Phenotypic and functional characteristics of monocyte subsets in the blood and bone marrow of Indian subjects with Visceral Leishmaniasis.

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-1ß 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.

Elucidating the role played by bone marrow in visceral leishmaniasis.

Leishmaniasis is a widespread group of infectious diseases that significantly impact global health. Despite high prevalence, leishmaniasis often receives inadequate attention in the prioritization of measures targeting tropical diseases. The causative agents of leishmaniasis are protozoan parasites of the Leishmania genus, which give rise to a diverse range of clinical manifestations, including cutaneous and visceral forms. Visceral leishmaniasis (VL), the most severe form, can be life-threatening if left untreated. Parasites can spread systemically within the body, infecting a range of organs, such as the liver, spleen, bone marrow and lymph nodes. Natural reservoirs for these protozoa include rodents, dogs, foxes, jackals, and wolves, with dogs serving as the primary urban reservoir for Leishmania infantum. Dogs exhibit clinical and pathological similarities to human VL and are valuable models for studying disease progression. Both human and canine VL provoke clinical symptoms, such as organ enlargement, fever, weight loss and abnormal gamma globulin levels. Hematologic abnormalities have also been observed, including anemia, leukopenia with lymphocytosis, neutropenia, and thrombocytopenia. Studies in dogs have linked these hematologic changes in peripheral blood to alterations in the bone marrow. Mouse models of VL have also contributed significantly to our understanding of the mechanisms underlying these hematologic and bone marrow abnormalities. This review consolidates information on hematological and immunological changes in the bone marrow of humans, dogs, and mice infected with Leishmania species causing VL. It includes findings on the role of bone marrow as a source of parasite persistence in internal organs and VL development. Highlighting gaps in current knowledge, the review emphasizes the need for future research to enhance our understanding of VL and identify potential targets for novel diagnostic and therapeutic approaches.

An in vivo humanized model to study homing and sequestration of Plasmodium falciparum transmission stages in the bone marrow.

Introduction: Recent evidence suggests that the bone marrow (BM) plays a key role in the diffusion of P. falciparum malaria by providing a "niche" for the maturation of the parasite gametocytes, responsible for human-to-mosquito transmission. Suitable humanized in vivo models to study the mechanisms of the interplay between the parasite and the human BM components are still missing. Methods: We report a novel experimental system based on the infusion of immature P. falciparum gametocytes into immunocompromised mice carrying chimeric ectopic ossicles whose stromal and bone compartments derive from human osteoprogenitor cells. Results: We demonstrate that immature gametocytes home within minutes to the ossicles and reach the extravascular regions, where they are retained in contact with different human BM stromal cell types. Discussion: Our model represents a powerful tool to study BM function and the interplay essential for parasite transmission in P. falciparum malaria and can be extended to study other infections in which the human BM plays a role.

Differential miRNA expression profiles in the bone marrow of Beagle dogs at different stages of Toxocara canis infection.

BACKGROUND: Toxocara canis is distributed worldwide, posing a serious threat to both human and dog health; however, the pathogenesis of T. canis infection in dogs remains unclear. In this study, the changes in microRNA (miRNA) expression profiles in the bone marrow of Beagle dogs were investigated by RNA-seq and bioinformatics analysis. RESULTS: Thirty-nine differentially expressed (DE) miRNAs (DEmiRNAs) were identified in this study. Among these, four DEmiRNAs were identified at 24 h post-infection (hpi) and all were up-regulated; eight DEmiRNAs were identified with two up-regulated miRNAs and six down-regulated miRNAs at 96 hpi; 27 DEmiRNAs were identified with 13 up-regulated miRNAs and 14 down-regulated miRNAs at 36 days post-infection (dpi). Among these DEmiRNAs, cfa-miR-193b participates in the immune response by regulating the target gene cd22 at 24 hpi. The novel_328 could participate in the inflammatory and immune responses through regulating the target genes tgfb1 and tespa1, enhancing the immune response of the host and inhibiting the infection of T. canis at 96 hpi. In addition, cfa-miR-331 and novel_129 were associated with immune response and self-protection mechanisms at 36 dpi. 20 pathways were significantly enriched by KEGG pathway analysis, most of which were related to inflammatory response, immune response and cell differentiation, such as Cell adhesion molecules (CAMs), ECM-receptor interaction and Focal adhesion. CONCLUSIONS: These findings suggested that miRNAs of Beagle dog bone marrow play important roles in the pathogenesis of T. canis infection in dogs and provided useful resources to better understand the interaction between T. canis and the hosts.

CD169+ macrophages orchestrate plasmacytoid dendritic cell arrest and retention for optimal priming in the bone marrow of malaria-infected mice.

Plasmacytoid dendritic cells (pDCs) are the most potent producer of type I interferon (IFN), but how pDC is primed in vivo is poorly defined. Using a mouse model of severe malaria, we have previously established that upon priming by CD169+ macrophages (MPs), pDC initiates type I IFN-I secretion in the bone marrow (BM) of infected mice via cell-intrinsic TLR7 sensing and cell-extrinsic STING sensing. Herein we show that CD169+ MP and TLR7 sensing are both required for pDC arrest during priming, suggesting CD169+ MP are the source of TLR7 ligands. We establish that TLR7 sensing in pDC and chemotaxis are both required for pDC arrest and functional communication with CD169+ MP in the BM. Lastly, we demonstrate that STING sensing in CD169+ MP control pDC initiation of type I IFN production while also regulating pDC clustering and retention/egress from the BM. Collectively, these results link pDC acquisition of type I IFN-secreting capacity with changes in their motility, homing and interactions with CD169+ MP during infection. Thus, targeting this cellular interaction may help modulate type I IFN to improve outcomes of microbial infections and autoimmune diseases.

Advancing Key Gaps in the Knowledge of Plasmodium vivax Cryptic Infections Using Humanized Mouse Models and Organs-on-Chips.

Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden.

Uncovering a Cryptic Site of Malaria Pathogenesis: Models to Study Interactions Between Plasmodium and the Bone Marrow.

The bone marrow is a critical site of host-pathogen interactions in malaria infection. The discovery of Plasmodium asexual and transmission stages in the bone marrow has renewed interest in the tissue as a niche for cellular development of both host and parasite. Despite its importance, bone marrow in malaria infection remains largely unexplored due to the challenge of modeling the complex hematopoietic environment in vitro. Advancements in modeling human erythropoiesis ex-vivo from primary human hematopoietic stem and progenitor cells provide a foothold to study the host-parasite interactions occurring in this understudied site of malaria pathogenesis. This review focuses on current in vitro methods to recapitulate and assess bone marrow erythropoiesis and their potential applications in the malaria field. We summarize recent studies that leveraged ex-vivo erythropoiesis to shed light on gametocyte development in nucleated erythroid stem cells and begin to characterize host cell responses to Plasmodium infection in the hematopoietic niche. Such models hold potential to elucidate mechanisms of disordered erythropoiesis, an underlying contributor to malaria anemia, as well as understand the biological determinants of parasite sexual conversion. This review compares the advantages and limitations of the ex-vivo erythropoiesis approach with those of in vivo human and animal studies of the hematopoietic niche in malaria infection. We highlight the need for studies that apply single cell analyses to this complex system and incorporate physical and cellular components of the bone marrow that may influence erythropoiesis and parasite development.

Cryptic erythrocytic infections in Plasmodium vivax, another challenge to its elimination.

Human malaria caused by Plasmodium vivax infection (vivax malaria) is a major global health issue. It is the most geographically widespread form of the disease, accounting for 7 million annual clinical cases, the majority of cases in America and Asia and an estimation of over 2.5 billion people living under risk of infection. The general perception towards vivax malaria has shifted recently, following a series of reports, from being viewed as a benign infection to the recognition of its potential for more severe manifestations including fatal cases. However, the underlying pathogenic mechanisms of vivax malaria remain largely unresolved. Asymptomatic carriers of malaria parasites are a major challenge for malaria elimination. In the case of P. vivax, it has been widely accepted that the only source of cryptic parasites is hypnozoite dormant stages. Here, we will review new evidence indicating that cryptic erythrocytic niches outside the liver, in particular in the spleen and bone marrow, can represent a major source of asymptomatic infections. The origin of such parasites is being controversial and many key gaps in the knowledge of such infections remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Last, we will glimpse into the role of reticulocyte-derived exosomes, extracellular vesicles of endocytic origin, as intercellular communicators likely involved in the formation of such cryptic erythrocytic 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.

Plasmodium in the bone marrow: case series from a hospital in Pakistan, 2007-2015.

BACKGROUND: Malaria is a life-threatening, multisystem disease caused by the plasmodial parasite with a global incidence of approximately 229 million annually. The parasites are known to have unique and crucial interactions with various body tissues during its life cycle, notably the liver, spleen, and recent work has shown the bone marrow to be a reservoir of infection. METHODS: This study is a case series of patients in whom examination of bone marrow revealed malarial parasites. A retrospective record review of 35 parasite-positive bone marrow specimens examined at Aga Khan University Hospital (AKUH), Karachi, Pakistan, over the years 2007 to 2015 was conducted. Bone marrow aspirates were collected as per International Council for Standardization in Haematology (ICSH) guidelines. RESULTS: The median age of patients was 22 years (range 1-75), and 60 % (n = 21) were male. 22 patients had evidence of Plasmodium falciparum, 12 had evidence of Plasmodium vivax and 1 patient had a mixed infection. Gametocytes and trophozoites were the most common stages identified on both peripheral blood and bone marrow examinations. Indications for bone marrow examination included fever of unknown origin and the workup of cytopenias and malignancies. CONCLUSIONS: The incidental finding of Plasmodium in samples of bone marrow suggests the reticuloendothelial system may be regularly harbour these parasites, be the infection acute or chronic in character.

Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study.

BACKGROUND: Plasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life. METHODS AND FINDINGS: To identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI -15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI -0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI -15.9% to +25.0%], P. falciparum: +3.2% [CI -3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI -8.7% to +21.1%], P. falciparum: -1.4% [CI -4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantly greater in participants with P. vivax infection compared to P. falciparum. The main limitations of this study are the small sample size and the inability of this tracer to differentiate between host and parasite metabolic activity. CONCLUSIONS: PET/MRI indicated greater splenic tropism and metabolic activity in early P. vivax infection compared to P. falciparum, supporting the hypothesis of splenic accumulation of P. vivax very early in infection. The absence of uptake in the bone marrow and liver suggests that, at least in early infection, these tissues do not harbor a large parasite biomass or do not provoke a prominent metabolic response. PET/MRI is a safe and noninvasive method to evaluate infection-associated organ changes in morphology and glucose metabolism.

Antileishmanial efficacy and tolerability of combined treatment with non-ionic surfactant vesicle formulations of sodium stibogluconate and paromomycin in dogs.

Infection with Leishmania infantum causes the disease visceral leishmaniasis (VL), which is a serious clinical and veterinary problem. The drugs used to treat canine leishmaniasis (CanL) do not cause complete parasite clearance; they can be toxic, and emerging drug resistance in parasite populations limits their clinical utility. Therefore, in this study we have evaluated the toxicity and efficacy of joint treatment with a 1:1 mixture of sodium stibogluconate-NIV (SSG-NIV, 10 mg Sbv/day) and paromomycin-NIV (PMM-NIV, 10 mg PMM/kg/day), given intravenously daily for seven days from day 270 post-infection, to nine-month-old female beagle dogs (n = 6) experimentally infected with Leishmania infantum. Treatment significantly improved the clinical symptoms of VL infection in all the treated dogs, reduced parasite burdens in lymph nodes and bone marrow, and all symptomatic treated dogs, were asymptomatic at 90 days post-treatment. Treatment was associated with a progressive and significant decrease in specific IgG anti-Leishmania antibodies using parasite soluble antigen (p < 0.01) or rK39 (p < 0.01) as the target antigen. In addition, all dogs were classified as parasite negative based on Leishmania nested PCR and quantitative real time PCR tests and as well as an inability to culture of promastigote parasites from lymph nodes and bone marrow tissue samples taken at day 90 post-treatment. However, treatment did not cure the dogs as parasites were detected at 10 months post-treatment, indicating that a different dosing regimen is required to cause long term cure or prevent relapse.

Resistance to Experimental Visceral Leishmaniasis in Mice Infected With Leishmania infantum Requires Batf3.

Unveiling the protective immune response to visceral leishmaniasis is critical for a rational design of vaccines aimed at reducing the impact caused by this fatal, if left untreated, vector-borne disease. In this study we sought to determine the role of the basic leucine zipper transcription factor ATF-like 3 (Batf3) in the evolution of infection with Leishmania infantum, the causative agent of human visceral leishmaniasis in the Mediterranean Basin and Latin America. For that, Batf3-deficient mice in C57BL/6 background were infected with an L. infantum strain expressing the luciferase gene. Bioluminescent imaging, as well as in vitro parasite titration, demonstrated that Batf3-deficient mice were unable to control hepatic parasitosis as opposed to wild-type C57BL/6 mice. The impaired microbicide capacities of L. infantum-infected macrophages from Batf3-deficient mice mainly correlated with a reduction of parasite-specific IFN-γ production. Our results reinforce the implication of Batf3 in the generation of type 1 immunity against infectious diseases.

Case Report: Confirmation by Metagenomic Sequencing of Visceral Leishmaniasis in an Immunosuppressed Returned Traveler.

There has been increased interest in using metagenomic next-generation sequencing as an unbiased approach for diagnosing infectious diseases. We describe a 61-year-old man on fingolimod therapy for multiple sclerosis with an extensive travel history who presented with 7 months of fevers, night sweats, and weight loss. Peripheral blood tests showed pancytopenia and abnormal acute phase reactants. A bone marrow aspirate showed the presence of numerous intracellular and extracellular amastigotes consistent with visceral leishmaniasis (VL). Metagenomic sequencing of the bone marrow aspirate confirmed Leishmania infantum, a species widely reported in the Mediterranean region. This correlated with acquisition of VL infection during the patient's most recent epidemiological exposure in southern Italy 12 months prior. This case demonstrates the potential application of metagenomic sequencing for identification and speciation of Leishmania in cases of VL; however, further assessment is required using other more readily obtained clinical samples such as blood.

Case Report: Autochthonous Case of Human Visceral Leishmaniasis in the West Bank, Palestine.

Human visceral leishmaniasis (HVL) is a parasitic disease infecting children in the Mediterranean region. Here, we portray a case of a 2-year-old child with an epidemiological description of the situation surrounding the case. The patient was suffering from recurrent fever, weakness, and abdominal discomfort associated with loss of appetite. Routine blood investigations showed pancytopenia, whereas examination revealed hepatomegaly. A diagnosis of HVL was made by demonstrating amastigotes in a Giemsa-stained smear from a bone marrow aspirate followed by genotyping by PCR and sequencing. In conclusion, early detection of VL infection followed by appropriate treatment protocols is essential to saving the patient.

A new Leishmania hypothetical protein can be used for accurate serodiagnosis of canine and human visceral leishmaniasis and as a potential prognostic marker for human disease.

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.

Pancytopenia: a rare cause for a common presentation.

Filariasis is a major public health hazard in tropical and subtropical countries and is endemic among the Indian population. Asymptomatic microfilariaemia, elephantiasis, acute adenolymphangitis, hydrocoele and chronic lymphatic disease are its common manifestations. We hereby report a case of microfilaria found in the bone marrow presenting as pancytopenia. There was no classical feature of elephantiasis or lymphoedema present.

Plasmodium falciparum sexual parasites develop in human erythroblasts and affect erythropoiesis.

Plasmodium falciparum gametocytes, the sexual stage responsible for malaria parasite transmission from humans to mosquitoes, are key targets for malaria elimination. Immature gametocytes develop in the human bone marrow parenchyma, where they accumulate around erythroblastic islands. Notably though, the interactions between gametocytes and this hematopoietic niche have not been investigated. Here, we identify late erythroblasts as a new host cell for P falciparum sexual stages and show that gametocytes can fully develop inside these nucleated cells in vitro and in vivo, leading to infectious mature gametocytes within reticulocytes. Strikingly, we found that infection of erythroblasts by gametocytes and parasite-derived extracellular vesicles delay erythroid differentiation, thereby allowing gametocyte maturation to coincide with the release of their host cell from the bone marrow. Taken together, our findings highlight new mechanisms that are pivotal for the maintenance of immature gametocytes in the bone marrow and provide further insights on how Plasmodium parasites interfere with erythropoiesis and contribute to anemia in malaria patients.