BACKGROUND:Leishmania infantum can be an opportunistic pathogen, with an immunocompromised status increasing the risk of converting asymptomatic infection to symptomatic visceral leishmaniasis (VL). VL has approximately 5% fatality rate; and HIV coinfection (AIDS/VL) increases this risk. We hypothesized that, relative to those with HIV alone, people with co-infection would have altered T cell activation which could impact on the risk of VL. METHODS:A cross-sectional study was performed between 2014 and 2016 to determine the prevalence of L. infantum infection in people living with HIV (PLHIV) residing in Brazil (n = 1,372). Subsequent incident cases of VL were ascertained from a public health database through 2018 and from a cohort of families with VL. Immune status of 69 participants was evaluated and comparisons made between those with and without HIV, with latent or with active Leishmania infection and those without HIV but with active or resolved Leishmania or T cell hypersensitivity to Leishmania antigen and healthy control subjects. RESULTS:A total of 24.2% of PLHIV had positive anti-IgG L. infantum antibodies. The relative risk of developing AIDS/VL was 2.27 (95% CI: 0.920 to 5.59; p = 0.07) to HIV/Leish coinfected subjects with positive leishmania serology compared to HIV subjects without leishmania serology. Poor adherence to antiretroviral therapy (p = 0.0008) or prior opportunistic infections (p = 0.0007) was associated with development of AIDS/VL in asymptomatic HIV/Leish. CD4+ and CD8+ T cells counts or viral load were similar between asymptomatic HIV/Leish and HIV subjects. However, activated CD8+CD38+HLA-DR+ T cells were higher in asymptomatic HIV/Leish than HIV. Likewise, senescent (CD57+) and PD1+ CD8+ T cells were higher in asymptomatic HIV/Leish than in AIDS/VL or HIV groups. CONCLUSION:Although asymptomatic HIV/Leish subjects had CD4+ and CD8+ T cells similar to HIV alone, their CD8+T cells had increased activation and senescence which could contribute to risk of developing VL.

Neutrophils, the first cells to arrive at infection sites, release neutrophil extracellular traps (NETs) comprising nuclear and/or mitochondrial DNA webs decorated with proteins. Similar to other parasites, Leishmania infantum induces NET extrusion. However, our understanding of NET formation and neutrophil fate after NET release in a Leishmania infection context is limited. Our study aimed to determine the DNA origin of the NET scaffolds released by human neutrophils in response to chemical or L. infantum stimulation. Neutrophils were incubated with PMA, PHA, LPS, or L. infantum, followed by DNA and elastase activity quantification; additionally, we evaluated the source of DNA that composes NETs. Neutrophil viability was evaluated by annexin-V/7AAd labeling. Expression of IL6, TNFA, IL10, CXCL1, CXCL8, and FPR1 in response to the L. infantum interaction was assessed. Neutrophils incubated with chemicals or L. infantum released NETs. However, neutrophils stimulated by the chemicals showed lower viability after 1 h in comparison to neutrophils incubated with parasites. NETs from chemically stimulated neutrophils were mainly composed of nuclear DNA. Conversely, the NET induced by the parasites was of mitochondrial DNA origin and had no leishmanicidal activity. After 4 h of parasite stimulation, neutrophils peak the expression of genes such as IL6, TNFA, CXCL1, CXCL8, and FPR1. Our study demonstrates that neutrophils produce NETs after chemical or L. infantum exposure. Although they are not toxic to the parasite, NETs are released as danger signals. These findings support the role of neutrophils in releasing signaling molecules, which influence the inflammatory environment in which the infectious process occurs.

ABSTRACT Visceral leishmaniasis (VL) is a vector-borne disease caused by the obligate intracellular protozoan Leishmania donovani in India. VL can be complicated by post-kala-azar dermal leishmaniasis (PKDL), a macular or nodular rash that develops in 10%–20% of patients after treatment of VL in India. Patients with PKDL are infectious to sand flies, promoting further transmission of the parasite. MicroRNAs (miRNAs) are 18–25 nt, non-coding RNAs that simultaneously regulate the expression of several or many target transcripts. This study was based on the hypothesis that the host response to L. donovani is modified by distinct sets of miRNAs in VL or PKDL and that these might differ from healthy controls. We investigated this hypothesis using a NanoString panel to profile the miRNAs expressed in the plasma of patients with VL or PKDL diagnosed at a hospital in Bihar, India. We compared these to plasma microRNAs of healthy control individuals from the same endemic villages. miRNAs hsa-miR-223-3p, hsa-miR-191-5p, hsa-miR-23a-3p, and hsa-1285-5p were significantly higher in the plasma samples from patients with VL compared to either PKDL or endemic controls. Prediction programs highlighted potential mRNA targeted by these miRNAs, among which we verified the down-modulation of several transcripts belonging to the NFκB and NLRP3 inflammasome pathways in circulating leukocytes of VL patients. By contrast, miRNA patterns in subjects with PKDL were similar to control subjects, possibly suggesting that the pathogenic immune response during PKDL is primarily localized in the skin. IMPORTANCE Infection of humans with the protozoan Leishmania donovani can be asymptomatic or it can cause fatal visceral leishmaniasis (VL), sometimes followed by the cutaneous complication PKDL. Parasites are spread through sand fly bites in endemic regions, and parasites in post-kala-azar dermal leishmaniasis (PKDL) skin lesions are a source of prolonged parasite transmission to sand flies, compromising disease eradication efforts. Since microRNAs can simultaneously modify the expression of multiple genes, we examined microRNAs in the blood that might be partial determinants of pathogenic responses leading to VL or PKDL. Our studies revealed several miRNAs expressed that are elevated in the plasma of patients with VL, which suppress some of the inflammatory responses that promote parasite killing. However, miRNA profiles were very similar between PKDL patients and controls, raising the possibility that major factors that lead to prolonged retention of parasites in the skin during PKDL are not systemic but are localized in the skin.

The demographics of leishmaniasis has changed dramatically in endemic regions of Brazil, concurrent with shifts towards a high-fat high-cholesterol diet (HFHC). With this shift, a novel presentation of cutaneous leishmaniasis due to L. braziliensis has emerged in obese patients, characterized by poorer treatment response with higher recurrence rates in obese individuals (Lago, 2021). A direct link between diet-induced changes in metabolic and immune status and the manifestations of visceral leishmaniasis (VL) has not been established. We hypothesize that diet-induced changes in immunometabolism may affect the progression of VL. We examined the effects of diet in a murine model of VL. BALB/c mice were maintained on HFHC, LP, or control diets for 4 weeks, then either infected or not with L. infantum. Inflammation and parasite loads were evaluated by histology, qPCR, and RNAscope. HFHC diet abrogated parasite loads in the liver and caused exacerbated parasite growth in spleens (P < 0.0001). LP diet developed higher parasite loads in livers (P < 0.0001). Control mice developed granulomas in the livers. HFHC mice failed to develop mature granulomas (P < 0.0001). LP mice had increased numbers of small granulomas in the liver (P < 0.0001). These findings suggested that the basal inflammation of the HFHC diet led to failure to contain the parasite and early dissemination of infection to the spleen. Dietary factors may influence the changing spectrum of VL. Microbial, Parasitic, and Fungal Immunology (MPF)

BACKGROUND:The mechanisms that mediate immune protection in individuals with subclinical (SC) or asymptomatic infection with Leishmania braziliensis are largely unknown. Neutrophils (polymorphonuclear leukocytes [PMNs]) have been implicated in progressive symptomatic cutaneous leishmaniasis (CL), but their potential participation in maintenance of subclinical infection is unexplored. The aim of this study was to compare the phenotypic and functional profiles of PMNs in individuals with SC infection versus patients with symptomatic CL due to L braziliensis. METHODS:Subjects were recruited in the endemic region of Corte de Pedra, Bahia, Brazil. Surface markers to define activation status were characterized by flow cytometry. Functional responses of PMNs including phagocytic capacity, production of oxidative species, and oxidative killing of intracellular parasites were studied in vitro. RESULTS:PMNs from individuals with SC infection displayed a more activated phenotype and greater ability to control the infection than PMNs from patients with CL. In contrast, PMNs from patients with CL exhibited higher expression of HLA-DR and higher production of oxidative species than PMNs from subjects with SC infection. CONCLUSIONS:PMNs from individuals with SC infection can control the infection more efficiently than PMNs from patients with CL, despite the lower production of oxidants. Our observations suggest that L braziliensis may evade microbicidal mechanisms of PMNs from patients with CL, contributing to parasite dissemination and the establishment of disease.

Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.

Background:The pursuit of selective therapeutic delivery to target tissue types represents a key goal in the treatment of a range of adverse health issues, including diseases afflicting the heart. The development of new cardiac-specific ligands is a crucial step towards effectively targeting therapeutics to the heart. Methods:Utilizing an ex vivo and in vivo SELEX approaches, we enriched a library of 2'-fluoro modified aptamers for ventricular cardiomyocyte specificity. Lead candidates were identified from this library, and their binding and internalization into cardiomyocytes was evaluated in both ex vivo and in vivo mouse studies. Results:The ex vivo and in vivo SELEX processes generated an aptamer library with significant cardiac specificity over non-cardiac tissues such as liver and skeletal muscle. Our lead candidate aptamer from this library, CA1, demonstrates selective in vivo targeting and delivery of a fluorophore cargo to ventricular cardiomyocytes within the murine heart, while minimizing off-target localization to non-cardiac tissues, including the liver. By employing a novel RNase-based assay to evaluate aptamer interactions with cardiomyocytes, we discovered that CA1 predominantly internalizes into ventricular cardiomyocytes; conversely, another candidate CA41 primarily binds to the cardiomyocyte cell surface. Conclusions:These findings suggest that CA1 and CA41 have the potential to be promising candidates for targeted drug delivery and imaging applications in cardiac diseases.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1β and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.

Visceral leishmaniasis (VL) is a neglected tropical disease that is globally distributed and has the potential to cause very serious illness. Prior literature highlights the emergence and spread of VL is influenced by multiple factors, such as socioeconomic status, sanitation levels or animal and human reservoirs. The study aimed to retrospectively investigate the presence and infectiousness of VL in Rio Grande do Norte (RN), Brazil between 2007 and 2020. We applied a hierarchical Bayesian approach to estimate municipality-specific relative risk of VL across space and time. The results show evidence that lower socioeconomic status is connected to higher municipality-specific VL risk. Overall, estimates reveal spatially heterogeneous VL risks in RN, with a high probability that VL risk for municipalities within the West Potiguar mesoregion are more than double the expected VL risk. Additionally, given the data available, results indicate there is a high probability of increasing VL risk in the municipalities of Natal, Patu and Pau dos Ferros. These findings demonstrate opportunities for municipality-specific public health policy interventions and warrant future research on identifying epidemiological drivers in at-risk regions.

Background: Leishmania infantum is an opportunistic parasitic infection. An immunocompromised state increases the risk of converting asymptomatic infection to symptomatic visceral leishmaniasis (VL), which has a ~5% fatality rate even with treatment. HIV coinfection increases the risk of death from VL. Methods: A cross-sectional study was performed between 2014 and 2016 to determine the prevalence of L. infantum infection in HIV positive subjects residing in the state of Rio Grande do Norte, Brazil (n=1,372) and of these a subgroup of subjects were followed longitudinally. Subsequent incident cases of VL were ascertained from a public health database through 2018. A subgroup (n=69) of the cross-sectional study subjects was chosen to assess immune status (T cell activation, senescence, exhaustion) and outcome. The data were compared between asymptomatic HIV+/L. infantum+ (HIV/Leish), symptomatic visceral leishmaniasis (VL), recovered VL, DTH+ (Delayed-Type Hypersensitivity response – Leishmanin skin test), AIDS/VL, HIV+ only (HIV+), and Non-HIV/Non L. infantum infection (control subjects). Results: The cross-sectional study showed 24.2% of HIV+ subjects had positive anti-IgG Leishmania antibodies. After 3 years, 2.4% (8 of 333) of these HIV/Leish coinfected subjects developed AIDS/VL, whereas 1.05% (11 of 1,039) of HIV subjects with negative leishmania serology developed AIDS/VL. Poor adherence to antiretroviral therapy (p=0.0008) or prior opportunistic infections (p=0.0007) was associated with development of AIDS/VL. CD4+ (p=0.29) and CD8+ (p=0.38) T cells counts or viral load (p=0.34) were similar between asymptomatic HIV/Leish and HIV subjects. However, activated CD8+CD38+HLA-DR+ T cells were higher in asymptomatic HIV/Leish than HIV group. Likewise, senescent (CD57+) or exhausted (PD1+) CD8+ T cells were higher in asymptomatic HIV/Leish than in AIDS/VL or HIV groups. Conclusion: Although asymptomatic HIV/Leish subjects had normal and similar CD4+ and CD8+ T cells counts, their CD8+T cells had increased activation, senescence, and exhaustion, which could contribute to risk of developing VL.

The NLR protein NLRP12 contributes to innate immunity, but the mechanism re-mains elusive. Infection of Nlrp12(-/-) or wild-type mice with Leishmania infantum led to aberrant parasite tropism. Parasites replicated to higher levels in livers of Nlrp12(-/-) mice than in the livers of WT mice and failed to disseminate to spleens. Most retained liver parasites resided in dendritic cells (DCs), with correspond-ingly fewer infected DCs in spleens. Furthermore, Nlrp12(-/-) DCs expressed lower CCR7 than WT DCs, failed to migrate toward CCL19 or CCL21 in chemo-taxis assays, and migrated poorly to draining lymph nodes after sterile inflamma-tion. Leishmania-infected Nlpr12(-/-) DCs were significantly less effective at trans-porting parasites to lymph nodes than WT DCs. Consistently, adaptive immune responses were also impaired in infected Nlrp12(-/-) mice. We hypothesize that Nlrp12-expressing DCs are required for efficient dissemination and immune clearance of L. infantum from the site of initial infection. This is at least partly due to the defective expression of CCR7.

Background Leishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil. Methodology/Principal findings Two temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008-2011) primary sample, N = 120; (1999-2001) validation sample, N = 35. Parasites were genotyped by Sanger's sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward's comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software. Conclusions/Significance These findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite's lifecycle.

No single intervention is highly effective in malaria control. Are combinations better? In a double-blind trial in Burkina Faso and Mali, investigators

Innovative strategies to curb malaria transmission represent an important approach to reducing disease burden. Lethal house lures are

Cancer is likened to a non-healing wound. There is limited evidence on the expression of wound healing tissue inflammatory markers, CD68(pan-macrophage marker), HO-1(tumour cell marker) and FAP(cancer-associated fibroblast marker) in human breast cancer. In 201 invasive breast cancer and 58 DCIS patients, CD68+TAM expression, tumour HO-1 and fibroblast FAP expression, quantified by immunohistochemistry(dichotomised: high/present vs low/absent), was correlated with tumour factors (grade, proliferation(Ki67), ER, HER2); demographic factors, behavioural factors (smoking, alcohol) and survival status(DFS, OS) High CD68+macrophage expression was increased in invasive breast cancer, compared to DCIS, and normal tissue distant from the tumour(59%,41%and 6% respectively; p<0.001).In invasive cancer,CD68+TAM expression increased with increasing tumour grade(Grade 1:42%, Grade 2:58%, Grade 3:72%; p=0.006), high Ki67(71%vs.47%; p=0.004), ER negativity(79.4%vs.55.4%; p=0.01) and HER2(HER2 positive 81.8% vs. HER2 negative 56.3%; p=0.03). CD68+TAM expression was higher in high compared to low/intermediate grade DCIS(44% % vs. 31% p=0.52). CD68+TAM expression was increased in patients who self-reported alcohol intake(non-drinker 43% vs. drinker 62%; p=0.01). HO-1 was associated with shorter DFS(HR:3.22,p=0.027) and OS(HR:2.86,p=0.029).FAP fibroblast expression correlated with longer DFS (HR:0.296,p=0.029) and OS (HR:0.271,p=0.008). Tumour inflammation as assessed by CD68+TAM expression shows utility in identifying aggressive breast cancer sub-types. The association reported between CD68+TAM density and alcohol intake suggests a possible mechanism for alcohol as a risk factor for breast cancer. The prognostic value of HO-1 and FAP expression demonstrated here suggests a functional role of these wound healing markers in breast cancer. HO-1:Heme-oxygenase-1; FAP:Fibroblast activation protein; TAM:Tumour associated macrophage; DCIS: Ductal carcinoma in situ Wound healing pathways of inflammation may be implicated in early breast cancer development

Visceral leishmaniasis is an infectious parasitic disease caused by the protozoan parasites Leishmania donovani and Leishmania infantum . The drugs currently used to treat visceral leishmaniasis suffer from toxicity and the emergence of parasite resistance, and so a better solution would be the development of an effective subunit vaccine; however, no approved vaccine currently exists. The comparative testing of a large number of vaccine candidates requires a quantitative and reproducible experimental murine infection model, but the parameters that influence infection pathology have not been systematically determined. To address this, we have established an infection model using a transgenic luciferase-expressing L. donovani parasite and longitudinally quantified the infections using in vivo bioluminescent imaging within individual mice. We examined the effects of varying the infection route, the site of adjuvant formulation administration, and standardised the parasite preparation and dose. We observed that the increase in parasite load within the liver during the first few weeks of infection was directly proportional to the parasite number in the initial inoculum. Finally, we show that immunity can be induced in pre-exposed animals that have resolved an initial infection. This murine infection model provides a platform for systematic subunit vaccine testing against visceral leishmaniasis.