Funding social innovation for health with research funds for development.

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?

A Mechanism for Reviewing Investments in Health Research Capacity Strengthening in Low- and Middle-Income Countries.

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.

New insights into leishmaniasis in the immunosuppressed.

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.

Cutaneous Leishmaniasis Due to Leishmania aethiopica.

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.

Worms and Humans: A Happy Divorce?

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-Guérin (BCG) against TB. In addition, we discuss briefly the putative relationship between chronic worm infestation and autoimmunity/allergy.

The interplay between Leishmania promastigotes and human Natural Killer cells in vitro leads to direct lysis of Leishmania by NK cells and modulation of NK cell activity by Leishmania promastigotes.

NK cells represent one of the first lines of defence in the immune reaction after invasion of Leishmania parasites. 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 between Leishmania promastigotes and naïve human NK cells. These contacts were associated with transfer of cytosol by membrane bridges and cytotoxicity of NK cells against Leishmania. However, in contrast to other target cells which allow repeated exocytosis of lytic granules, contact with Leishmania causes immediate destruction of NK cells in a non-apoptotic way. Our results give a reasonable explanation for ex vivo observations 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.

An outbreak of suspected cutaneous leishmaniasis in Ghana: lessons learnt and preparation for future outbreaks.

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?

Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

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.

Systemic FasL and TRAIL neutralisation reduce leishmaniasis induced skin ulceration.

Cutaneous leishmaniasis (CL) is caused by Leishmania infection of dermal macrophages and is associated with chronic inflammation of the skin. L. aethiopica infection displays two clinical manifestations, firstly ulcerative disease, correlated to a relatively low parasite load in the skin, and secondly non-ulcerative disease in which massive parasite infiltration of the dermis occurs in the absence of ulceration of epidermis. Skin ulceration is linked to a vigorous local inflammatory response within the skin towards infected macrophages. Fas ligand (FasL) and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expressing cells are present in dermis in ulcerative CL and both death ligands cause apoptosis of keratinocytes in the context of Leishmania infection. In the present report we show a differential expression of FasL and TRAIL in ulcerative and non-ulcerative disease caused by L. aethiopica. In vitro experiments confirmed direct FasL- and TRAIL-induced killing of human keratinocytes in the context of Leishmania-induced inflammatory microenvironment. Systemic neutralisation of FasL and TRAIL reduced ulceration in a model of murine Leishmania infection with no effect on parasitic loads or dissemination. Interestingly, FasL neutralisation reduced neutrophil infiltration into the skin during established infection, suggesting an additional proinflammatory role of FasL in addition to direct keratinocyte killing in the context of parasite-induced skin inflammation. FasL signalling resulting in recruitment of activated neutrophils into dermis may lead to destruction of the basal membrane and thus allow direct FasL mediated killing of exposed keratinocytes in vivo. Based on our results we suggest that therapeutic inhibition of FasL and TRAIL could limit skin pathology during CL.

Low-cost liquid medium for in vitro cultivation of Leishmania parasites in low-income countries.

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 approximately 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.

Tracing immunity to human leishmaniasis.

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.

Poor immunogenicity of BCG in helminth infected population is associated with increased in vitro TGF-beta production.

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-gamma (129 vs 191, p=0.03) and IL-12 (149 vs 243, p=0.013) producing cells per 2 x 10(5) 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-beta 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-beta production but not with enhanced Th2 immune response.

Chronic helminth infections may negatively influence immunity against tuberculosis and other diseases of public health importance.

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.

FasL and TRAIL induce epidermal apoptosis and skin ulceration upon exposure to Leishmania major.

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.

Surrogate markers of immunity to Leishmania major in leishmanin skin test negative individuals from an endemic area re-visited.

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, IFNgamma production, or source of IFNgamma 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 IFNgamma 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 IFNgamma 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.

Are intestinal helminths risk factors for developing active tuberculosis?

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 = 4.2, 95% CI 2.7-5.9, P < 0.001), and between TB and HIV infection (OR = 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.

The contribution of the Fas/FasL apoptotic pathway in ulcer formation during Leishmania major-induced cutaneous Leishmaniasis.

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.

Schistosoma mansoni infection reduces the protective efficacy of BCG vaccination against virulent Mycobacterium tuberculosis.

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.