immunomodulation characterized by dominant Th2 type immune responses, chronic immune activation as well as up-regulated regulatory T cell activity. Such a wide range of immunomodulation caused by helminthes may have an impact on the host's ability to cope with subsequent infections and/or may affect the efficacy of vaccination. Indeed studies conducted in humans living in helminth-endemic areas and in animal models showed that helminth infection makes the host more permissive to mycobacterial infections and less able to benefit from vaccination. These observations have fundamental practical consequences if confirmed by large and appropriately controlled clinical studies. Eradication of worms could offer an affordable, simple and novel means to reduce the burden of the tuberculosis problem that at the moment seems to be getting out of control in sub-Saharan Africa. This information would also be of great relevance in the design of vaccines against diseases of major public health importance, including malaria and HIV/AIDS.

Summary objectives To determine the prevalence of intestinal helminth infections in active tuberculosis patients and their healthy household contacts and to assess its association with active TB in an area endemic for both types of infections. methods Smear-positive pulmonary TB patients and healthy household contacts were tested for intestinal helminths using direct microscopy and the formol-ether concentration techniques. Three consecutive stool samples were examined before the start of TB chemotherapy. Sputum microscopy was done using the sodium hypochlorite concentration techniques. Participants were also tested for HIV by commercial sandwich enzyme linked immunosorbent assay. results The study population consisted of 230 smear-positive TB patients and 510 healthy household contacts. The prevalence of intestinal helminths was 71% in patients and 36% in controls. HIV seroprevalence was significantly higher in patients than in controls (46.7% vs. 11.6%, P < 0.001). Conditional logistic regression analysis showed a strong association between TB and intestinal helminth infection (OR ¼ 4.2, 95% CI 2.7‐5.9, P < 0.001), and between TB and HIV infection (OR ¼ 7.8, 95% CI 4.8‐12.6, P < 0.0001). The odds of being a TB patient increased with the number of helminth species per person: in individuals with mono-infection it was 4.3 (95% CI 2.8‐6.8); in people infected with two species was 4.7 (95% CI 2.5‐8.7), and in patients infected with three or more helminths was 12.2 (3.9‐52.6). conclusion Intestinal helminth infection may be one of the risk factors for the development of active pulmonary TB in addition to HIV infection. This finding may have important implications in the control of TB in helminth endemic areas of the world.

Correlates of protection against tuberculosis are crucial for the evaluation of new vaccine candidates and for the demonstration of their potential efficacy. Such correlates can be proposed on the basis of animal models. In this study, we hypothesized that protection against tuberculosis (TB) induced by bacillus Calmette-Guerin (BCG) correlates with in vitro TB antigen-specific IFN-gamma production. BCG vaccination, known to provide effective protection against TB in animals, was used to investigate the use of in vitro IFN-gamma production as a marker of BCG-induced protection against TB. Our results show that BCG vaccination does provide substantial protection against challenge with Mycobacterium tuberculosis. However, despite previous compelling evidence that Th1 type immune responses are essential for TB immunity, the magnitude of in vitro purified protein derivative (PPD)-specific IFN-gamma production assessed during the course of TB infection did not correlate with protection. This emphasizes the need to identify further correlates of protection, in addition to IFN-gamma, to be used as markers of protective immunity against M. tuberculosis and/or to identify M. tuberculosis antigens inducing IFN-gamma that correlate with protective immunity.

SUMMARYLymphoproliferative 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.

We describe a highly immunogenic low molecular weight fraction of proteins obtained from Onchocerca volvulus. It is comprised of a mixture of native proteins ranging in molecular weight from 10 to 40 kDa and is collectively designated PakF. Unlike other Onchocerca (and other filarial) antigens previously described, IgG4 antibody responses to this protein mixture were absent or low. In a simple, IgG-based dot blot assay, this mixture of native proteins showed high sensitivity, comparable to or better than that of other previously described recombinant proteins, when used to detect infected individuals in endemic areas of Ghana and Guatemala. The specificity was also high when evaluated using sera from patients with other filarial infections. Arguments are put forward to suggest that such a native fraction could present an interesting alternative to the use of well-defined molecules, which are expensive, labile or prone to denaturation, in endemic areas.

SUMMARY The protective efficacy of BCG vaccination against pulmonary tuberculosis (TB) is highly variable in different populations. The reason remains to be elucidated. This study aims to investigate the possible effect of intestinal helminths on the immune response to PPD in naturally immunized or BCG-vaccinated humans. The study population was assessed for helminthic infection and those found to be positive were randomly assigned to either an albendazole treatment group or a control group who received a placebo. The immune response to PPD was compared between the two groups. In addition, subjects who were tuberculin skin test-negative in both groups were BCG vaccinated and later on tested for PPD-specific responses. Albendazole induced elimination/or reduction in intestinal worms resulting in a significant improvement in T cell proliferation and in interferon-gamma production by peripheral blood mononuclear cells (PBMC) stimulated with PPD. Moreover, BCG vaccination significantly improved PPD-specific immune responses in the treated group but not in the placebo group. The differences in the in vivo skin test responses were not significant. The data show that cellular immune responses to PPD are reduced in persons with concurrent helminthic infections, perhaps reflecting a lowered resistance to mycobacterial infections. This could explain, at least in part, the reduced efficacy of BCG against TB in helminth-endemic areas of the world.

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.

OBJECTIVE To investigate the in vitro and in vivo effect of HIV-1 on lymphoproliferative and T helper (Th) cytokine responses in leishmaniasis. METHODS Th1 [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2 (IL-4 and IL-10) as well as IFN-gamma-inducing cytokines (IL-12 and IL-18) were measured in antigen and mitogen-stimulated culture supernatants of peripheral blood mononuclear cells (PBMC) of healthy donors, HIV-infected and visceral leishmaniasis (VL) patients with or without HIV co-infection. RESULTS Proliferative responses to phytohaemagglutinin (PHA) were significantly lower in PBMC from VL and asymptomatic HIV-infected persons compared with responses in healthy individuals. VL-HIV co-infected patients showed the lowest responses. Although there was no significant difference in the Leishmania-induced proliferative responses among the healthy group and those infected with HIV only, VL patients (with or without HIV) exhibited very low proliferation. When cultured with PHA or Leishmania, PBMC from healthy donors produced high levels of a Th1 cytokine (IFN-gamma) and low levels of Th2 cytokines (IL-4 and IL-10). In addition, co-culturing PBMC from healthy donors with a killed HIV preparation abrogated the production of IFN-gamma induced by Leishmania and augmented IL-4 and IL-10 production. Cells from HIV-infected patients produced low levels of IFN-gamma, but high levels of IL-10. The addition of anti-IL-10 did not increase Leishmania-induced proliferative responses or IFN-gamma production. Both IL-12 and/or IL-18 responses were lower in VL patients, HIV-infected, or VL-HIV co-infected patients as compared with those of healthy donors. CONCLUSION The data suggest that the inhibitory effect of HIV and VL on proliferation and IFN-gamma production is not due to IL-10 alone, but that the defect induced by HIV and VL probably operates at the level of regulation of IFN-gamma-inducing factors, such as IL-12 and IL-18.

Chronic immune activation by coinfecting pathogens has been suggested as a cofactor in human immunodeficiency virus (HIV) disease progression, particularly in the setting of developing countries. Here, we used in vivo-infected mononuclear cells to examine the role of the protozoan parasite Leishmania donovani and its major membrane constituent, lipophosphoglycan (LPG), in mediating CD4 + T-lymphocyte activation-induced HIV replication and CD4 + T-cell death. We found that Leishmania antigens upregulated HIV replication in CD8-depleted peripheral blood mononuclear cells from asymptomatic HIV-infected donors compared to unstimulated cells. L. donovani -induced viral replication was associated with cellular proliferation, increased expression of the cellular immune activation markers CD25 and HLA-DR within the CD4 + subpopulation, and enhanced secretion of tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), and IL-6. LPG induced TNF-α secretion in the absence of increased expression of cellular activation markers. Moreover, in a few cases we observed that L. donovani induced HIV replication without significant cellular activation but with cytokine secretion. The rate of apoptosis was accelerated in these latently infected CD4 + T cells primed with Leishmania antigens compared to controls, and TNF-α production appeared to be the central event necessary for this effect. Furthermore, we demonstrate that thalidomide inhibited Leishmania -induced virus replication coupled with abrogated Leishmania -induced TNF-α secretion but not IL-2 or IL-6 production. Furthermore, thalidomide did not affect Leishmania -induced apoptosis. The results suggest that Leishmania and its product, LPG, up-regulate HIV replication in latently infected cells through distinct antigen-specific and non-antigen-specific cellular immune activation mechanisms and that TNF-α secretion is pivotal in this process. The immunomodulatory role of thalidomide raises interest as a potential adjuvant to reduce HIV disease progression in Leishmania -HIV coinfected individuals.

Both Leishmania and the human immunodeficiency virus (HIV) can infect and multiply in macrophages, and both can dysregulate the immune system. Recent studies indicate that Leishmania can induce the activation of HIV in latently infected monocytic and T cells. Moreover, HIV can enhance intracellular growth of Leishmania in macrophages. Here, Dawit Wolday and colleagues examine the mechanisms that might be involved in the immunopathogenesis of Leishmania – HIV co-infection.

Diffuse Cutaneous Leishmaniasis (DCL) is a rare complication of Leishmania aethiopica-induced cutaneous leishmaniasis which is associated with non-self healing and in vivo and in vitro antigen-specific non-responsiveness. Such antigen-specific unresponsiveness is also observed in visceral leishmaniasis (VL). The non-responsiveness seen in VL disease is believed to be due, in part, to serum-derived factors, including raised serum soluble IL-2 receptor (sIL-2R). Raised sIL-2R in serum was not a consistent feature of DCL in our study (range: 787-4546 U/ml) but was frequently observed in sera of patients with other dermatological disorders (range: 474-3313 U/ml) and some patients with the simple local cutaneous leishmaniasis (LCL; range: 556-4247 U/ml). The level of sIL-2R in the sera of DCL patients was not indicative of the disease state. Sera from DCL patients did not reduce proliferation of the IL-2-dependent CTLL cell line nor reduce PHA-driven mononuclear cell proliferation, although sera from VL patients could. Both DCL and VL sera could reduce the L. aethiopica-driven proliferation. Furthermore addition of serial dilutions of recombinant IL-2 to CTLL cultured in VL or DCL sera containing high sIL-2R levels did not alter the effect of such sera on proliferation. We conclude therefore, that raised sIL-2R in serum is not associated with the immunosuppression in DCL.

The leishmanin skin test (LST) is used in immunogenicity studies. The effect of multiple LSTs on immune responses was assessed. None of the volunteers converted to LST positive. IFN-gamma and IL-10 levels remained unchanged. Repetition of LST does not modulate the in vivo or in vitro immune responses to Leishmania antigen.

SUMMARYNK cells represent one of the first lines of defence in the immune reaction after invasion ofLeishmaniaparasites. Depletion of mouse natural killer (NK) cells dramatically enhances susceptibility of normally resistant mice. In this study we evaluated the fate of NK cells and parasites after contact formation. The hydrophilic fluorescent dye CMFDA (chloro-methylfluorescin diacetate) that allows analysis of cytotoxicity in flow cytometry and microscopy was used. Furthermore, these findings were confirmed with scanning and transmission electron microscopy. Direct contact points were found betweenLeishmaniapromastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells againstLeishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact withLeishmaniacauses immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation forex vivoobservations of reduced NK cell numbers and impaired NK response in patients with acute cutaneous leishmaniasis. Animal models have clearly shown that NK cells play a key role in the induction and direction of the immune response. Thus inhibition of NK cells at the onset of infection would be advantageous for the survival of the parasite.

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

SUMMARYLymphocytes of individuals from a country non-endemic for Leishmania (Sweden), responded with a vigorous interferon-gamma (IFN-γ) and IL-6 response when exposed to live or dead promastigotes of Leishmania aethiopica. This response was sometimes as strong as when the same cells were exposed to the mitogen (phytohaemagglutinin (PHA)). Furthermore, supernatants of cells exposed to Leishmania promastigotes were able to inhibit the amastigote form of the same parasite. In some few instances there was no such reactivity to Leishmania parasites. It is proposed that most individuals have such a first line cytokine response which is enough to prevent further spread and growth of the parasites. In exposed individuals who display disease symptoms, this non-Leishmania-specific response is overcome (by dose) or is weak (for genetic reasons). In the latter instances curbing of parasite growth would depend on acquired immunity.

Background: Prompt laboratory diagnosis and initiation of treatment are effective components of leishmaniasis control. Detection of Leishmania parasites by ex-vivo culture of lesion scrapings is considered a definitive diagnostic method preceding initiation of treatment. Objective: A pilot study to find alternative medium that could reduce the cost of culturing from patient lesions for diagnosing leishmaniasis. Method: GALF-1 medium was formulated in our lab from locally available inexpensive solutions and powders in the presence of urine from healthy individuals. Amastigote to promastigote transformation, recovery of parasites after cryopreservation, cost and mass cultivation was compared using the following media: GALF-1, RPMI 1640, and conventional Locke’s semi-solid medium (LSSM), a modifications of Novy-MacNeal-Nicolle culture media, which uses Locke’s solution as an overlay. Results: GALF-1 preparation was cheap and the components available in low-income countries such as Ethiopia. Preparation was simple, not requiring autoclaving and extra distilled water. GALF-1 was able to transform amastigotes from Ethiopian patients’ samples and could be used to cultivate promastigotes in large quantities. GALF-1 decreased Leishmania culture costs by ~80-95% compared to LSSM and RPMI 1640, respectively. Promastigotes cultured with GALF-1 could be cryopreserved in liquid nitrogen with comparable re-culture potential. Conclusion: Affordability of diagnostic assays is a key issue for endemic resource-poor countries and the possibility to cut the cost of the efficient culture method for diagnosis through the use of inexpensive, locally formulated reagents could improve the diagnosis of leishmaniasis in Ethiopia and in other low-income countries. Keywords: Leishmania culture; urine; liquid medium; low-income countries (Published: 22 October 2009) Citation: Global Health Action 2009. DOI: 10.3402/gha.v2i0.2046

Three patients with disseminated cutaneous leishmaniasis received three intranodular injections of 10 micrograms of recombinant interleukin 2 (rIL-2) at 48-h intervals. After 7 and 14 days, 4-mm punch biopsies were taken of control and injected nodules and processed for histology, electron microscopy, immunocytochemistry, and parasite culture. Control sites exhibited loose infiltrates of parasitized macrophages and T cells predominantly of the CD8+ phenotype. Amastigotes were present in large numbers and were found distributed within tightly apposed endosomes and larger vacuoles. After the administration of rIL-2, there was a prominent influx of T cells, predominantly of the CD4+ phenotype, and an increased number of plasma cells. At 7 days, leishmanial amastigotes were present in either the same or somewhat reduced numbers but predominantly within large, lucent vacuoles. By 14 days the number of amastigotes was strikingly lower. Lymphokine-treated skin sites became sterile in two patients, as evaluated by parasite culture after rIL-2 injection. The results suggest that the local administration of rIL-2 induces a beneficial enhancement of the cellular immunity with a consequent disposal of parasites in the cutaneous site.

Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL.

To determine the prevalence of intestinal helminth infections in active tuberculosis patients and their healthy household contacts and to assess its association with active TB in an area endemic for both types of infections.Smear-positive pulmonary TB patients and healthy household contacts were tested for intestinal helminths using direct microscopy and the formol-ether concentration techniques. Three consecutive stool samples were examined before the start of TB chemotherapy. Sputum microscopy was done using the sodium hypochlorite concentration techniques. Participants were also tested for HIV by commercial sandwich enzyme linked immunosorbent assay.The study population consisted of 230 smear-positive TB patients and 510 healthy household contacts. The prevalence of intestinal helminths was 71% in patients and 36% in controls. HIV seroprevalence was significantly higher in patients than in controls (46.7%vs. 11.6%, P0.001). Conditional logistic regression analysis showed a strong association between TB and intestinal helminth infection (OR = 4.2, 95% CI 2.7-5.9, P0.001), and between TB and HIV infection (OR = 7.8, 95% CI 4.8-12.6, P0.0001). The odds of being a TB patient increased with the number of helminth species per person: in individuals with mono-infection it was 4.3 (95% CI 2.8-6.8); in people infected with two species was 4.7 (95% CI 2.5-8.7), and in patients infected with three or more helminths was 12.2 (3.9-52.6).Intestinal helminth infection may be one of the risk factors for the development of active pulmonary TB in addition to HIV infection. This finding may have important implications in the control of TB in helminth endemic areas of the world.

Background In the screening of vaccine candidates it is important to select candidates that evoke immune responses associated with protection. Valid surrogate markers against human leishmaniasis are still lacking. Methods A controlled injection of live Leishmania known as leishmanization, (LZ), was used to evaluate vaccine (alum-precipitated autoclaved Leishmania major with BCG) efficacy and more accurately define surrogate markers of immunity to leishmaniasis in humans. Cellular immune responses to this artificial infection were monitored in the volunteers prior to and 9 months post infection. Comparisons were made between those who developed a lesion after infection and those who did not. Results In the volunteers monitored there was no significant difference in LST, IFNγ production, or source of IFNγ between those who developed a lesion and those who did not after LZ, with the exception that ulcer development was associated with an enhanced number of IFNγ secreting CD4 + CD45RA − (memory) T cells. Discussion Ulcer development following LZ was lower than anticipated by a pilot study (47% versus 78%) using the same stabilate several years earlier. While this may be an effect of low viability/virulence of the LZ inocula, alternative explanations are also possible. The IFNγ responses in the study subjects were significantly lower compared to volunteers with previous history of cutaneous leishmaniasis. The findings raise the possibility that the selection of LST-negative volunteers in an endemic area may bias the study towards potentially non/low L. major -reactive volunteers.

Cutaneous leishmaniasis (CL), caused by the intracellular protozoan Leishmania major, is characterized by lesion formation and ulceration at the site of infection. The mechanism of ulcer formation during CL is not fully understood. The expression of Fas and FasL and the levels of apoptosis in skin biopsies and in restimulated blood mononuclear cells from patients with 1 to 7 months of L. major-induced CL were analyzed using immunohistochemistry and fluorescence-activated cell sorting analysis. The levels of soluble Fas and FasL were also analyzed by enzyme-linked immunosorbent assay. A substantial number of apoptotic keratinocytes were observed mainly in the superficial epidermis of morphologically active and healing CL skin samples. Fas expression was increased on epidermis in active CL, whereas Fas expression was similar in healing and healthy epidermis. FasL-expressing macrophages and T cells were found in subepidermal infiltrate, mainly in active disease. When CL peripheral blood mononuclear cells were restimulated with L. major, Fas was up-regulated on effector T cells, and high levels of sFasL were secreted. Supernatants from restimulated cultures induced apoptosis in human keratinocytes (HaCaT), possibly through Fas/FasL interactions. Our results indicate that FasL-expressing effector T cells and macrophages may act to induce apoptosis and ulcer formation in Fas-expressing keratinocytes during L. major infection.

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 alpha-MHC class II antibodies markedly inhibited proliferation and interferon-gamma (IFN-gamma) production, whereas interleukin-10 production was not affected. Experimental evidence indicates that CD4(+) cells were not necessary for the proliferative and IFN-gamma 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.

We hypothesized that the ability of BCG vaccination to protect against Mycobacterium tuberculosis is less in hosts exposed to chronic helminthes infection compared to unexposed individuals. To test this hypothesis we evaluated the efficacy of BCG vaccination in protecting against M. tuberculosis challenge in Schistosoma mansoni pre-infected mice by analyzing their ability to limit the replication of TB bacilli in the lung and liver and the histology of lung sections. The results show that BCG vaccinated mice with prior S. mansoni infection show significantly higher number of colony forming units of TB bacilli as well as significant reduction in air exchange area in the lung compared to controls. In addition, spleen cells from S. mansoni infected mice were found to produce significantly less IFN-gamma and nitric oxide when stimulated in vitro with PPD and several fold higher soluble egg antigen (SEA) and Concanavalin A induced IL-4 and IL-5 secretion. Taken together, our data show that S. mansoni infection reduces the protective efficacy of BCG vaccination against M. tuberculosis possibly by attenuation of protective immune responses to mycobacterial antigens and/or by polarizing the general immune responses to the Th2 profile.