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Background/aims: Chagasic megacolon is caused by Trypanosoma cruzi , which promotes in several cases, irreversible segmental colonic dilation. This alteration is the major anatomic-clinical disorder, characterized by the enteric nervous system and muscle wall structural damage. Herein, we investigate how T. cruzi -induced progressive colonic structural changes modulate the colonic contractile pattern activity., Methods: We developed a murine model of T. cruzi -infection that reproduced long-term modifications of the enlarged colon. We evaluated colonic and total intestinal transit time in animals. The patterns of motor response at several time intervals between the acute and chronic phases were evaluated using the organ bath assays. Enteric motor neurons were stimulated by electric field stimulation. The responses were analyzed in the presence of the nicotinic and muscarinic acetylcholine receptor antagonists. Western blot was performed to evaluate the expression of nicotinic and muscarinic receptors. The neurotransmitter expression was analyzed by real-time polymerase chain reaction., Results: In the chronic phase of infection, there was decreased intestinal motility associated with decreased amplitude and rhythmicity of intestinal contractility. Pharmacological tests suggested a defective response mediated by acetylcholine receptors. The contractile response induced by acetylcholine was decreased by atropine in the acute phase while the lack of its action in the chronic phase was associated with tissue damage, and decreased expression of choline acetyltransferase, nicotinic subunits of acetylcholine receptors, and neurotransmitters., Conclusions: T. cruzi -induced damage of smooth muscles was accompanied by motility disorders such as decreased intestinal peristalsis and cholinergic system response impairment. This study allows integration of the natural history of Chagasic megacolon motility disorders and opens new perspectives for the design of effective therapeutic.

Acute chagasic encephalitis is a clinically severe central nervous system (CNS) manifestation. However, the knowledge of the nervous form of Chagas disease is incomplete. The role of the muscarinic acetylcholine receptor (mAChR) on mice behavior and brain lesions induced by Trypanosoma cruzi (Colombian strain) was herein investigated in mice treated with the mAChR agonist and antagonist (carbachol and atropine), respectively. Immunosuppressed or non-immunosuppressed mice were intracerebroventricularly (icv) or intraperitoneally (ip) infected. All groups were evaluated 15 d.p.i. (days post infection). Intraperitoneally infected animals had subpatent parasitemia. Patent parasitemia occurred only in icv infected mice. The blockade of mAChR increased the parasitemia, parasitism and lesions compared to its activation. Infected not treated (INT ip) mice did not present meningitis and encephalitis, regardless of immunosuppression. INT icv brains presented higher cellularity, discrete signs of cellular degeneration, frequent presence of parasites and focal meningitis. The immunosuppressed atropine + icv mice presented increased intracellular parasitism associated with degenerative parenchymal changes, while carbachol + icv mice presented discrete meningitis, preservation of the cortex and absence of relevant parasitism. Cholinergic receptor blockage increased impairment of coordination vs. receptor activation. Muscarinic cholinergic pathway seems to be involved in immune mediated cell invasion events while its blockade favored infection evolution, brain lesions, and behavioral alterations.

There is a growing consensus that the balance between the persistence of infection and the host immune response is crucial for chronification of Chagas heart disease. Extrapolation for chagasic megacolon is hampered because research in humans and animal models that reproduce intestinal pathology is lacking. The parasite-host relationship and its consequence to the disease are not well-known. Our model describes the temporal changes in the mice intestine wall throughout the infection, parasitism, and the development of megacolon. It also presents the consequence of the infection of primary myenteric neurons in culture with Trypanosoma cruzi ( T. cruzi ). Oxidative neuronal damage, involving reactive nitrogen species induced by parasite infection and cytokine production, results in the denervation of the myenteric ganglia in the acute phase. The long-term inflammation induced by the parasite's DNA causes intramuscular axonal damage, smooth muscle hypertrophy, and inconsistent innervation, affecting contractility. Acute phase neuronal loss may be irreversible. However, the dynamics of the damages revealed herein indicate that neuroprotection interventions in acute and chronic phases may help to eradicate the parasite and control the inflammatory-induced increase of the intestinal wall thickness and axonal loss. Our model is a powerful approach to integrate the acute and chronic events triggered by T. cruzi , leading to megacolon., (Copyright © 2020 Ricci, Béla, Moraes, Bahia, Mazzeti, Oliveira, Andrade, Radí, Piacenza and Arantes.)

In the present study we have evaluated the performance of several immunological biomarkers for early diagnosis and prognosis of congenital toxoplasmosis. Our results showed that ex vivo serum levels of CXCL9, and the frequencies of circulating CD4 + CD25 + T-cells and T. gondii-specific IFN-γ + CD4 + T-cells measured 30-45 days after birth presented high accuracy to distinguish T. gondii-infected infants from healthy age-matched controls (Global Accuracy/AUC = 0.9; 0.9 and 0.8, respectively). Of note was the enhanced performance (Accuracy = 96%) achieved by using a combined stepwise analysis of CD4 + CD25 + T-cells and CXCL9. In addition, high global accuracy (AUC = 0.9) with elevated sensitivity (Se = 98%) was also reached by using the total frequency of in vitro IFN-γ-producing T. gondii-specific T-cells (∑ IFN-γ + CD4 + & CD8 + ) as a biomarker of congenital toxoplasmosis. Furthermore, the analysis of in vitro T. gondii-specific IL5 + CD4 + T-cells and IFN-γ + NK-cells displayed a high accuracy for early prognosis of ocular lesion in infant with congenital toxoplasmosis (Global Accuracy/AUC = 0.8 and 0.9, respectively). Together, these findings support the relevance of employing the elements of the cell-mediated immune response as biomarkers with potential to endorse early diagnosis and prognosis of congenital ocular toxoplasmosis to contribute for a precise clinical management and effective therapeutic intervention.

The present study characterized the early changes in the serum chemokines/cytokine signatures and networks in infants with congenital-toxoplasmosis/(TOXO) as compared to non-infected-controls/(NI). TOXO were subgrouped according to the retinochoroidal lesion status as no-lesion/(NL), active-lesion/(ARL), active/cicatricial-lesion/(ACRL) and cicatricial-lesion/(CRL). The results showed that TOXO display prominent chemokine production mediated by IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and RANTES/CCL5. Additionally, TOXO is accompanied by mixed proinflammatory/regulatory cytokine pattern mediated by IL-6, IFN-γ, IL-4, IL-5 and IL-10. While TNF appears as a putative biomarker for NL and IFN-γ/IL-5 as immunological features for ARL, IL-10 emerges as a relevant mediator in ACRL/CRL. IL-8/CXCL8 and IP-10/CXCL10 are broad-spectrum indicators of ocular disease, whereas TNF is a NL biomarker, IFN-γ and MIG/CXCL9 point out to ARL; and IL-10 is highlighted as a genuine serum biomarker of ACRL/CRL. The network analysis demonstrated a broad chemokine/cytokine crosstalk with divergences in the molecular signatures in patients with different ocular lesions during congenital toxoplasmosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The incidence of anal cancer has increased over the past 25 years, and HIV/HPV coinfection is the most important risk factor for anal squamous cell carcinoma. In this study, we demonstrated that the evaluation of systemic and compartmentalized anal mucosa immune response is relevant to differentiating HIV(+) patients at risk of anal intraepithelial neoplasia (AIN)., Methods: A systems biology approach was used to integrate different immunological parameters from anal mucosal tissue and peripheral blood assessed by phenotypic and intracytoplasmic analysis of lymphocytes and dendritic cell subsets., Results: Our data demonstrated that anal mucosal mononuclear cells from AIN(+)HIV(+) patients showed a robust capacity in producing proinflammatory/regulatory cytokines, mainly mTNF-α > IL-4 > IL-10 > IL-6 = IL-17A. Mucosal TNF-α/IFN-γ/IL-17A are selective high-grade squamous intraepithelial lesion (HSIL)-related biomarkers. Higher levels of circulating CD11cCD123cells and CD1a cells along with elevated levels of IFN-γCD4 T cells are major features associated with HSIL in AIN(+)HIV(+) patients. Regardless of the presence of AIN, HIV(+) patients presented a complex biomarker network, rich in negative connections. Among those patients, however, HSIL+ patients displayed stronger positive links between peripheral blood and anal mucosa environments, exemplified by the subnet of IL-17A/TNF-α/CD4IFN-γ/CD11cCD123 cells., Conclusions: The significant association between HSIL and the levels of TNF-α/IL-17A/IFN-γ along with the different subsets of DCs present in the anal mucosa milieu should be studied in more detail as a way to identify and categorize HIV(+) patients vis à vis the high risk of anal cancer outcome.

Toxoplasma gondii is the main infectious cause of human posterior retinochoroiditis, the most frequent clinical manifestation of congenital toxoplasmosis. This investigation was performed after neonatal screening to identify biomarkers of immunity associated with immunopathological features of the disease by flow cytometry. The study included infected infants without NRL and with retinochoroidal lesions (ARL, ACRL, and CRL) as well as noninfected individuals (NI). Our data demonstrated that leukocytosis, with increased monocytes and lymphocytes, was a relevant hematological biomarker of ARL. Immunophenotypic analysis also revealed expansion of CD14(+)CD16(+)HLA-DR(high) monocytes and CD56(dim) cytotoxic NK-cells in ARL. Moreover, augmented TCRγ δ (+) and CD8(+) T-cell counts were apparently good biomarkers of morbidity. Biomarker network analysis revealed that complex and intricated networks underscored the negative correlation of monocytes with NK- and B-cells in NRL. The remarkable lack of connections involving B-cells and a relevant shift of NK-cell connections from B-cells toward T-cells observed in ARL were outstanding. A tightly connected biomarker network was observed in CRL, with relevant connections of NK- and CD8(+) T-cells with a broad range of cell subsets. Our findings add novel elements to the current knowledge on the innate and adaptive immune responses in congenital toxoplasmosis.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a member of the IRAK family and has an important role in inducing the production of inflammatory mediators. This kinase is downstream of MyD88, an adaptor protein essential for Toll-like receptor (TLR) function. We investigated the role of this kinase in IRAK4-deficient mice orally infected with the cystogenic ME49 strain of Toxoplasma gondii. IRAK4(-/-) mice displayed higher morbidity, tissue parasitism, and accelerated mortality than the control mice. The lymphoid follicles and germinal centers from infected IRAK4(-/-) mice were significantly smaller. We consistently found that IRAK4(-/-) mice showed a defect in splenic B cell activation and expansion as well as diminished production of gamma interferon (IFN-γ) by T lymphocytes. The myeloid compartment was also affected. Both the frequency and ability of dendritic cells (DCs) and monocytes/macrophages to produce IL-12 were significantly decreased, and resistance to infection with Toxoplasma was rescued by treating IRAK4(-/-) mice with recombinant IL-12 (rIL-12). Additionally, we report the association of IRAK4 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital toxoplasmosis in infected individuals (rs1461567 and rs4251513, P < 0.023 and P < 0.045, respectively). Thus, signaling via IRAK4 is essential for the activation of innate immune cells, development of parasite-specific acquired immunity, and host resistance to infection with T. gondii.

Herpes simplex virus 1 (HSV-1) is a neurotropic DNA virus that is responsible for several clinical manifestations in humans, including encephalitis. HSV-1 triggers toll-like receptors (TLRs), which elicit cytokine production. Viral multiplication and cytokine expression in C57BL/6 wild-type (WT) mice infected with HSV-1 were evaluated. Virus was found in the trigeminal ganglia (TG), but not in the brains of animals without signs of encephalitis, between 2 and 6 days postinfection (d.p.i.). Cytokine expression in the TG peaked at 5 d.p.i. TLR9-/- and TLR2/9-/- mice were more susceptible to the virus, with 60% and 100% mortality, respectively, as opposed to 10% in the WT and TLR2-/- mice. Increased levels of both CXCL10/IP-10 and CCL2/MCP-1, as well as reduced levels of interferon-γ and interleukin 1-β transcripts, measured in both the TG and brains at 5 d.p.i., and the presence of virus in the brain were correlated with total mortality in TLR2/9-/- mice. Cytokine alterations in TLR2/9-/- mice coincided with histopathological changes in their brains, which did not occur in WT and TLR2-/- mice and occurred only slightly in TLR9-/- mouse brain. Increased cellularity, macrophages, CD8 T cells producing interferon-γ, and expression levels of TLR2 and TLR9 were detected in the TG of WT-infected mice. We hypothesize that HSV-1 infection is controlled by TLR-dependent immune responses in the TG, which prevent HSV-1 encephalitis.

The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.