Anatomical Vascular Differences and Leishmania-Induced Vascular Morphological Changes Are Associated with a High Parasite Load in the Skin of Dogs Infected with Leishmania infantum.

Canine visceral leishmaniasis (CVL), caused by the protozoan Leishmania infantum, affects several organs, including the skin. Dogs are considered the major domestic reservoir animals for leishmaniasis, and through their highly parasitized skin, they can serve as a source of infection for sandfly vectors. Therefore, studies of the skin parasite-host relationship can contribute to the understanding of the infectious dissemination processes of parasites in the dermis and help to identify targets for diagnosis and treatment. Thus, the aim of this study was to evaluate the association of anatomical vascular differences and Leishmania-induced vascular morphological changes with clinical signs and parasite load by analyzing the ear and abdominal skin from dogs naturally infected with L. infantum. Paired samples of ear and abdominal skin from L. infantum-positive dogs (n = 26) were submitted for histological and immunohistochemistry analyses. The ear skin samples showed a more intense and more diffusely distributed granulomatous inflammatory reaction, a higher number and larger diameter of blood vessels, increased parasite load, higher expression of VEGF+ (vascular endothelial growth factor) and MAC 387+ (calprotectin) recently infiltrating cells, and more intense collagen disruption compared to the abdominal skin samples. Intracellular amastigotes were observed in blood vessels and inside endothelial cells and were diffusely distributed throughout the dermis in the ear skin samples. The NOS2/MAC387+ cell ratio was lower in the ear skin samples than in those of the abdomen, suggesting that in the ear dermis, the inflammatory infiltrate was less capable of producing NO and thereby control the parasite load. Together, these findings indicate how parasites and immune cells are distributed in the skin and suggest an important role for dermal vascularization in cellular influx and thereby in parasite dissemination through the skin of naturally infected dogs.

Lectin-based carbohydrate profile of megakaryocytes in murine fetal liver during development.

Hematopoiesis is the process by which blood cells are generated. During embryonic development, these cells migrate through different organs until they reach the bone marrow, their definitive place in adulthood. Around E10.5, the fetal liver starts budding from the gut, where first hematopoietic cells arrive and expand. Hematopoietic cell migration occurs through cytokine stimulation, receptor expression, and glycosylation patterns on the cell surface. In addition, carbohydrates can modulate different cell activation states. For this reason, we aimed to characterize and quantify fetal megakaryocytic cells in mouse fetal liver according to their glycan residues at different gestational ages through lectins. Mouse fetuses between E11.5 and E18.5 were formalin-fixed and, paraffin-embedded, for immunofluorescence analysis using confocal microscopy. The results showed that the following sugar residues were expressed in proliferating and differentiating megakaryocytes in the fetal liver at different gestational ages: α-mannose, α-glucose, galactose, GlcNAc, and two types of complex oligosaccharides. Megakaryocytes also showed three proliferation waves during liver development at E12.5, E14.5, and E18.5. Additionally, the lectins that exhibited high and specific pattern intensities at liver capsules and vessels were shown to be a less time-consuming and robust alternative alternative to conventional antibodies for displaying liver structures such as capsules and vessels, as well as for megakaryocyte differentiation in the fetal liver.

The hepatic extramedullary hematopoiesis during experimental murine Schistosomiasis mansoni.

Many years ago, our research group has demonstrated extramedullary hematopoiesis in the peripheral zone of murine hepatic schistosomal granulomas. In the present study, we revisit this phenomenon using new technical and conceptual approaches. Therefore, newborn mice were percutaneously infected by Schistosoma mansoni cercariae and euthanized between 35- and 60-days post infection. Liver samples were submitted to histopathology and immunohistochemical analyses. Cells under mitosis and/or expressing Ki67 demonstrated the proliferation of hematopoietic cells both around the parasite's eggs trapped in the liver and around hepatic vessels. After 50 days post infection, proliferating cells at different levels on differentiation were located preferentially in the peripheral zone of the granulomas, around the vessels and inside the sinusoids. The presence of acidic and sulfated glycoconjugates, reticular fibers and the absence of fibronectin characterized the microenvironment for attraction and maintenance of hematopoiesis. Some neutrophils secreted MMP9 from the earliest points of infection, indicating degradation of the extracellular matrix in regions of histolysis and a possible chemoattraction of hematopoietic stem cells to the liver. Fall-3+ cells and Sca-1+ cells indicated that early hematopoietic progenitors could be mobilized to the liver. Groups of vWF+ megakaryocytes suggest chemoattraction of these cells and/or migration, proliferation, and differentiation of very immature progenitors to this organ. The increase of blood vessels and extramedullary hematopoiesis in this environment, where markers of immature hematopoietic and endothelial cells have been identified, points to the possibility of the presence of progenitors for endothelial and hematopoietic cells in the liver during the infection. There is also the possibility of concomitant migration of more differentiated hematopoietic progenitors, that proliferate and differentiate in the liver, and the occurrence of angiogenesis caused by inflammation or release of ovular antigens that stimulate the activation and proliferation of endothelial cells. Altogether, these data increase knowledge about a murine model that is of interest for investigating the pathology of the schistosomiasis and also the dynamics of hematopoiesis.

Host cholesterol influences the activity of sterol biosynthesis inhibitors in Leishmania amazonensis.

A significant percentage of exogenous cholesterol was found in promastigotes and amastigotes of all studied species of Leishmania, suggesting a biological role for this molecule. Previous studies have shown that promastigotes of Leishmania uptake more low-density lipoprotein (LDL) particles under pharmacological pressure and are more susceptible to ergosterol inhibition in the absence of exogenous sources of cholesterol. This work shows that the host's LDL is available to intracellular amastigotes and that the absence of exogenous cholesterol enhances the potency of sterol biosynthesis inhibitors in infected macrophages. A complete understanding of cholesterol transport to the parasitophorous vacuole can guide the development of a new drug class to be used in combination with sterol biosynthesis inhibitors for the treatment of leishmaniases.

Oral Trypanosoma cruzi Acute Infection in Mice Targets Primary Lymphoid Organs and Triggers Extramedullary Hematopoiesis.

During the acute phase of Chagas disease, Trypanosoma cruzi circulation through the bloodstream leads to high tissue parasitism in the host. In primary lymphoid organs, progenitor cell reduction paralleled transient immunosuppression. Herein we showed that acute oral infection in mice promotes diffuse parasitism in bone marrow cells at 14 and 21 days post-infection (dpi), with perivascular regions, intravascular regions, and regions near the bone being target sites of parasite replication. Phenotypic analysis of hematopoietic differentiation in the bone marrow of infected mice showed that the cell number in the tissue is decreased (lineage-negative and lineage-positive cells). Interestingly, analysis of hematopoietic branching points showed that hematopoietic stem and progenitor cells (HSPCs) were significantly increased at 14 dpi. In addition, the pool of progenitors with stem plasticity (HSC-MPP3), as well as multipotent progenitors (MPPs) such as MPP4, also showed this pattern of increase. In contrast, subsequent progenitors that arise from MPPs, such as common lymphoid progenitors (CLPs), lymphoid-primed MPPs (LMPPs), and myeloid progenitors, were not enhanced; conversely, all presented numeric decline. Annexin V staining revealed that cell death increase in the initial hematopoietic branching point probably is not linked to CLPs and that myeloid progenitors decreased at 14 and 21 dpi. In parallel, our investigation provided clues that myeloid progenitor decrease could be associated with an atypical expression of Sca-1 in this population leading to a remarkable increase on LSK-like cells at 14 dpi within the HSPC compartment. Finally, these results led us to investigate HSPC presence in the spleen as a phenomenon triggered during emergency hematopoiesis due to mobilization or expansion of these cells in extramedullary sites. Splenocyte analysis showed a progressive increase in HSPCs between 14 and 21 dpi. Altogether, our study shows that the bone marrow is a target tissue in T. cruzi orally infected mice, leading to a hematopoietic disturbance with LSK-like cell bias accounting on HSPCs possibly affecting myeloid progenitor numbers. The LMPP and CLP reduction converges with defective thymocyte development. Lastly, it is tempting to speculate that the extramedullary hematopoiesis seen in the spleen is a mechanism involved in the hematological maintenance reported during the acute phase of oral T. cruzi infection.

Should COVID-19 be branded to viral thrombotic fever?

Coronaviruses can cause a diverse array of clinical manifestations, from fever with symptoms of the common cold to highly lethal severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS). SARS-CoV-2, the coronavirus discovered in Hubei province, China, at the end of 2019, became known worldwide for causing coronavirus disease 2019 (COVID-19). Over one year's time period, the scientific community has produced a large bulk of knowledge about this disease and countless reports about its immune-pathological aspects. This knowledge, including data obtained in postmortem studies, points unequivocally to a hypercoagulability state. However, the name COVID-19 tells us very little about the true meaning of the disease. Our proposal is more comprehensive; it intends to frame COVID-19 in more clinical terminology, making an analogy to viral haemorrhagic fever (VHF). Thus, we found irrefutable evidence in the current literature that COVID-19 is the first viral disease that can be branded as a viral thrombotic fever. This manuscript points out that SARS-CoV-2 goes far beyond pneumonia or SARS. COVID-19 infections promote remarkable interactions among the endothelium, coagulation, and immune response, building up a background capable of promoting a "thrombotic storm," much more than a "cytokine storm." The importance of a viral protease called main protease (Mpro) is highlighted as a critical component for its replication in the host cell. A deeper analysis of this protease and its importance on the coagulation system is also discussed for the first time, mainly because of its similarity with the thrombin and factor Xa molecules, as recently pointed out by structural comparison crystallographic structures.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification.

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain-Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

Amentoflavone as an Ally in the Treatment of Cutaneous Leishmaniasis: Analysis of Its Antioxidant/Prooxidant Mechanisms.

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reported in vitro, the mechanisms involved in the parasite death, as well as its action in vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC50 2.3 ± 0.93 µM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro. The increase of ROS in vitro, associated with the reduction of NO and iNOS expression in vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.

Host Genetics Background Influence in the Intragastric Trypanosoma cruzi Infection.

Background: Considering the complexity of the factors involved in the immunopathology of Chagas disease, which influence the Chagas' disease pathogenesis, anti-T. cruzi immune response, and chemotherapy outcome, further studies are needed to improve our understanding about these relationships. On this way, in this article we analyzed the host genetic influence on hematological, histopathological and immunological aspects after T. cruzi infection. Methods: BALB/c and A mice were intragastrically infected with T. cruzi SC2005 strain, isolated from a patient of an outbreak of Chagas disease. Parameters such as parasite load, survival rates, cytokines production, macrophages, T and B cell frequencies, and histopathology analysis were carried out. Results: BALB/c mice presented higher parasitemia and mortality rates than A mice. Both mouse lineages exhibited hematological alterations suggestive of microcytic hypochromic anemia and histopathological alterations in stomach, heart and liver. The increase of CD8+ T cells, in heart, liver and blood, and the increase of CD19+ B cells, in liver, associated with a high level of proinflammatory cytokines (IL-6, TNF-α, IFN-γ), confer a resistance profile to the host. Although BALB/c animals exhibited the same findings observed in A mice, the response to infection occurred later, after a considerable parasitemia increase. By developing an early response to the infection, A mice were found to be less susceptible to T. cruzi SC2005 infection. Conclusions: Host genetics background shaping the response to infection. The early development of a cytotoxic cellular response profile with the production of proinflammatory cytokines is important to lead a less severe manifestation of Chagas disease.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Antiparasitic and anti-inflammatory activities of ß-lapachone-derived naphthoimidazoles in experimental acute Trypanosoma cruzi infection.

BACKGROUND: Chagas disease, which is caused by the protozoan Trypanosoma cruzi, is endemic to Latin America and mainly affects low-income populations. Chemotherapy is based on two nitrocompounds, but their reduced efficacy encourages the continuous search for alternative drugs. Our group has characterised the trypanocidal effect of naphthoquinones and their derivatives, with naphthoimidazoles derived from ß-lapachone (N1, N2 and N3) being the most active in vitro. OBJECTIVES: In the present work, the effects of N1, N2 and N3 on acutely infected mice were investigated. METHODS: in vivo activity of the compounds was assessed by parasitological, biochemical, histopathological, immunophenotypical, electrocardiographic (ECG) and behavioral analyses. FINDINGS: Naphthoimidazoles led to a decrease in parasitaemia (8 dpi) by reducing the number of bloodstream trypomastigotes by 25-50% but not by reducing mortality. N1 protected mice from heart injury (15 dpi) by decreasing inflammation. Bradycardia was also partially reversed after treatment with N1 and N2. Furthermore, the three compounds did not reverse hepatic and renal lesions or promote the improvement of other evaluated parameters. MAIN CONCLUSION: N1 showed moderate trypanocidal and promising immunomodulatory activities, and its use in combination with benznidazole and/or anti-arrhythmic drugs as well as the efficacy of its alternative formulations must be investigated in the near future.

Howler monkeys are the reservoir of malarial parasites causing zoonotic infections in the Atlantic forest of Rio de Janeiro.

BACKGROUND: Although malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015-2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts. METHODOLOGY: A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques. PRINCIPAL FINDINGS: In total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms-the only available genetic markers that differentiate P. simium from P. vivax-were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum. CONCLUSIONS/SIGNIFICANCE: Our data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics.

Generation of Yellow Fever virus vaccine in skeletal muscle cells of chicken embryos.

BACKGROUND: The Yellow Fever (YF) vaccine is produced by the inoculation of embryonated chicken eggs with YF17DD virus on the ninth day of development. Full embryos are collected on the twelfth day of development for vaccine formulation. Skeletal muscle tissue is the main site where biosynthesis of viral particles occurs. OBJECTIVES: This study aimed to analyse the experimental infection of skeletal muscle cells of chicken embryos by the 17DD Yellow Fever virus (YFV) in vivo and in vitro. METHODS: Chicken embryos infected with YF17DD virus were analysed by immunofluorescence using confocal and super-resolution microscopes. Primary cultures of skeletal muscle cells of non-infected chicken embryos were evaluated for susceptibility and permissiveness to YF17DD virus using different protocols. This evaluation was performed based on morphological, viral titration, molecular biology, and colorimetric techniques. FINDINGS: The present work phenotypically characterises embryonic chicken skeletal muscle cells as myogenic precursors expressing the Pax7 transcription factor in some cases. We demonstrated that these cells are susceptible to in vitro infection at different multiplicities of infection (MOIs), reproducing the same infection pattern observed in vivo. Furthermore, myogenic precursors and myoblasts are preferred infection targets, but establishment of infection does not depend on the presence of these cells. The peak of viral production occurred at 48 hpi, with decay occurring 72 hpi, when the cytopathic effect can be observed. MAIN CONCLUSIONS: In conclusion, the primary culture of chicken skeletal muscle cells is a good model for studying muscle cells infected with YF17DD virus. This culture system displays satisfactory emulation of the in vitro phenomenon observed, contributing to our understanding of virus infection dynamics and leading to the development of alternative methods of vaccine production.

Evidences of HEV genotype 3 persistence and reactivity in liver parenchyma from experimentally infected cynomolgus monkeys (Macaca fascicularis).

Hepatitis E virus genotype 3 (HEV-3) is an emerging zoonotic pathogen, responsible for sporadic cases of acute hepatitis E worldwide. Primate models have proven to be an essential tool for the study of HEV pathogenesis. Here we describe the outcomes of HEV infection in Macaca fascicularis (cynomolgus) inoculated experimentally with genotype 3. Eight adult cynomolgus macaques were inoculated intravenously with HEV-3 viral particles isolated from swine and human samples. Liver, spleen, duodenum, gallbladder and bile were sequential assessed up to the end-point of this study, 67 days post-inoculation (dpi). Our previously published findings showed that biochemical parameters return gradually to baseline levels at 55 dpi, whereas anti-HEV IgM and HEV RNA become undetectable in the serum and feces of all animals, indicating a non-viremic phase of recovery. Nevertheless, at a later stage during convalescence (67 dpi), the presence of HEV-3 RNA and antigen persist in central organs, even after peripheral viral clearance. Our results show that two cynomolgus inoculated with swine HEV-3 (animals I3 and O1) presented persistence of HEV RNA low titers in liver, gallbladder and bile. At this same stage of infection, HEV antigen (HEV Ag) could be detected in all infected animals, predominantly in non-reactive Kupffer cells (CD68+iNOS-) and sinusoidal lining cells. Simultaneously, CD4+, CD3+CD4+, and CD3+CD8+ immune cells were identified in hepatic sinusoids and small inflammatory clusters of lobular mononuclear cells, at the end-point of this study. Inability of HEV clearance in humans can result in chronic hepatitis, liver cirrhosis, with subsequent liver failure requiring transplantation. The results of our study support the persistence of HEV-3 during convalescence at 67 dpi, with active immune response in NHP. We alert to the inherent risk of viral transmission through liver transplantation, even in the absence of clinical and biochemical signs of acute infection. Thus, besides checking conventional serological markers of HEV infection, we strongly recommend HEV-3 RNA and antigen detection in liver explants as public health measure to prevent donor-recipient transmission and spread of hepatitis E.

Mechanism of hematopoiesis and vasculogenesis in mouse placenta.

The placenta plays a major role in the development of blood cells in the mouse and human embryo. Hematopoiesis and vasculogenesis in placenta are two closely interconnected processes. The mouse model has been widely used to study placental hematopoiesis. During mid-gestation, mouse placenta generate blood cells and support the proliferation, maturation and erythroid differentiation of hematopoietic cells within two major vascular niches in the labyrinth. Here, we review the vasculogenesis in chorioallantoic mouse placenta and the current knowledge on hematopoietic activity, niche and origin in mice placenta.

Increased Plasmodium falciparum Parasitemia in Non-splenectomized Saimiri sciureus Monkeys Treated with Clodronate Liposomes.

A major constraint in the study of Plasmodium falciparum malaria, including vaccine development, lies on the parasite's strict human host specificity and therefore the shortage of animal experimental models able to harbor human plasmodia. The best experimental models are neo-tropical primates of the genus Saimiri and Aotus, but they require splenectomy to reduce innate defenses for achieving high and consistent parasitemias, an important limitation. Clodronate-liposomes (CL) have been successfully used to deplete monocytes/macrophages in several experimental models. We investigated whether a reduction in the numbers of phagocytic cells by CL would improve the development of P. falciparum parasitemia in non-splenectomized Saimiri sciureus monkeys. Depletion of S. sciureus splenocytes after in vitro incubation with CL was quantified using anti-CD14 antibodies and flow cytometry. Non-infected and P. falciparum-infected S. sciureus were injected intravenously twice a week with either CL at either 0.5 or 1 mL (5 mg/mL) or phosphate buffered saline (PBS). Animals were monitored during infection and treated with mefloquine. After treatment and euthanasia, spleen and liver were collected for histological analysis. In vitro CL depleted S. sciureus splenic monocyte/macrophage population in a dose- and time-dependent manner. In vivo, half of P. falciparum-infected S. sciureus treated with CL 0.5 mL, and two-thirds of those treated with CL 1 mL developed high parasitemias requiring mefloquine treatment, whereas all control animals were able to self-control parasitemia without the need for antimalarial treatment. CL-treated infected S. sciureus showed a marked decrease in the degree of splenomegaly despite higher parasitemias, compared to PBS-treated animals. Histological evidence of partial monocyte/macrophage depletion, decreased hemozoin phagocytosis and decreased iron recycling was observed in both the spleen and liver of CL-treated infected S. sciureus. CL is capable of promoting higher parasitemia in P. falciparum-infected S. sciureus, associated with evidence of partial macrophage depletion in the spleen and liver. Macrophage depletion by CL is therefore a practical and viable alternative to surgical splenectomy in this experimental model.

Outbreak of human malaria caused by Plasmodium simium in the Atlantic Forest in Rio de Janeiro: a molecular epidemiological investigation.

BACKGROUND: Malaria was eliminated from southern and southeastern Brazil over 50 years ago. However, an increasing number of autochthonous episodes attributed to Plasmodium vivax have recently been reported from the Atlantic Forest region of Rio de Janeiro state. As the P vivax-like non-human primate malaria parasite species Plasmodium simium is locally enzootic, we performed a molecular epidemiological investigation to determine whether zoonotic malaria transmission is occurring. METHODS: We examined blood samples from patients presenting with signs or symptoms suggestive of malaria as well as from local howler monkeys by microscopy and PCR. Samples were included from individuals if they had a history of travel to or resided in areas within the Rio de Janeiro Atlantic Forest, but not if they had malaria prophylaxis, blood transfusion or tissue or organ transplantation, or had travelled to known malaria endemic areas in the preceding year. Additionally, we developed a molecular assay based on sequencing of the parasite mitochondrial genome to distinguish between P vivax and P simium, and applied this assay to 33 cases from outbreaks that occurred in 2015, and 2016. FINDINGS: A total of 49 autochthonous malaria cases were reported in 2015-16. Most patients were male, with a mean age of 44 years (SD 14·6), and 82% lived in urban areas of Rio de Janeiro state and had visited the Atlantic Forest for leisure or work-related activities. 33 cases were used for mitochondrial DNA sequencing. The assay was successfully performed for 28 samples, and all were shown to be P simium, indicative of zoonotic transmission of this species to human beings in this region. Sequencing of the whole mitochondrial genome of three of these cases showed that P simium is most closely related to P vivax parasites from South America. The malaria outbreaks in this region were caused by P simium, previously considered to be a monkey-specific malaria parasite, related to but distinct from P vivax, and which has never conclusively been shown to infect people before. INTERPRETATION: This unequivocal demonstration of zoonotic transmission, 50 years after the only previous report of P simium in people, leads to the possibility that this parasite has always infected people in this region, but that it has been consistently misdiagnosed as P vivax because of an absence of molecular typing techniques. Thorough screening of local non-human primates and mosquitoes (Anopheline) is required to evaluate the extent of this newly recognised zoonotic threat to public health and malaria elimination in Brazil. FUNDING: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado de Rio de Janeiro, The Brazilian National Council for Scientific and Technological Development (CNPq), JSPS Grant-in-Aid for scientific research, Secretary for Health Surveillance of the Brazilian Ministry of Health, Global Fund, Fundaçao de amparo à pesquisa do estado de Minas Gerais (Fapemig), and PRONEX Program of the CNPq.

Characterisation of the vascular pathology in Sigmodon hispidus (Rodentia: Cricetidae) following experimental infection with Angiostrongylus costaricensis (Nematoda: Metastrongylidae)

BACKGROUND Angiostrongylus costaricensis is a nematode that causes human abdominal angiostrongyliasis, a disease found mainly in Latin American countries and particularly in Brazil and Costa Rica. Its life cycle involves exploitation of both invertebrate and vertebrate hosts. Its natural reservoir is a vertebrate host, the cotton rat Sigmodon hispidus. The adult worms live in the ileo-colic branches of the upper mesenteric artery of S. hispidus, causing periarteritis. However, there is a lack of data on the development of vasculitis in the course of infection. OBJECTIVE To describe the histopathology of vascular lesions in S. hispidus following infection with A. costaricensis. METHODS Twenty-one S. hispidus were euthanised at 30, 50, 90 and 114 days post-infection (dpi), and guts and mesentery (including the cecal artery) were collected. Tissues were fixed in Carson’s Millonig formalin, histologically processed for paraffin embedding, sectioned with a rotary microtome, and stained with hematoxylin-eosin, resorcin-fuchsin, Perls, Sirius Red (pH = 10.2), Congo Red, and Azan trichrome for brightfield microscopy analysis. FINDINGS At 30 and 50 dpi, live eggs and larvae were present inside the vasa vasorum of the cecal artery, leading to eosinophil infiltrates throughout the vessel adventitia and promoting centripetal vasculitis with disruption of the elastic layers. Disease severity increased at 90 and 114 dpi, when many worms had died and the intensity of the vascular lesions was greatest, with intimal alterations, thrombus formation, iron accumulation, and atherosclerosis. CONCLUSION In addition to abdominal angiostrongyliasis, our data suggest that this model could be very useful for autoimune vasculitis and atherosclerosis studies.

Characterisation of the vascular pathology in Sigmodon hispidus (Rodentia: Cricetidae) following experimental infection with Angiostrongylus costaricensis (Nematoda: Metastrongylidae).

BACKGROUND: Angiostrongylus costaricensis is a nematode that causes human abdominal angiostrongyliasis, a disease found mainly in Latin American countries and particularly in Brazil and Costa Rica. Its life cycle involves exploitation of both invertebrate and vertebrate hosts. Its natural reservoir is a vertebrate host, the cotton rat Sigmodon hispidus. The adult worms live in the ileo-colic branches of the upper mesenteric artery of S. hispidus, causing periarteritis. However, there is a lack of data on the development of vasculitis in the course of infection. OBJECTIVE: To describe the histopathology of vascular lesions in S. hispidus following infection with A. costaricensis. METHODS: Twenty-one S. hispidus were euthanised at 30, 50, 90 and 114 days post-infection (dpi), and guts and mesentery (including the cecal artery) were collected. Tissues were fixed in Carson's Millonig formalin, histologically processed for paraffin embedding, sectioned with a rotary microtome, and stained with hematoxylin-eosin, resorcin-fuchsin, Perls, Sirius Red (pH = 10.2), Congo Red, and Azan trichrome for brightfield microscopy analysis. FINDINGS: At 30 and 50 dpi, live eggs and larvae were present inside the vasa vasorum of the cecal artery, leading to eosinophil infiltrates throughout the vessel adventitia and promoting centripetal vasculitis with disruption of the elastic layers. Disease severity increased at 90 and 114 dpi, when many worms had died and the intensity of the vascular lesions was greatest, with intimal alterations, thrombus formation, iron accumulation, and atherosclerosis. CONCLUSION: In addition to abdominal angiostrongyliasis, our data suggest that this model could be very useful for autoimune vasculitis and atherosclerosis studies.

Localization of transient immature hematopoietic cells to two distinct, potential niches in the developing mouse placenta.

Previous studies have shown that human and mouse placentas have hematopoietic potential during mid-gestation. In this investigation, we used histological and immunohistological approaches to visualize hematopoietic cells in mouse placenta between 9.5 and 12.5 days of gestation (gd), identifying their topography and niche. Putative hematopoietic foci were present on 10.5 and 11.5 gd but not 9.5 or 12.5 gd and was restricted to the placental labyrinth. Two major niches each with distinctive hematopoietic cell clusters were present. One type of hematopoietic cell cluster involved the chorioallantoic vasculature and fetal vessels near the chorionic plate. These clusters resembled the hematopoietic stem cells produced by large embryonic arteries such as aorta that persist in postnatal marrow. The other type of hematopoietic cell cluster identified was at the opposite side of labyrinth next to the junctional zone and was composed of erythropoietic foci. Our results suggest that mouse placenta not only produces hematopoietic stem/progenitor cells but also a second wave of primitive erythrocytes that may support a rapid, mid-pregnancy, fetal growth trajectory. Our data also point to a close relationships in the origins of hematopoietic and endothelial cells within placenta.