Role of IL-17 gene polymorphism in Indian kala-azar.

BACKGROUND & OBJECTIVES: Visceral leishmaniasis or kala-azar is a fatal protozoan disease caused by an obligate intracellular parasite, Leishmania donovani. Susceptibility, establishment of infection and severity of this disease depend upon many factors, but it is the host immune system that plays decisive role in disease progression. Keeping this view into consideration, we investigated the probable relationship between polymorphisms rs2275913 and rs8193036 in IL-17 gene, and its association as a risk factor for kala-azar in an endemic population of Assam, India. METHODS: A total of 209 subjects, 76 kala-azar cases (male: 46, female: 30, mean age ± SD: 34.60 ± 12.61) and 133 controls (male: 66, female: 67, mean age ± SD: 33.35 ± 14.48) were included in this study. We analysed the polymorphisms, rs2275913 and rs8193036 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The data were analysed using logistic regression analysis and SPSS software. RESULTS: The results revealed that the mutant rs8193036 TT genotype conferred 4.7-fold higher risk for kala-azar (p = 0.00991, OR = 4.72, 95% CI = 1.330-16.911). A significant difference was found between the allele frequencies of rs8193036 (p = 0.029, OR = 1.64, 95% CI = 1.04-2.57) when comparisons were done using the genetic models of association. When stratification analysis was done on the basis of active and past cases we found that during active infection rs2275913 A allele was significantly associated with increased risk of kala-azar (p = 0.016, OR = 3.95, 95% CI = 1.21-12.87). INTERPRETATION & CONCLUSION: The findings revealed that IL-17 genetic variant, rs8193036 is an independent risk factor for kala-azar infection and may contribute in pathogenesis of the disease. Further, rs2275913 polymorphism of IL-17 gene is associated with kala-azar during active infection.

Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania.

Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.

Leishmaniasis.

Leishmaniasis is a poverty-related disease with two main clinical forms: visceral leishmaniasis and cutaneous leishmaniasis. An estimated 0·7-1 million new cases of leishmaniasis per year are reported from nearly 100 endemic countries. The number of reported visceral leishmaniasis cases has decreased substantially in the past decade as a result of better access to diagnosis and treatment and more intense vector control within an elimination initiative in Asia, although natural cycles in transmission intensity might play a role. In east Africa however, the case numbers of this fatal disease continue to be sustained. Increased conflict in endemic areas of cutaneous leishmaniasis and forced displacement has resulted in a surge in these endemic areas as well as clinics across the world. WHO lists leishmaniasis as one of the neglected tropical diseases for which the development of new treatments is a priority. Major evidence gaps remain, and new tools are needed before leishmaniasis can be definitively controlled.

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Growth factor and Th2 cytokine signaling pathways converge at STAT6 to promote arginase expression in progressive experimental visceral leishmaniasis.

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.

VISCERAL LEISHMANIASIS FROM AN AREA PREVIOUSLY NOT KNOWN TO BE ENDEMIC; DANGUR, BENSHANGUL-GUMUZ, REGIONAL STATE, NORTHWEST ETHIOPIA: A CASE REPORT.

Visceral leishmaniasis (VL) is a ftial and growing public health problem in Ethiopia. VL is recently reported outside the major endemic foci, the lowlands in the northwest and the Omo and Abaroba-plain, Segen and Woito valleys in the southwest. Here, we report a visceral leishmaniasis case from Benishangul-Gumuz Regional state near the Guba area. The patient had no history of travel to known VL endemic areas. The patient is a temporary farm laborer from West Go'jam Zone, Wanbermna District in Amhara Regional State. While in Benishangul-Gumuz, the patient was diagnosed with prolonged and intermittentfever, epistaxis, splenomegaly, skin pallor, diarrhea, cough and oedema. Laboratory diagnosis results showed that he had marked leucopenia, thrombocytopenia and anemia. The patient was suspected of having VL and checked with rK39 immunochromnatography and direct agglutination tests which were positive for anti leishmanial antibodies. After getting full dose of sodium stibogluconate as per the national visceral leishmaniasis treatment guideline, was clinically cured. As the area in Benshangul-Gumuz where this patient contracted visceral leishmaniasis is under social and ecological transformation with large scale projects attracting huge influx of temporary laborers and settlers, due attention is needed with respect to introduction or emergence of VL transmission.

[Visceral leishmaniasis concurrent with splenic marginal zone B-cell lymphoma].

Splenic marginal zone B-cell lymphoma (SMZBCL) is a rare non-Hodgkin B-cell lymphoma that presents with morphologically mature lymphoid cells corresponding in their immunological characteristics to secondary follicular marginal zone lymphocytes. It is clinically characterized by splenomegaly, moderate lymphocytosis, usually focal bone marrow lesion, sometimes moderate of monoclonal immunoglobulin in the serum (generally IgM or IgG) and/or urea, and a relatively benign course. Leishmaniasis is a transmissible natural focal infectious endemic disease that has a great diversity of clinical manifestations. The authors describe Russia's first case of SMZBCL concurrent with visceral leishmaniasis in a 52-year-old female patient admitted to a hematology hospital with weakness, splenomegaly, and lymphadenopathy. The simultaneous detection of lymphoma and leishmaniasis in the same biopsy specimen is extremely rare. Visceral leishmaniasis should be borne in mind as an opportunistic infection in patients with malignancies, particularly in immunocompromised persons who live or have stayed in the endemic areas.

Visceral leishmaniasis as an independent cause of high immune activation, T-cell senescence, and lack of immune recovery in virologically suppressed HIV-1-coinfected patients.

OBJECTIVES: Different immune alterations have been described in HIV-infected patients with visceral leishmaniasis (VL). We aimed to identify the immunological factors involved in the lack of immunological recovery and VL relapses in HIV-infected patients with VL, by comparison with other HIV-infected patients. METHODS: We carried out a cross-sectional study of 55 patients receiving suppressive combination antiretroviral therapy (cART) for at least 1 year: nine with previous relapsing VL, 20 with an immunodiscordant response (IDR) to cART (CD4 count < 200 cells/µL) and no previous VL, and 26 with a concordant response (CR) to cART (CD4 count > 350 cells/µL) without VL. Immunosenescence was investigated by analysing CD57(+) CD28(-) levels, immune activation by analysing CD38(+) HLA-DR(+) levels, inflammation by analysing interleukin (IL)-6 levels, and microbial translocation by analysing lipopolysaccharide (LPS) and soluble CD14 (sCD14) levels. RESULTS: In VL patients, the median time since VL diagnosis was 42 months, and all patients had had at least one relapse despite suppressive cART for a median time of 43 months. Patients with previously diagnosed VL had a higher CD8 T-cell activation level (P < 0.001) than those with IDR. Also, levels of IL-6, LPS and especially sCD14, associated with bacterial translocation and additional monocyte activation, were significantly increased in patients with previous VL compared with patients with IDR (P = 0.048, P = 0.049 and P < 0.001, respectively). In addition, patients with previous VL had higher levels of CD8 T-cell senescence. Notably, the levels of immune activation and inflammation in patients with previous VL were not related to the time of VL diagnosis, the number of VL relapses, or hepatitis C virus (HCV) coinfection. CONCLUSIONS: Our data demonstrate that VL patients had an even worse immunological status than patients with IDR, which was probably associated with increased microbial translocation and additional monocyte/macrophage activation. These data explain the observed lack of immunological recovery and the occurrence of VL relapses in HIV-infected patients with previous VL.

Efficacy of intravenous cyclosporine in a case of cytophagic histiocytic panniculitis complicated by haemophagocytic syndrome after visceral leishmania infection.

Cytophagic histiocytic panniculitis (CHP) is a rare panniculitis characterized by systemic features, due to histiocytic infiltration along with haemophagocytosis, which may also appear in bone marrow, spleen, lymph nodes, and liver. Haemophagocytic lymphohistiocytosis (HLH) is a group of autoinflammatory disorders, which include macrophage activation syndrome, sometimes observed in the course of systemic autoimmune diseases, such as juvenile chronic polyarthritis, systemic lupus erythematosus or vasculitis, and infection-associated haemophagocytic syndrome; if not promptly recognised and treated, HLH can be fatal. Visceral leishmaniasis (VL) is a systemic disease caused by different forms of Leishmania spp., an intracellular protozoa. VL is endemic in tropical countries such as in the Middle East and the Mediterranean. The typical clinical and laboratory features are fever, hepato-splenomegaly, hypergammaglobulinaemia and pancytopenia. The features of VL may mimic some haematologic diseases. We report a case of cytophagic histiocytic panniculitis and HLH, triggered by a previous visceral leishmania infection. Cyclosporine was quickly effective in this case, after failure of high-dose glucocorticoids, anakinra and etoposide.

[VISCERAL LEISHMANIASIS IN A THREE-YEAR-OLD CHILD].

The paper describes a clinical case of visceral leishmaniasis in a three-year-old child who Was born and is a permanent resident in Penm. Its clinical symptomatology; laboratory and instrumental findings; stages of a diagnostic search for fever of unknown origin concurrent with hepatosplenomegaly and pancytopenia; differential diagnosis with hemoblastosis; and an epidemiological history are detailed. Visceral leishmaniasis was diagnosed at examination of bone marrow specimens. The paper presents the positive results of combination etiopathogenetic treatment, which are confirmed by the time course of clinical changes and laboratory findings.

Clinico-hematological profile of visceral leishmaniasis among immunocompetent patients.

We studied cases of visceral leishmaniasis (VL) over a 2-year period among immunocompetent patients who presented to a rural medical college in West Bengal, India to determine a clinical and hematological profile among these patients. We studied a total of 36 cases of VL; the male to female ratio of the cases was 1.6:1 and the mean age was 20.1+/-11.1 years. A detailed history, physical examination, hemogram, bone marrow or splenic aspiration and chest x-ray were conducted on all cases. A CT-scan of the thorax and fiberoptic bronchoscopy were performed in selected cases. Fever and splenomegaly were present in all cases. Weakness, abdominal pain, bleeding, and hepatomegaly were seen in 63.9, 27.8, 8.3 and 58.3% of cases, respectively. Pancytopenia, bicytopenia, leukopenia and thrombocytopenia were seen in 58.3, 41.7, 61.1 and 83.3% of cases, respectively. Five patients (13.9%) had cough, 2 (5.6%) had hemoptysis, 6 (16.7%) had an abnormal chest x-ray and 3 (8.3%) had localized reticulo-nodular opacities on a CT-scan of the thorax. Bronchoalveolar lavage showed gram-positive cocci in 2 cases (5.6%). One patient died of acute respiratory distress syndrome. Cytopenia was common among the series of VL patients. Pulmonary complications, usually secondary infection, were less frequent (found in 13.9% cases) but was fatal in one patient.

Role of endothelial dysfunction in modulating the plasma redox homeostasis in visceral leishmaniasis.

BACKGROUND: Evidence in the literature suggests that down-regulation of nitric oxide (NO) is associated with the pathophysiological conditions during visceral leishmaniasis (VL). Here we have investigated the mechanism that leads to the down regulation of systemic NO in the infected condition. Moreover, we have determined whether down regulation of NO is associated with increased generation of reactive oxygen species (ROS) during this disease. Therapeutic strategy targeting signaling molecules of these events was evaluated. METHODS: Plasma protein-nitrotyrosine was examined by ELISA kit. Generation of superoxides and peroxynitrites was investigated by flow cytometry. NO bioavailability in endothelial cells was evaluated using DAF-2DA fluorescence. Ceramide contents were evaluated using FACS analysis, HPTLC and HPLC. RESULTS: L. donovani infected reticulo-endothelial cells regulated the activity of eNOS and NAD(P)H oxidase in the endothelial cells through the generation of intercellular messenger, ceramide. Activation of SMases played an important role in the generation of ceramide in animals during chronic infection. These events led to generation of ROS within endothelial cells. Modulation of redox status of plasma and accumulation of ROS in endothelial cells were critically involved in the regulation of NO bioavailability in plasma of the infected animal. Endothelial dysfunction and decline of NO were resulted from an increased production of superoxide where upregulation of eNOS expression appeared as an ineffective compensatory event. Inhibition of ceramide generation increased NO bioavailability, prevented endothelial dysfunction and concomitant oxidative stress. CONCLUSION AND GENERAL SIGNIFICANCE: Decreased NO bioavailability and endothelial dysfunction were the downstream of ceramide signaling cascade. ROS accumulation promoted peroxynitrite generation and reduced NO bioavailability. Inhibition of ceramide generation may be a potential therapeutic option in preventing the co-morbidity associated with VL.

Immunological factors, but not clinical features, predict visceral leishmaniasis relapse in patients co-infected with HIV.

Visceral leishmaniasis (VL) has emerged as a clinically important opportunistic infection in HIV patients, as VL/HIV co-infected patients suffer from frequent VL relapse. Here, we follow cohorts of VL patients with or without HIV in Ethiopia. By the end of the study, 78.1% of VL/HIV-but none of the VL patients-experience VL relapse. Despite a clinically defined cure, VL/HIV patients maintain higher parasite loads, lower BMI, hepatosplenomegaly, and pancytopenia. We identify three immunological markers associated with VL relapse in VL/HIV patients: (1) failure to restore antigen-specific production of IFN-γ, (2) persistently lower CD4+ T cell counts, and (3) higher expression of PD1 on CD4+ and CD8+ T cells. We show that these three markers, which can be measured in primary hospital settings in Ethiopia, combine well in predicting VL relapse. The use of our prediction model has the potential to improve disease management and patient care.

TGF-ß-regulated tyrosine phosphatases induce lymphocyte apoptosis in Leishmania donovani-infected hamsters.

Visceral leishmaniasis, which is caused by Leishmania donovani, is one of the major health problems of the Indian subcontinent. Infected hosts have been reported to have impaired lymphoproliferation. However, the fate of anergic cells is still elusive. In the present investigation, L. donovani-infected hamsters were used to study the mechanism of lymphocyte cell death. Lymph node-derived lymphocytes were analysed for apoptotic death through mitochondrial abnormality, caspase activity and DNA degradation. The data demonstrate that the disease progression leads to a gradual impairment of lymphocyte proliferation in the presence of Concanavalin A. The fate of the anergic lymphocytes is intrinsic apoptosis, which is evident by the depolarization of the mitochondrial membrane potential, cytosolic release of cytochrome c, caspase activation and DNA fragmentation. Tumour growth factor (TGF)-ß, which is secreted by macrophages, was significantly upregulated in the lymph node compartment of infected hamsters. Adding a neutralizing TGF-ß antibody and a recombinant TGF-ß resulted in the downregulation and induction of lymphocyte apoptosis, respectively. Furthermore, it has been observed that TGF-ß triggers the apoptotic death of lymphocytes through the upregulation of tyrosine phosphatase activity and that the use of sodium orthovanadate (Na(3)VO(4), a tyrosine phosphatase inhibitor) reduces the apoptotic frequency. Thus, this study clearly reports the novel involvement of tyrosine phosphatases in TGF-ß-induced lymphocyte apoptosis in Leishmania-infected hamsters.

Clinical expression of autoimmune hepatitis in a nine-year-old girl with visceral leishmaniasis.

Autoimmune hepatitis (AIH) is a chronic disease of unknown aetiology, which usually progresses to cirrhosis if not diagnosed and treated promptly. In childhood, autoimmune hepatitis prevalently presents with non-specific indications. The cause of entrance to hospital of the 9-year-old child was fever for 6 days (39°C) and intense cough, with other clinical symptoms and signs: abdominal distention, paleness, lack of appetite. In our case we report experience with AIH presenting with atypical clinical features of visceral leishmaniasis.

Association between Hypertriglyceridemia and Disease Severity in Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a tropical disease endemic to Brazil. The clinical manifestations of the infection range from asymptomatic to severe. In VL, changes in lipid metabolism, such as hypocholesterolemia and hypertriglyceridemia, occur that are believed to be related to its progression and severity. This study investigated the associations between serum levels of cholesterol, triglycerides, and lipoproteins (high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein) with clinical and hematological parameters that predict severity in a case series of 83 VL patients. Severely ill patients had higher mean serum triglyceride levels than non-severely ill patients. There was a significant positive correlation between disease severity score and serum triglyceride levels, very low-density lipoprotein, international normalized ratio for prothrombin time test, total bilirubin, and age. An inverse correlation was detected between the disease severity score and mean platelet and neutrophil counts. Hypertriglyceridemia can be a prognostic indicator of severity in patients diagnosed with VL.

Acute kidney injury in patients with Visceral Leishmaniasis in Northwest Ethiopia.

BACKGROUND: Visceral Leishmaniasis (VL) is a neglected tropical disease endemic to several countries including Ethiopia. Outside of Africa, kidney involvement in VL is frequent and associated with increased mortality. There is however limited data on acute kidney injury (AKI) in VL patients in East-Africa, particularly in areas with high rates of HIV co-infection. This study aims to determine the prevalence, characteristics and associated factors of AKI in VL patients in Northwest Ethiopia. METHODS: A hospital based retrospective patient record analysis was conducted including patients treated for VL from January 2019 to December 2019 at the Leishmaniasis Research and Treatment Center (LRTC), Gondar, Ethiopia. Patients that were enrolled in ongoing clinical trials at the study site and those with significant incomplete data were excluded. Data was analyzed using SPSS version 20. P values were considered significant if < 0.05. RESULTS: Among 352 VL patients treated at LRTC during the study period, 298 were included in the study. All were male patients except two; the median age was 23 years (IQR: 20-27). The overall prevalence of AKI among VL patients was 17.4% (confidence interval (CI): 13.6%-22.2%). Pre-renal azotemia (57%) and drug-induced AKI (50%) were the main etiologies of AKI at admission and post-admission respectively. Proteinuria and hematuria occurred in 85% and 42% of AKI patients respectively. Multivariate logistic regression revealed HIV co-infection (adjusted odds ratio (AOR): 6.01 95% CI: 1.99-18.27, p = 0.001) and other concomitant infections (AOR: 3.44 95% CI: 1.37-8.65, p = 0.009) to be independently associated with AKI. CONCLUSION: AKI is a frequent complication in Ethiopian VL patients. Other renal manifestations included proteinuria, hematuria, and pyuria. HIV co-infection and other concomitant infections were significantly associated with AKI. Further studies are needed to quantify proteinuria and evaluate the influence of AKI on the treatment course, morbidity and mortality in VL patients.