House dust mite sensitization drives cross-reactive immune responses to homologous helminth proteins.

The establishment of type 2 responses driven by allergic sensitization prior to exposure to helminth parasites has demonstrated how tissue-specific responses can protect against migrating larval stages, but, as a consequence, allow for immune-mediated, parasite/allergy-associated morbidity. In this way, whether helminth cross-reacting allergen-specific antibodies are produced and play a role during the helminth infection, or exacerbate the allergic outcome awaits elucidation. Thus, the main objective of the study was to investigate whether house dust mite (HDM) sensitization triggers allergen-specific antibodies that interact with Ascaris antigens and mediate antibody-dependent deleterious effects on these parasites as well as, to assess the capacity of cross-reactive helminth proteins to trigger allergic inflammation in house dust mite presensitized mice. Here, we show that the sensitization with HDM-extract drives marked IgE and IgG1 antibody responses that cross-react with Ascaris larval antigens. Proteomic analysis of Ascaris larval antigens recognized by these HDM-specific antibodies identified Ascaris tropomyosin and enolase as the 2 major HDM homologues based on high sequence and structural similarity. Moreover, the helminth tropomyosin could drive Type-2 associated pulmonary inflammation similar to HDM following HDM tropomyosin sensitization. The HDM-triggered IgE cross-reactive antibodies were found to be functional as they mediated immediate hypersensitivity responses in skin testing. Finally, we demonstrated that HDM sensitization in either B cells or FcγRIII alpha-chain deficient mice indicated that the allergen driven cell-mediated larval killing is not antibody-dependent. Taken together, our data suggest that aeroallergen sensitization drives helminth reactive antibodies through molecular and structural similarity between HDM and Ascaris antigens suggesting that cross-reactive immune responses help drive allergic inflammation.

Eosinophils mediate SIgA production triggered by TLR2 and TLR4 to control Ascaris suum infection in mice.

Human ascariasis is the most prevalent but neglected tropical disease in the world, affecting approximately 450 million people. The initial phase of Ascaris infection is marked by larval migration from the host's organs, causing mechanical injuries followed by an intense local inflammatory response, which is characterized mainly by neutrophil and eosinophil infiltration, especially in the lungs. During the pulmonary phase, the lesions induced by larval migration and excessive immune responses contribute to tissue remodeling marked by fibrosis and lung dysfunction. In this study, we investigated the relationship between SIgA levels and eosinophils. We found that TLR2 and TLR4 signaling induces eosinophils and promotes SIgA production during Ascaris suum infection. Therefore, control of parasite burden during the pulmonary phase of ascariasis involves eosinophil influx and subsequent promotion of SIgA levels. In addition, we also demonstrate that eosinophils also participate in the process of tissue remodeling after lung injury caused by larval migration, contributing to pulmonary fibrosis and dysfunction in re-infected mice. In conclusion, we postulate that eosinophils play a central role in mediating host innate and humoral immune responses by controlling parasite burden, tissue inflammation, and remodeling during Ascaris suum infection. Furthermore, we suggest that the use of probiotics can induce eosinophilia and SIgA production and contribute to controlling parasite burden and morbidity of helminthic diseases with pulmonary cycles.

Nitric oxide contributes to liver inflammation and parasitic burden control in Ascaris suum infection.

BACKGROUND: Human ascariasis is one of the most prevalent neglected tropical diseases worldwide. The immune response during human ascariasis is characterized by Th2 polarization and a mixed Th2/Th17 response during the pathogenesis of experimental larval ascariasis. Cytokines and other pro-inflammatory mediators, such as nitric oxide (NO), are involved in helminthic infections. However, the role of NO in ascariasis remains unclear. OBJECTIVES: Given the importance of NO in inflammation, we aimed to determine the immunological and histopathological alterations in the livers of C57BL/6 iNOS-/- mice during A. suum infection. METHODS: In this study, parasitic load was evaluated in the livers of wild type C57BL/6 and C57BL/6 iNOS-/- mice infected with A. suum. Histopathological and morphometric analyses and analysis of serum cytokines via Cytometric Bead Array were performed, and the activity of eosinophil peroxidase and myeloperoxidase of neutrophils in the tissues were determined. RESULTS: The results showed that NO is important for controlling parasitic load during infection by A. suum. C57BL/6iNOS-/- mice showed reduced inflammatory processes and less tissue damage during liver larval migration of A. suum, which is associated with a reduction in serum levels of pro-inflammatory cytokines. CONCLUSIONS: We demonstrated that NO is a crucial inflammatory molecule during Ascaris sp. infection and controls the establishment of the parasite and the development of the host immune response in the liver.

Regulatory monocytes in helminth infections: insights from the modulation during human hookworm infection.

BACKGROUND: While the macrophage polarization is well characterized in helminth infections, the natural heterogeneity of monocytes with multiple cell phenotypes might influence the outcome of neglected diseases, such hookworm infection. Here, we report the profile of monocytes in human hookworm infections as a model to study the regulatory subpopulation of monocytes in helminth infections. METHODS: Blood samples were collected from 19 Necator americanus-infected individuals and 13 healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated, and immunophenotyping was conducted by flow cytometry. The expressions of genes encoding human nitric oxide synthase (iNOS), interleukin 4 (IL-4), arginase-1 (Arg-1) and glyceraldehyde 3-phosphate dehydrogenase were quantified by qPCR. Plasma levels of IL-4 were determined by sandwich ELISA. Unpaired t-tests or Mann-Whitney tests were used depending on the data distribution. RESULTS: Hookworm infected individuals (HWI) showed a significant increase in the number of monocytes/mm3 (555.2 ± 191.0) compared to that of the non-infected (NI) individuals (120.4 ± 44.7) (p < 0.0001). While the frequencies of CD14+IL-10+ and CD14+IL-12+ cells were significantly reduced in the HWI compared to NI group (p = 0.0289 and p < 0.0001, respectively), the ratio between IL-10/IL-12 producing monocytes was significantly elevated in HWI (p = 0.0004), indicating the potential regulatory activity of these cells. Measurement of IL-4 levels and gene expression of IL-4 and Arg-1 (highly expressed in alternatively activated macrophages) revealed no significant differences between the NI and HWI groups. Interestingly, individuals from the HWI group had higher expression of the iNOS gene (associated with a regulatory profile) (20.27 ± 2.97) compared to the NI group (11.28 ± 1.18, p = 0.0409). Finally, individuals from the HWI group had a significantly higher frequency of CD206+CD23+IL-10+ (7.57 ± 1.96) cells compared to individuals from the NI group (0.35 ± 0.09) (p < 0.001), suggesting that activated monocytes are a potential source of regulatory cytokines during hookworm infection. CONCLUSIONS: Natural hookworm infection induces a high frequency of circulating monocytes that present a regulatory profile and promote the downmodulation of the proinflammatory response, which may contribute to prolonged survival of the parasite in the host.

Immunoregulatory mechanisms in Chagas disease: modulation of apoptosis in T-cell mediated immune responses.

BACKGROUND: Chronic Chagas disease presents different clinical manifestations ranging from asymptomatic (namely indeterminate) to severe cardiac and/or digestive. Previous results have shown that the immune response plays an important role, although no all mechanisms are understood. Immunoregulatory mechanisms such as apoptosis are important for the control of Chagas disease, possibly affecting the morbidity in chronic clinical forms. Apoptosis has been suggested to be an important mechanism of cellular response during T. cruzi infection. We aimed to further understand the putative role of apoptosis in Chagas disease and its relation to the clinical forms of the disease. METHODS: Apoptosis of lymphocytes, under antigenic stimuli (soluble T. cruzi antigens - TcAg) where compared to that of non-stimulated cells. Apoptosis was evaluated using the expression of annexin and caspase 3(+) by T cells and the percentage of cells positive evaluated by flow cytometry. In addition activation and T cell markers were used for the identification of TCD4(+) and TCD8(+) subpopulations. The presence of intracellular and plasma cytokines were also evaluated. Analysis of the activation status of the peripheral blood cells showed that patients with Chagas disease presented higher levels of activation determined by the expression of activation markers, after TcAg stimulation. PCR array were used to evaluate the contribution of this mechanism in specific cell populations from patients with different clinical forms of human Chagas disease. RESULTS: Our results showed a reduced proliferative response associated a high expression of T CD4(+)CD62L(-) cells in CARD patients when compared with IND group and NI individuals. We also observed that both groups of patients presented a significant increase of CD4(+) and CD8(+) T cell subsets in undergoing apoptosis after in vitro stimulation with T. cruzi antigens. In CARD patients, both CD4(+) and CD8(+) T cells expressing TNF-α were highly susceptible to undergo apoptosis after in vitro stimulation. Interestingly, the in vitro TcAg stimulation increased considerably the expression of cell death TNF/TNFR superfamily and Caspase family receptors genes in CARD patients. CONCLUSIONS: Taken together, our results suggest that apoptosis may be an important mechanism for the control of morbidity in T. cruzi infection by modulating the expression of apoptosis genes, the cytokine environment and/or killing of effector cells.

Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis.

OBJECTIVES: To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. METHODS: Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. RESULTS: Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. CONCLUSIONS: Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens.

Phenotypic profiling of CD8(+) T cells during Plasmodium vivax blood-stage infection.

BACKGROUND: For a long time, the role of CD8(+) T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8(+) T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8(+) T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8(+) T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8(+) T cells subsets from patients with uncomplicated symptomatic P. vivax malaria. METHODS: Blood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann-Whitney test was used depending on the data distribution. RESULTS: P. vivax-infected subjects had lower percentages and absolute numbers of CD8(+)CD45RA(+) and CD8(+)CD45RO(+) T cells when compared to uninfected individuals (p ≤ 0.0002). A significantly lower absolute number of circulating CD8(+)CD45(+)CCR7(+) cells (p = 0.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ(+) (p = 0.001), TNF-α(+) (p < 0.0001) and IL-10(+) (p < 0.0001) CD8(+) T cells. Despite the reduction in the number of CD8(+) memory T cells producing IFN-γ (p < 0.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8(+)TNF-α(+) (p = 0.016) and CD8(+)IL-10(+) (p = 0.004) cells. Positive correlations were observed between absolute numbers of CD8(+)IL-10(+) and numbers of CD8(+)IFN-γ(+) (p < 0.001) and CD8(+)TNF-α(+) T cells (p ≤ 0.0001). Finally, an increase in the plasma levels of TNF-α (p = 0.017) and IL-10 (p = 0.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals. CONCLUSIONS: P. vivax infection reduces the numbers of different subsets of CD8(+) T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8(+) memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ.

Exploring the antiviral potential of justicidin B and four glycosylated lignans from Phyllanthus brasiliensis against Zika virus: A promising pharmacological approach.

BACKGROUND: Zika virus (ZIKV) is an emerging arbovirus that in recent years has been associated with cases of severe neurological disorders, such as microcephaly in newborns and Guillain-Barré syndrome in adults. As there is no vaccine or treatment, the search for new therapeutic targets is of great relevance. In this sense, plants are extremely rich sources for the discovery of new bioactive compounds and the species Phyllanthus brasiliensis (native to the Amazon region) remains unexplored. PURPOSE: To investigate the potential antiviral activity of compounds isolated from P. brasiliensis leaves against ZIKV infection. METHODS: In vitro antiviral assays were performed with justicidin B (a lignan) and four glycosylated lignans (tuberculatin, phyllanthostatin A, 5-O-ß-d-glucopyranosyljusticidin B, and cleistanthin B) against ZIKV in Vero cells. MTT colorimetric assay was used to assess cell viability and plaque forming unit assay to quantify viral load. In addition, for justicidin B, tests were performed to investigate the mechanism of action (virucidal, adsorption, internalization, post-infection). RESULTS: The isolated compounds showed potent anti-ZIKV activities and high selectivity indexes. Moreover, justicidin B, tuberculatin, and phyllanthostatin A completely reduced the viral load in at least one of the concentrations evaluated. Among them, justicidin B stood out as the main active, and further investigation revealed that justicidin B exerts its antiviral effect during post-infection stages, resulting in a remarkable 99.9 % reduction in viral load when treatment was initiated 24 h after infection. CONCLUSION: Our findings suggest that justicidin B inhibits endosomal internalization and acidification, effectively interrupting the viral multiplication cycle. Therefore, the findings shed light on the promising potential of isolated compounds isolated from P. brasiliensis, especially justicidin B, which could contribute to the drug development and treatments for Zika virus infections.

BACH1 promotes tissue necrosis and Mycobacterium tuberculosis susceptibility.

Oxidative stress triggers ferroptosis, a form of cellular necrosis characterized by iron-dependent lipid peroxidation, and has been implicated in Mycobacterium tuberculosis (Mtb) pathogenesis. We investigated whether Bach1, a transcription factor that represses multiple antioxidant genes, regulates host resistance to Mtb. We found that BACH1 expression is associated clinically with active pulmonary tuberculosis. Bach1 deletion in Mtb-infected mice increased glutathione levels and Gpx4 expression that inhibit lipid peroxidation. Bach1-/- macrophages exhibited increased resistance to Mtb-induced cell death, while Mtb-infected Bach1-deficient mice displayed reduced bacterial loads, pulmonary necrosis and lipid peroxidation concurrent with increased survival. Single-cell RNA-seq analysis of lungs from Mtb-infected Bach1-/- mice revealed an enrichment of genes associated with ferroptosis suppression. Bach1 depletion in Mtb-infected B6.Sst1S mice that display human-like necrotic lung pathology also markedly reduced necrosis and increased host resistance. These findings identify Bach1 as a key regulator of cellular and tissue necrosis and host resistance in Mtb infection.

Prednisolone and cyclosporine A: effects on an experimental model of ancylostomiasis.

Corticosteroids and cyclosporine A (CsA) are important clinical immunosuppressive drugs used in the maintenance of organ transplants and in suppressing undesired autoimmune or allergic immune responses. To study the effect of CsA and prednisolone on the course of an Ancylostoma ceylanicum infection, hamsters were treated with commercially available prednisolone or CsA. For both drugs, half the recommended dose was sufficient to inhibit the proliferation of more than 70% of hamster lymph node cells. There was no difference in the recovery of adult worms; however, animals treated with prednisolone presented with low egg counts in the feces. Infection with A. ceylanicum resulted in an increase in specific antibodies against adult worm antigens, but hamsters treated with either drug presented with lower IgG titers. We observed that A. ceylanicum infection caused peripheral cellular immune suppression, which is characterized by a reduction in the total white cell count, neutropenia and lymphopenia. We also observed a lymphoplasmacytic pattern and few eosinophils in the mucosal inflammatory infiltrate for all the animals. The animals treated with prednisolone showed changes in the architecture of the intestine, including the loss of the mucosa, intense congestion and inflammation. In spleen, we observed hyperplasia of white pulp in all infected animals; in addition, there was a loss of tissue architecture in the animals treated with prednisolone. In conclusion, this work shows that an A. ceylanicum infection leads to acute peripheral cellular immune suppression in hamsters but not humoral immune suppression and that CsA treatment does not interfere with the process of infection. However, prednisolone treatment causes intestinal injury, what could hamper the parasite attachment to the intestinal wall, and as a result affects copulation and, consequently, decreases the number of eggs eliminated in the feces. Moreover, the possibility that the drug can also be exerting an effect on female fertility should be considered.

New naphthoquinones and an alkaloid with in vitro activity against Toxoplasma gondii RH and EGS strains.

The efficacy of three amino-terpenyl naphthoquinones and the alkaloid liriodenine were examined against tachyzoites and tissues cysts of the RH and EGS strains, respectively. Monolayers of 2C4 fibroblasts infected with tachyzoites of the RH strain were incubated with different concentrations of the compounds for 48 h. Specifically, 7-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-5), 6-(4-methyl-3-pentenyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-6), 6-(4-methylpentyl)-2-pyrrolidine-[1,4]-naphthoquinone (QUI-11), and 8 h-benzo[g]-1,3-benzodioxolo[6,5,4-de]quinolin-8-one,9Cl-1,2-methylene dioxiaporfina (liriodenine) inhibited intracellular replication of T. gondii. The IC(50) values obtained for compounds QUI-5 and QUI-6 were 69.35 and 172.81 µM (i.e., 21.4 and 53.4 µg/mL), respectively. The naphthoquinone QUI-11 and liriodenine significantly inhibited intracellular replication of T. gondii. The IC(50) values obtained with these experiments were 0.32 and 0.07 µM (i.e., 0.1 and 0.02 µg/mL), respectively. Compounds QUI-5, QUI-6, QUI-11 and liriodenine demonstrated lower toxicity for 2C4 fibroblasts compared to atovaquone. In addition, cysts isolated from the brains of mice chronically infected with the EGS strain were exposed to the compounds. Infectivity of the cysts after incubation with the compounds was assessed by infection of mice. The data obtained showed that in vitro incubation with QUI-6, QUI-11 and liriodenine inhibited the infectivity of the bradyzoites. This activity was time- and concentration-dependent.

Seric chemokines and chemokine receptors in eosinophils during acute human schistosomiasis mansoni.

The recruitment of circulating eosinophils by chemokines and chemokine receptors plays an important role in the inflammation process in acute human schistosomiasis. Our main focus has been on the plasma chemokines (CXCL8/CCL2/CCL3/CCL24) and chemokine receptors (CCR2/CCR3/CCR5/CXCR1/CXCR2/CXCR3/CXCR4) expressed by circulating eosinophils from acute Schistosoma mansoni infected patients (ACT). Our studies compared ACT patients and healthy individuals as a control group. Our major findings demonstrated a plethora of chemokine secretion with significantly increased secretion of all chemokines analysed in the ACT group. Although no differences were detected for beta-chemokine receptors (CCR2, CCR3 and CCR5) or alpha-chemokine receptors (CXCR3 and CXCR4), a significantly lower frequency of CXCR1+ and CXCR2+ eosinophils in the ACT group was observed. The association between chemokines and their chemokine receptors revealed that acutely infected schistosome patients displaying decreased plasma levels of CCL24 are the same patients who presented enhanced secretion of CCL3, as well as increased expression of both the CCR5 and CXCR3 chemokine receptors. These findings suggest that CCL24 may influence the kinetics of chemokines and their receptors and eosinophils recruitment during human acute schistosomiasis mansoni.

Impacto da pandemia da COVID-19 na doação de córnea e atuação do Banco de Olhos na Região da Zona da Mata Mineira / Impact of the COVID-19 pandemic on cornea donation and Eye Bank operation in the Zona da Mata Mineira Region

RESUMO Objetivo: Avaliar o impacto da pandemia da COVID-19 nas doações de córnea e na atuação de um banco de olhos na região da Zona da Mata Mineira. Métodos: Análise retrospectiva de prontuários de todas as doações obtidas pelo Banco de Olhos Hospital Regional Dr. João Penido, de Juiz de Fora (MG), entre 2017 e 2022, comparando-se os períodos pré (janeiro de 2017 a 11 de março de 2020) e pós-pandemia (12 de março de 2020 a dezembro de 2022). Resultados: Verificou-se uma redução nas doações de córnea e no número de tecidos corneanos liberados para transplantes no período pós pandemia, 68,2 e 67,3% respectivamente. Não houve diferença estatística no sexo, idade média, causa básica de óbito dos doadores, nos números de globos oculares não preservados e de córneas não preservadas entre os períodos pré e pós-pandemia. As taxas de liberação de córneas preservadas e aproveitamento das doações foram maiores no período pós-pandemia: 86,5 versus 79,0% e 68,1 versus 63,0%, respectivamente. O número de tecidos corneanos liberados para transplantes óptico e tectônico foram estatisticamente maiores no período pré-pandemia (p<0,001). Conclusão: A pandemia da COVID-19 impactou negativamente nas doações de córnea e na atuação do banco de olhos na região da Zona da Mata Mineira.

ABSTRACT Objective: To assess the impact of the COVID-19 pandemic on corneal donations and the operation of the eye bank in the Zona da Mata Mineira Region. Methods: Retrospective analysis of medical records of all donations obtained by the Banco de Olhos Hospital Regional Dr. João Penido/FHEMIG, Juiz de Fora - MG between 2017 and 2022, comparing pre (January/2017 - March 11/2020) and post-pandemic (12/March/2020 - December/2022) periods. Results: There was a reduction in cornea donations and the number of corneal tissues released for transplants in the post-pandemic period, 68.2% and 67.3%, respectively. There was no statistical difference in gender, average age, basic cause of death of donors, in the number of non-preserved eyeballs and non-preserved corneas between the pre- and post-pandemic periods. The rates of release of preserved corneas and use of donations were higher in the post-pandemic period, 86.5% vs 79.0%, and 68.1% vs 63.0%, respectively. The number of corneal tissues released for optical and tectonic transplants were statistically higher in the pre-pandemic period (P<0.001). Conclusion: The COVID-19 pandemic had a negative impact on cornea donations and the eye bank's operations in the Zona da Mata Mineira region.

Discrepancy between batches and impact on the sensitivity of point-of-care circulating cathodic antigen tests for Schistosoma mansoni infection.

The Kato-Katz (KK) technique is the mainstay mapping tool for the diagnosis of Schistosoma mansoni infection, despite showing poor sensitivity in cases of low-intensity infections. As an alternative, a rapid point-of-care circulating cathodic antigen diagnostic test (POC-CCA) has been commercially developed that involves a simple urine assay to detect S. mansoni, rather than a stool-based parasitological examination. Although POC-CCA has proven to be a more sensitive test than KK, it is not yet clear how to interpret discordant results between the two tests, particularly for situations in which the KK result is positive and the POC-CCA result is negative. Thus, the objective of this study was to evaluate the degree of diagnostic variability between different POC-CCA batches with respect to results obtained with KK. For this purpose, we collected urine and stool samples of school-aged children from areas of low and moderate endemicity in Brazil, and compared different POC-CCA batches results with those of KK-positive individuals. We found a statistically significant difference between the results obtained from various POC-CCA batches using the same urine samples, regardless of the degree of endemicity and the intensity of infection in positive KK samples. In addition, there was poor agreement between the KK and POC-CCA results in some batches of the rapid test, resulting in false negatives. These findings raise concerns around quality control checks of POC-CCA, especially in light of the high cost and increasing reliance on this new diagnostic method as control programs move towards a goal of elimination.

Chronic helminth infection does not impair immune response to malaria transmission blocking vaccine Pfs230D1-EPA/Alhydrogel® in mice.

INTRODUCTION: Malaria transmission blocking vaccines (TBV) are innovative approaches that aim to induce immunity in humans against Plasmodium during mosquito stage, neutralizing the capacity of the infected vectors to transmit malaria. Pfs230D1-EPA/Alhydrogel®, a promising protein-protein conjugate malaria TBV, is currently being tested in human clinical trials in areas where P. falciparum malaria is coendemic with helminth parasites. Helminths are complex metazoans that share the master capacity to downregulate the host immune response towards themselves and also to bystander antigens, including vaccines. However, it is not known whether the activity of a protein-based malaria TBV may be affected by a chronic helminth infection. METHODS: Using an experimental murine model for a chronic helminth infection (Heligmosomoides polygyrus bakeri - Hpb), we evaluated whether prior infection alters the activity of Pfs230D1-EPA/Alhydrogel® TBV in mice. RESULTS: After establishment of a chronic infection, characterized by a marked increase of parasite antigen-specific IgG1, IgA and IgE antibody responses, concomitant with an increase of systemic IL-10, IL-5 and IL-6 levels, the Hpb-infected mice were immunized with Pfs230D1-EPA/Alhydrogel® and the vaccine-specific immune response was compared with that in non-infected immunized mice. TBV immunizations induced an elevated vaccine specific-antibody response, however Pfs230D1 specific-IgG levels were similar between infected and uninfected mice at days 15, 25 and 35 post-vaccination. Absolute numbers of Pfs230D1-activated B cells generated in response to the vaccine were also similar among the vaccinated groups. Finally, vaccine activity assessed by reduction of oocyst number in P. falciparum infected mosquitoes was similar between Hpb-infected and immunized mice with non-infected immunized mice. CONCLUSION: Pfs230D1-EPA/Alhydrogel® efficacy is not impaired by a chronic helminth infection in mice.

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection.

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity.

Concomitant helminth infection downmodulates the Vaccinia virus-specific immune response and potentiates virus-associated pathology.

The aim of this work was to elucidate the immunopathological mechanisms of how helminths may influence the course of a viral infection, using a murine model. Severe virulence, a relevant increase in the virus titres in the lung and a higher mortality rate were observed in Ascaris and Vaccinia virus (VACV) co-infected mice, compared with VACV mono-infected mice. Immunopathological analysis suggested that the ablation of CD8+ T cells, the marked reduction of circulating CD4+ T cells producing IFN-γ, and the robust pulmonary inflammation were associated with the increase of morbidity/mortality in co-infection and subsequently with the negative impact of concomitant pulmonary ascariasis and respiratory VACV infection for the host. On the other hand, when evaluating the impact of the co-infection on the parasitic burden, co-infected mice presented a marked decrease in the total number of migrating Ascaris lung-stage larvae in comparison with Ascaris mono-infection. Taken together, our major findings suggest that Ascaris and VACV co-infection may potentiate the virus-associated pathology by the downmodulation of the VACV-specific immune response. Moreover, this study provides new evidence of how helminth parasites may influence the course of a coincident viral infection.

Multiple Exposures to Ascaris suum Induce Tissue Injury and Mixed Th2/Th17 Immune Response in Mice.

Ascaris spp. infection affects 800 million people worldwide, and half of the world population is currently at risk of infection. Recurrent reinfection in humans is mostly due to the simplicity of the parasite life cycle, but the impact of multiple exposures to the biology of the infection and the consequences to the host's homeostasis are poorly understood. In this context, single and multiple exposures in mice were performed in order to characterize the parasitological, histopathological, tissue functional and immunological aspects of experimental larval ascariasis. The most important findings revealed that reinfected mice presented a significant reduction of parasite burden in the lung and an increase in the cellularity in the bronchoalveolar lavage (BAL) associated with a robust granulocytic pulmonary inflammation, leading to a severe impairment of respiratory function. Moreover, the multiple exposures to Ascaris elicited an increased number of circulating inflammatory cells as well as production of higher levels of systemic cytokines, mainly IL-4, IL-5, IL-6, IL-10, IL-17A and TNF-α when compared to single-infected animals. Taken together, our results suggest the intense pulmonary inflammation associated with a polarized systemic Th2/Th17 immune response are crucial to control larval migration after multiple exposures to Ascaris.

New insights into the immunopathology of early Toxocara canis infection in mice.

BACKGROUND: Nematodes of the genus Toxocara are cosmopolitan roundworms frequently found in dogs and cats. Toxocara spp. can accidentally infect humans and cause a zoonosis called human toxocariasis, which is characterized by visceral, ocular or cerebral migration of larval stages of the parasite, without completing its life cycle. In general, chronic nematode infections induce a polarized TH2 immune response. However, during the initial phase of infection, a strong pro-inflammatory response is part of the immunological profile and might determine the outcome and/or pathology of the infection. METHODS: Parasitological aspects and histopathology during larval migration were evaluated after early T. canis experimental infection of BALB/c mice, which were inoculated via the intra-gastric route with a single dose of 1000 fully embryonated eggs. Innate immune responses and systemic cytokine patterns (TH1, TH2, TH17 and regulatory cytokines) were determined at different times after experimental challenge by sandwich ELISA. RESULTS: We found that experimental infection with T. canis induced a mix of innate inflammatory/TH17/TH2 responses during early infection, with a predominance of the latter. The TH2 response was evidenced by significant increases in cytokines such as IL-4, IL-5, IL-13 and IL-33, in addition to increasing levels of IL-6 and IL-17. No significant increases were observed for IL-10, TNF-α or IFN-γ levels. In parallel, parasitological analysis clearly revealed the pattern of larval migration through the mouse organs, starting from the liver in the first 24 h of infection, reaching the peak in the lungs on the 3rd day of infection and finally being found numerously in the brain after 5 days of infection. Peripheral leukocytosis, characterized by early neutrophilia and subsequent eosinophilia, was remarkable during early infection. The tissue damage induced by larvae was evidenced by histopathological analysis of the organs at different time points of infection. In all of the affected organs, larval migration induced intense inflammatory infiltrate and hemorrhage. CONCLUSION: In conclusion, these new insights into early T. canis infection in mice presented here enabled a better understanding of the immunopathological events that might also occur during human toxocariasis, thus contributing to future strategies of diagnosis and control.