Blood monocytes: differing effector role in experimental visceral versus cutaneous leishmaniasis

Irrespective of the tissue infected or the strain involved, all Leishmania species selectively parasitize and replicate within the resident tissue macrophage. Henry Murray here discusses the role of a second mononuclear phagocyte, the blood monocyte, which is also attracted to leishmanial lesions, but which appears to play quite different roles in experimental visceral versus cutaneous infection: in visceral disease, the monocyte is a critical host defense effector cell, in cutaneous disease, it may, paradoxically, serve to perpetuate intracellular infection.     

Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives

Visceral leishmaniasis is a major health problem in Latina America, as well as the Mediterranean region of Europe and Asia. We aimed to develop a vaccine against visceral leishmaniasis targeting the intracellular amastigotes, which is the parasite stage that persists throughout infections with Leishmania parasites. With this in mind, we identified an amastigote specific antigen (A2) that contains an immunogenic epitope for CD4+ T helper (Th) cells and multiple repetitive units encoding CD8+ cytotoxic T lymphocyte (CTL) epitopes. Vaccine formulations containing the recombinant A2 associated with saponin, alum and IL-12 or expressed by attenuated adenovirus were shown to be protective in mice, dogs and nonhuman-primates. We are currently identifying novel amastigote specific immunogenic proteins that could be aggregated to A2 to further improve the level of vaccine-induced cell-mediated immunity and protection against visceral leishmaniasis.     

水痘减毒活疫苗的研究进展

水痘是由水痘-带状疱疹病毒( VZV) 引起的一种高传染性疾病。接种疫苗是预防和控制VZV 流行的最有效手段。美国从1995 年开始实施儿童接种水痘疫苗的政策, 显著降低了水痘相关疾病的发病率和病死率。近年来, 水痘疫苗在我国应用越来越广泛。本文就水痘减毒活疫苗Oka 株的研发, 疫苗的有效性、安全性, 病毒减毒的分子生物学特征和免疫策略等方面进行综述, 以期能更全面地认识水痘减毒疫苗在预防和控制VZV 传播方面所起的作用。     

Balancing immunity and pathology in visceral leishmaniasis

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ-specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell-mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)-10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38(+) stromal cells, and IL-10 promotes impaired DC migration into T-cell areas with consequent ineffective T-cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL-10-producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.     

Experimental visceral leishmaniasis in golden hamsters

As a result of a long passage of L. donovani isolate on golden hamsters (21 passages were observed), in transplanting the agent from animals with a distinct clinical picture there was formed a highly virulent strain "G" of L. donovani for this species of animals. The weight arrest and then body mass losses were the most early signs of the disease. Parasites were regularly accumulated in spleen and liver and to a less extent in bone marrow. The main manifestations of visceral leishmaniasis in hamsters are cachexia, lienal syndrome, polyglandular deficiency on the background of hypoplasia of lymphoid tissue and defects of the system of monocytic phagocytes. Such symptom-complex can be a result of neuroendocrine deficiency during visceral leishmaniasis. Pathohistological picture of experimental visceral leishmaniasis is similar to that of man, so L. donovani infection in hamsters can serve as a model for studies of different medical and biological aspects of leishmaniasis.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

First visceral leishmaniasis focus in Argentina

An eight-year old boy from Posadas (27 masculine 23'S, 55 masculine 54'W) was diagnosed with visceral leishmaniasis (VL) during 2006. Lutzomyia longipalpis was discovered in the backyard of his house, while the spread of canine visceral leishmaniasis was confirmed in Posadas. This is the southernmost report of a VL transmission focus and the first in Argentina.     

Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis

The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmaniadonovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund’s adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1 × 10⁷L.donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.     

Epitopic suppression in synthetic vaccine models: analysis of the effector mechanisms

The induction of an immune response against synthetic peptides usually requires the use of an immunogenic carrier. The use of tetanus toxoid (TT) has been proposed for this purpose as it is highly immunogenic and has been used extensively in humans. Previous studies have demonstrated that an epitope-specific suppression of IgG antibody responses occurs when mice previously primed with TT are subsequently immunized with SODP, a haptenic epitope linked to TT. In the present investigation, we characterized the effector populations which regulate anti-SODP antibody responses in TT/TT-SODP immunized mice. In vitro studies showed that epitopic suppression did not arise due to nonspecific suppressor phenomena. Coculture experiments demonstrated that epitopic suppression was partially mediated by suppressor T cells which specifically inhibited the anti-hapten but not the anti-carrier antibody response. The majority of these T cells were shown to possess the Lyt-2+ phenotype. Apart from the T suppressor population we demonstrated a deficiency at the B-cell level which contributed to the total suppressive effect. Epitopic suppression, therefore, resulted from the effects of dual specific suppressor mechanisms.     

Genes and environment in susceptibility to visceral leishmaniasis

Kala azar (KA) is a lethal disease caused by Leishmania parasites (Leishmania donovani s.l.) that multiply in large numbers in deep organs such as spleen and liver. The host immunological response to these organisms is complex and experimental studies in animals have detected a large number of genetic loci involved in the control of infection and disease. We report here on a study in a human population of Sudan carried out during an outbreak of KA. The following conclusions are presented: (1) environmental factors that could have affected the distribution of the insect vector, influenced progression of KA in the initial phase of the epidemics - but they became less important later at the peak of transmission, probably after infected phlebotomies had spread to all parts of the village -; (2) Leishmania population during the epidemics was heterogeneous, suggesting a possible parasite evolution during the outbreak; (3) the incidence of KA varied markedly among age groups, families and ethnic groups. Susceptibility to KA was shown to depend on a locus on chromosomes 22q12 and on NRAMP1 on chromosome 2q35; the data also suggested a third locus in the region 2q23-q24. Overall, this study indicates complex interactions between host genes and environment in the spreading of KA in that population. It is also suspected that the large parasite diversity observed in the outbreak has contributed to disease spreading across host genetic barriers.     

Prevention of relapse after chemotherapy in a chronic intracellular infection: mechanisms in experimental visceral leishmaniasis

In visceral leishmaniasis, chemotherapy probably seldom eradicates all parasites in tissue macrophages; nevertheless, most T cell-intact patients show long-lasting clinical cure after treatment despite residual intracellular infection. To characterize prevention of posttreatment relapse, amphotericin B was used to kill approximately 90-95% of Leishmania donovani in livers of mice deficient in mechanisms of acquired antileishmanial resistance. Recrudescence subsequently developed 1) in animals deficient in both CD4 and CD8 T cells as well as CD40L-mediated T cell costimulation, but not in a) CD4 or CD8 cells alone, b) NK cell lytic activity, or c) ICAM-1-recruited monocytes; and 2) in mice deficient in IFN-gamma, but not in the IFN-gamma-inducing cytokines, a) IL-12, b) IL-12 and IL-23, or c) IL-18. Posttreatment recrudescence also did not develop in animals deficient in macrophage phagocyte NADPH oxidase (phox) or inducible NO synthase (iNOS) alone or, surprisingly, in those deficient in both phox and iNOS. Therefore, regulation of the intracellular replication of residual Leishmania donovani that escape chemotherapy evolves to a host mechanism distinguishable from initial acquired resistance at the T cell, cytokine, and macrophage levels. Posttreatment, either CD8 or CD4 cells can direct the response, IL-12 is not required, and iNOS and phox, the activated macrophage's primary IFN-gamma-inducible leishmanicidal pathways, both become dispensable.     

Bacterial infection in patients with visceral leishmaniasis

In an analysis of 63 hospitalized cases with visceral leishmaniasis, the clinical or post-mortem diagnosis of bacterial infection was performed in 33; 13 (39.3%) patients had respiratory infection, 4 (12.1%) had skin infection, 4 had urinary tract infection, 3 (9.0%) showed ear infection and 2 (6.6%) had infection of the oral cavity. It is worth mentioning that in 7 (21%) cases there was infection in multiple sites. Gram positive and/or Gram negative organisms were isolated from 10 patients. In only two (autopsied) cases, infection with less common organisms was recorded, one with disseminated candidiasis and another with disseminated tuberculosis. Death occurred in 9 of the 63 cases, and in 8 of these, concomitant bacterial infection of importance was documented. Patients who had serum globulins lower than 4 g% had significantly more infection (p less than 0.05) than patients with globulin levels higher than 4 g%; there was no significant difference when the number of leucocytes and neutrophils in patients with associated infection was compared with those in patients without bacterial infection. The present study demonstrates that bacterial infection frequently occurs in patients with visceral leishmaniasis, and indicates an unfavourable prognosis. Even though the mechanism of increased susceptibility to infection in this condition was unclear, the widespread range of infections and of infective agents, suggests a multifactorial process.     

Urban parasitology: visceral leishmaniasis in Brazil

Since the early 1980s, visceral leishmaniasis (VL) which is, in general, a rural zoonotic disease, has spread to the urban centers of the north, and now the south and west of Brazil. The principal drivers differ between cities, though human migration, large urban canid populations (animal reservoir), and a decidedly peripatetic and adaptable sand fly vector are the primary forces. The exact number of urban cases remains unclear as a result of challenges with surveillance. However, the number of urban cases registered continues to increase annually. Most control initiatives (e.g. culling infected dogs and household spraying to kill the sand fly) could be effective, but have proven hard to maintain at large scales due to logistical, financial and other reasons. In this article, the urbanization of VL in Brazil is reviewed, touching on these and other topics related to controlling VL within and outside Brazil.     

Present situation of visceral leishmaniasis in China

Visceral leishmaniasis or kala azar is a disease that is distributed world-wide from countries around the Mediterranean Sea to Africa, the Middle East, Asia and South America (Fig. 1). Visceral leishmaniasis was highly prevalent in China but since 1958, after a nationwide campaign, it has been brought under control. Only sporadic cases occur in the hilly and newly reclaimed desert areas in NW China.     

Exploring sand fly salivary proteins to design multiepitope subunit vaccine to fight against visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by the parasites of Leishmania donovani complex, leads to the death of 20 000 to 40 000 people from 56 affected countries, worldwide. Till date, there is not a single available vaccine candidate to prevent the VL infection, and treatment only relies upon expensive and toxic chemotherapeutic options. Consequently, immunoinformatics approach was applied to design a multiepitope-based subunit vaccine to enhance the humoral as well as cell-mediated immunity. Constructed vaccine candidate was further subjected to evaluation on allergenicity and antigenicity and physiochemical parameters. Later on, disulfide engineering was performed to increase the stability of vaccine construct. Also, molecular docking and molecular dynamics simulation study were performed to check the binding affinity and stability of toll-like receptor-4 to vaccine construct complex. Finally, codon optimization and in silico cloning were performed to ensure the expression of proposed vaccine construct in a microbial expression system.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

Intranasal vaccine from whole Leishmania donovani antigens provides protection and induces specific immune response against visceral leishmaniasis

Visceral leishmaniasis is a protozoan disease associated with high fatality rate in developing countries. Although the drug pipeline is constantly improving, available treatments are costly and live-threatening side effects are not uncommon. Moreover, an approved vaccine against human leishmaniasis does not exist yet. Using whole antigens from Leishmania donovani promastigotes (LdAg), we investigated the protective potential of a novel adjuvant-free vaccine strategy. Immunization of mice with LdAg via the intradermal or the intranasal route prior to infection decreases the parasitic burden in primary affected internal organs, including the liver, spleen, and bone marrow. Interestingly, the intranasal route is more efficient than the intradermal route, leading to better parasite clearance and remarkable induction of adaptive immune cells, notably the helper and cytotoxic T cells. In vitro restimulation experiments with Leishmania antigens led to significant IFN-γ secretion by splenocytes; therefore, exemplifying specificity of the adaptive immune response. To improve mucosal delivery and the immunogenic aspects of our vaccine strategy, we used polysaccharide-based nanoparticles (NP) that carry the antigens. The NP-LdAg formulation is remarkably taken up by dendritic cells and induces their maturation in vitro, as revealed by the increased expression of CD80, CD86 and MHC II. Intranasal immunization with NP-LdAg does not improve the parasite clearance in our experimental timeline; however, it does increase the percentage of effector and memory T helper cells in the spleen, suggesting a potential induction of long-term memory. Altogether, this study provides a simple and cost-effective vaccine strategy against visceral leishmaniasis based on LdAg administration via the intranasal route, which could be applicable to other parasitic diseases.     

Anemia in experimental visceral leishmaniasis in hamsters.

Experimental infection of hamsters with Leishmania donovani caused visceral leishmaniasis in which hematological changes occurred. The infected hamsters were anemic and reticulocyte counts were high. No significant change in the serum erythropoietin level was noted. Red cell membrane Na(+)-K(+)-ATPase and acetylcholinesterase activities increased. Osmotic fragility of the erythrocytes from infected animals increased. The level of 2,3-diphosphoglycerate of the red cells increased with the degree of anemia.     

Evidence-based diagnostic algorithm for visceral leishmaniasis in Bangladesh

Evidence-based diagnostic algorithm is highly recommended for the visceral leishmaniasis (VL). This cross-sectional study was performed in Bangladesh to evaluate VL diagnostic tools including serology, buffy coat smear microscopy for LD body and various DNA-based techniques using buffy coat in 100 confirmed VL cases and 100 controls. The performance of tools against spleen smear (gold standard) was evaluated using kappa coefficient. Diagnostic precision and other inherent indicators were considered for index scoring (IS) of performance of tools using factor analysis. A diagnostic algorithm was formulated based on the IS and availability of the tools at different health care facilities of Bangladesh. A high level of agreement (kappa ≥  0.80) was observed for all the diagnostic tools. The highest kappa coefficients were found for rK39 RDT and rK39 ELISA (0.95), followed by ssuRNA-PCR (0.94), Buffy coat smear (0.93), rK28 ELISA (0.92), rK28 RDT (0.89), LAMP (0.89), Mini-exon PCR (0.86), ITS1 (0.85), and ITS2 PCR (0.80). rK39 RDT was found to be the best diagnostic test (IS: 1.7) followed by rK28 RDT (IS: 1.5), buffy coat smear microscopy (IS: 0.5), rK39 & rK28 ELISA (IS: 0.3), ssuRNA-PCR (IS: ‐0.7) and LAMP, Mini-exon, ITS1, & ITS2 PCR (IS: ‐0.9). rK39 RDT has been proposed as the best option for primary health care facilities, while buffy coat smear microscopy was found to be a good adjunct for confirmation of serology-positive cases and proposed for secondary and tertiary facilities. ssuRNA-PCR or LAMP can be an alternate confirmation tool only applicable to the tertiary facilities.     

Fine needle aspiration of lymphadenopathy in visceral leishmaniasis

OBJECTIVE: To illustrate the cytomorphologic features of Leishmania lymphadenitis associated with visceral leishmaniasis (V/L) and post-kala-azar dermal leishmaniasis (PKDL) and to highlight the fact that Leishmania lymphadenitis must he included in the differential diagnosis of patients presenting with lymphadenopathy, particularly in areas endemic for the disease. STUDY DESIGN: Fine needle aspiration (FNA) was routinely done in 21 cases of lymphadenopathy in VL (18 cases) and PKDL (3 cases), and the detailed cytomorphologic features were correlated with the respective histopathologic findings. RESULTS: Amastigote forms of Leishman-Donovan (LD) bodies were seen in 19 cases both intracellularly, in histiocytes and multinucleate giant cells, and extracellularly. The FNA smears revealed a polymorphous population of cells composed of lymphocytes, histiocytes, plasma cells, giant cells and tingible body macrophages. In a few cases, epithelioid cell granulomas were also seen. The cytomorphologic features were confirmed and correlated on histopathology. CONCLUSION: Not all lymphadenopathy in VL and PKDL is due to Leishmania lymphadenitis. Demonstration of LD bodies on FNA smears helps with the early diagnosis of VL and PKDL with lymphadenopathy where the diseases are endemic.