P2X7 receptor drives Th1 cell differentiation and controls the follicular helper T cell population to protect against Plasmodium chabaudi malaria.

A complete understanding of the mechanisms underlying the acquisition of protective immunity is crucial to improve vaccine strategies to eradicate malaria. However, it is still unclear whether recognition of damage signals influences the immune response to Plasmodium infection. Adenosine triphosphate (ATP) accumulates in infected erythrocytes and is released into the extracellular milieu through ion channels in the erythrocyte membrane or upon erythrocyte rupture. The P2X7 receptor senses extracellular ATP and induces CD4 T cell activation and death. Here we show that P2X7 receptor promotes T helper 1 (Th1) cell differentiation to the detriment of follicular T helper (Tfh) cells during blood-stage Plasmodium chabaudi malaria. The P2X7 receptor was activated in CD4 T cells following the rupture of infected erythrocytes and these cells became highly responsive to ATP during acute infection. Moreover, mice lacking the P2X7 receptor had increased susceptibility to infection, which correlated with impaired Th1 cell differentiation. Accordingly, IL-2 and IFNγ secretion, as well as T-bet expression, critically depended on P2X7 signaling in CD4 T cells. Additionally, P2X7 receptor controlled the splenic Tfh cell population in infected mice by promoting apoptotic-like cell death. Finally, the P2X7 receptor was required to generate a balanced Th1/Tfh cell population with an improved ability to transfer parasite protection to CD4-deficient mice. This study provides a new insight into malaria immunology by showing the importance of P2X7 receptor in controlling the fine-tuning between Th1 and Tfh cell differentiation during P. chabaudi infection and thus in disease outcome.

In vivo approaches reveal a key role for DCs in CD4+ T cell activation and parasite clearance during the acute phase of experimental blood-stage malaria.

Dendritic cells (DCs) are phagocytes that are highly specialized for antigen presentation. Heterogeneous populations of macrophages and DCs form a phagocyte network inside the red pulp (RP) of the spleen, which is a major site for the control of blood-borne infections such as malaria. However, the dynamics of splenic DCs during Plasmodium infections are poorly understood, limiting our knowledge regarding their protective role in malaria. Here, we used in vivo experimental approaches that enabled us to deplete or visualize DCs in order to clarify these issues. To elucidate the roles of DCs and marginal zone macrophages in the protection against blood-stage malaria, we infected DTx (diphtheria toxin)-treated C57BL/6.CD11c-DTR mice, as well as C57BL/6 mice treated with low doses of clodronate liposomes (ClLip), with Plasmodium chabaudi AS (Pc) parasites. The first evidence suggesting that DCs could contribute directly to parasite clearance was an early effect of the DTx treatment, but not of the ClLip treatment, in parasitemia control. DCs were also required for CD4+ T cell responses during infection. The phagocytosis of infected red blood cells (iRBCs) by splenic DCs was analyzed by confocal intravital microscopy, as well as by flow cytometry and immunofluorescence, at three distinct phases of Pc malaria: at the first encounter, at pre-crisis concomitant with parasitemia growth and at crisis when the parasitemia decline coincides with spleen closure. In vivo and ex vivo imaging of the spleen revealed that DCs actively phagocytize iRBCs and interact with CD4+ T cells both in T cell-rich areas and in the RP. Subcapsular RP DCs were highly efficient in the recognition and capture of iRBCs during pre-crisis, while complete DC maturation was only achieved during crisis. These findings indicate that, beyond their classical role in antigen presentation, DCs also contribute to the direct elimination of iRBCs during acute Plasmodium infection.

The role of clinical pharmacists in treatment adherence: fast impact in suppression of chronic myeloid leukemia development and symptoms.

PURPOSE: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease, accounting for 15 to 20% of leukemias, with an incidence of one to two cases/100,000 inhabitants. In Brazil, the estimated incidence of leukemia is six cases/100,000 men and 4.28 cases/100,000 women. CML is characterized by the presence of the Philadelphia chromosome. At present, three types of tyrosine kinase inhibitors (TKI) are administered to treat CML patients in the Brazilian public national health system (NHS), called the Unified Health System (in Portuguese, "Sistema Único de Saúde", SUS). Such treatments are only effective if patients adhere to strict dosage regimens; protocol improvements that increase patient adherence to treatment would have economic and health benefits for overburdened health care systems. Here, pharmacist-monitored treatment is assessed. METHODS: In our study, we applied two questionnaires, one to assess the adherence to pharmacological treatment and another to assess the quality of life. All patients studied (n = 23) were diagnosed with CML at a local hospital in "Espírito Santo" State, the "Hospital Evangélico Vila Velha" (HEVV). RESULTS: Treatment adherence was significantly higher in pharmacist-monitored patients than in nonmonitored patients (p = 0.0135). The quality of life of CML patients was also analyzed, indicating that monitored patients had a lower number of symptoms/complaints during treatment periods than nonmonitored patients. Finally, improved treatment adherence also translated into better clinical conditions, particularly during the early stage of treatment (e.g., the first 4 months). CONCLUSIONS: The intervention of a clinical pharmacist is significant to obtain positive clinical results. Therefore, it is recommended that this protocol be included in the standard NHS treatment protocol CML patient outcomes to reduce the indirect and recurring costs to the health care system caused by nonadherence.

Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes.

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE.

PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous Leishmaniasis.

Patients infected by Leishmania braziliensis develop debilitating skin lesions. The role of inhibitory checkpoint receptors (ICRs) that induce T cell exhaustion during this disease is not known. Transcriptional profiling identified increased expression of ICRs including PD-1, PDL-1, PDL-2, TIM-3, and CTLA-4 in skin lesions of patients that was confirmed by immunohistology where there was increased expression of PD-1, TIM-3, and CTLA-4 in both CD4+ and CD8+ T cell subsets. Moreover, PDL-1/PDL-2 ligands were increased on skin macrophages compared to healthy controls. The proportions PD1+, but not TIM-3 or CTLA-4 expressing T cells in the circulation were positively correlated with those in the lesions of the same patients, suggesting that PD-1 may regulate T cell function equally in both compartments. Blocking PD-1 signaling in circulating T cells enhanced their proliferative capacity and IFN-γ production, but not TNF-α secretion in response to L. braziliensis recall antigen challenge in vitro. While we previously showed a significant correlation between the accumulation of senescent CD8+CD45RA+CD27- T cells in the circulation and skin lesion size in the patients, there was no such correlation between the extent of PD-1 expression by circulating on T cells and the magnitude of skin lesions suggesting that exhausted-like T cells may not contribute to the cutaneous immunopathology. Nevertheless, we identified exhausted-like T cells in both skin lesions and in the blood. Targeting this population by PD-1 blockade may improve T cell function and thus accelerate parasite clearance that would reduce the cutaneous pathology in cutaneous leishmaniasis.

Inhale, exhale: Why particulate matter exposure in animal models are so acute?

Ecotoxicological studies that try to describe the effects of particulate matter (PM) on human health are important in order to gain a deeper understanding of their effects in disease outcomes. Because exposure protocols are not easily comparable, evaluating human PM exposure is a difficult task. Thus, interpreting ambiguous or conflicting results from different experiments could lead to misleading conclusions about the true nature of PM effects. To address these issues, we compiled a collection of relevant research articles in order to compare present PM exposure methods and extract data related to concentration, inhalation rates (IR), and doses. We also compare the experimental exposure levels reported in these articles to PM levels around the world. In particular, our dataset covers reported results from 75 research articles. To allow for comparison between protocols, we used this data to fit a normalization equation that depends upon concentration, exposure time, dose, inhalability, and physiological parameters. Based on the collected research papers, instillation is the prevalent exposure method. Also, the median PM IR from these experiments is three orders of magnitude higher than the PM IR found in environmental conditions (EAP). Experiments employing inhalation of concentrated PM show IR results that are two orders of magnitude higher than EAP; these results are cause for concerns, since the PM exposure were acute, sudden, and higher than the worst-case exposure scenarios reported by the world megacities. We also found that different PM exposure protocols are sources for the observed variability in physiological response results found from animal models. We discuss these findings and make suggestions for future exposure methodologies. Such considerations should be valuable for quantifying PM exposure in disease outcomes.

Inhale, exhale: Why particulate matter exposure in animal models are so acute? Data and facts behind the history.

We present a dataset obtained by extracting information from an extensive literature search of toxicological experiments using mice and rat animal models to study the effects of exposure to airborne particulate matter (PM). Our dataset covers results reported from 75 research articles considering paper published in 2017 and seminal papers from previous years. The compiled data and normalization were processed with an equation based on a PM dosimetry model. This equation allows the comparison of different toxicological experiments using instillation and inhalation as PM exposure protocols with respect to inhalation rates, concentrations and PM exposure doses of the toxicological experiments performed by different protocols using instillation and inhalation PM as exposure methods. This data complements the discussions and interpretations presented in the research article "Inhale, exhale: why particulate matter exposure in animal models are so acute?" Curbani et al., 2019.

Purpuriocillium lilacinum infection in captive loggerhead sea turtle hatchlings.

This paper reports a case of Purpureocillium lilacinum infection in seven loggerhead sea turtle (Caretta caretta) hatchlings kept in an aquarium under inadequate condition. The fungus was isolated from skin and pulmonary lesions. Metilene blue and NaCl solutions, Schinus terebinthifolius and eucalyptus essential oils Minimum Inhibitory Concentrations were determined indicating new possibilities for treatment.

B220 expression as an immunological marker for differentiation of Feline Leukemia Virus carrying cats / A expressão de B220 como um marcador imunológico para diferenciação de gatos portadores do vírus da Leucemia Felina

ABSTRACT: Feline leukemia virus (FeLV) causes an infection in cats that, in some cases, can also be reported with other pathologies, such as infection with feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), and lymphoma. Although, a compromised immune response is reported in these animals, little is known about the immunological state of their cells. To shed some light in this area, we studied peripheral blood samples from both infected and non-infected cats with FeLV, with or without FIV, FIP, and lymphoma. We tested a panel of monoclonal antibodies (n=11) against mouse and human antigens and we reported that cat leukocytes can be stained with anti-mouse B220 monoclonal antibody; therefore, percentages of B cells were evaluated in different cat groups. Our results showed that cats with FeLV and FIP, or with leukemia, presented a large decrease in B220+ mononuclear cells. However, FeLV+ cats without clinical signs, or with unspecific clinical signs, had the same amount of B220+ mononuclear cells as healthy cats (control cats). Since the expression of B220 is exclusively restricted to the naïve B cell population, we inferred that the absence of these B cells in FeLV+ cats is related to other conditions that affect B cell numbers, such as viral infections and leukemias. Therefore, the amount of naïve B cells in peripheral blood (i.e., B220+ cells) can be used to identify FeLV+ cats concomitantly carrying FIP or leukemia, from FeLV+ cats with lymphoma or without any clinical signs.

RESUMO: O vírus da leucemia felina (FeLV) causa de uma infecção em gatos, que também podem ter outras patologias, como a imunodeficiência felina (FIV), a peritonite infecciosa felina (FIP) e linfoma. Embora uma resposta imune comprometida seja encontrada nestes animais, pouco se sabe sobre o estado imunológico de suas células. Para ampliar o número de testes com a finalidade de avaliar o estado imunológico destes animais, estudamos amostras de sangue periférico de gatos infectados, ou não, com FeLV, e que apresentavam (concomitantemente) FIV, FIP e linfoma. Para isto, amostras de sangue foram marcadas com um painel de anticorpos monoclonais contra antígenos de camundongos e humanos (n = 11), para avaliar seu potencial para estudos imunológicos em gatos. De todo o painel de anticorpos testados, apenas o anticorpo anti-B220 de camundongo foi capaz de marcar leucócitos de gato. Nossos resultados mostraram que os gatos com FeLV e FIP, ou com leucemia, apresentaram uma grande diminuição nas células mononucleares B220+. No entanto, gatos FeLV+ sem sinais clínicos, ou com sinais clínicos inespecíficos, tiveram a mesma quantidade de células B220+ que os gatos saudáveis (gatos controle). Como a expressão de B220 é restrita à população de células B naïve, podemos inferir que a ausência dessas células B em gatos FeLV+ está relacionada a outras condições que afetam o número destas células, como infecções virais e leucemias. Portanto, a quantidade de células B naïve no sangue periférico pode ser usada para identificar gatos FeLV+ concomitantemente portadores de PIF ou leucemia, de gatos FeLV+ com linfoma ou sem sinais clínicos.

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection.

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.

Involvement of nitric oxide (NO) and TNF-alpha in the oxidative stress associated with anemia in experimental Trypanosoma cruzi infection.

Trypanosoma cruzi infection in mice is associated with severe hematological changes, including anemia, which may contribute to mortality. TNF-alpha and nitric oxide (NO) play a critical role in establishing host resistance to this pathogen. We hypothesized that phagocyte-derived NO damages erythrocytes and contributes to the anemia observed during T. cruzi infection. To test this hypothesis, two strains of mice that differed in susceptibility and NO response to T. cruzi infection were used in these studies. We also blocked endogenous NO production by aminoguanidine (AG) treatment or blocked TNF-alpha with a neutralizing antibody and used mice that cannot produce phagocyte-derived NO (C57BL/6 iNOS(-/-)). Following infection with T. cruzi, resistant (C57BL/6) and susceptible (Swiss) mice displayed a parasitemia that peaked at the same time (i.e., day 9), yet parasitemia was 3-fold higher in Swiss mice (P < 0.05). All Swiss mice were dead by day 23 post-infection, while no C57BL/6 mice died during the study. At 14 days post-infection anemia in C57BL/6 mice was more severe than in Swiss mice. Treatment of both strains with the NO inhibitor, AG (50 mg/kg), and the use of iNOS(-/-) mice, revealed that the anemia in T. cruzi-infected mice is not caused by NO. However, the reticulocytosis that occurs during infection was significantly reduced after treatment with AG in both Swiss and C57BL/6 mice (P < 0.05). In addition, we showed that neutralization of TNF-alpha in vivo induced a significant increase in circulating reticulocytes in T. cruzi-infected C57BL/6 mice (P < 0.05), but did not modify other hematologic parameters in these mice. The evaluation of the oxidative stress after induction by t-butyl hydroperoxide (t-BHT) revealed that the treatment with AG completely protected against NO-mediated haemoglobin oxidation. Further, treatment with AG, but not with anti-TNF-alpha, protected against the infection-induced reduction of antioxidant capacity of erythrocytes as assessed by oxygen uptake and induction time. In summary, this is the first report showing the participation of NO and TNF-alpha in the oxidative stress to erythrocytes in acute T. cruzi infection. Further, our data suggest that NO does not play a direct role in development of the anemia. However, NO may contribute to other hematological changes noted during T. cruzi infection, such as the elevation of circulating reticulocytes and the reduction in circulating leukocytes and neutrophils.

Salivary epidermal growth factor concentration in adults with reflux laryngitis.

OBJECTIVE: The mechanisms involved in the mucosal alterations of laryngopharyngeal reflux (LPR) have not been well established. Reports indicate a decrease in the salivary epidermal growth factor (EGF) of patients with reflux esophagitis, but there are no reports of its behavior in LPR. Our objective was to determine the salivary concentration of EGF in adults with LPR. STUDY DESIGN AND SETTING: Salivary EGF concentration of 26 patients with LPR and 20 healthy controls was determined using a commercially available ELISA kit. Patients with LPR were graded according to endoscopic and laryngoscopic criteria. RESULTS: Salivary EGF concentration was significantly lower in the LPR group when compared with controls (P = 0.002). No correlation between the severity of laryngeal findings or esophagitis and salivary EGF concentration could be determined. CONCLUSIONS: The decreased salivary concentration of EGF in adults with LPR suggests that a deficiency in this polypeptide could be associated to the disease.

Control of uterine microenvironment by foxp3(+) cells facilitates embryo implantation.

Implantation of the fertilized egg into the maternal uterus depends on the fine balance between inflammatory and anti-inflammatory processes. Whilst regulatory T cells (Tregs) are reportedly involved in protection of allogeneic fetuses against rejection by the maternal immune system, their role for pregnancy to establish, e.g., blastocyst implantation, is not clear. By using 2-photon imaging we show that Foxp3(+) cells accumulated in the mouse uterus during the receptive phase of the estrus cycle. Seminal fluid further fostered Treg expansion. Depletion of Tregs in two Foxp3.DTR-based models prior to pairing drastically impaired implantation and resulted in infiltration of activated T effector cells as well as in uterine inflammation and fibrosis in both allogeneic and syngeneic mating combinations. Genetic deletion of the homing receptor CCR7 interfered with accumulation of Tregs in the uterus and implantation indicating that homing of Tregs to the uterus was mediated by CCR7. Our results demonstrate that Tregs play a critical role in embryo implantation by preventing the development of a hostile uterine microenvironment.

Early IL-10 production is essential for syngeneic graft acceptance.

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival.

CTLA-4 blockage increases resistance to infection with the intracellular protozoan Trypanosoma cruzi.

Recent studies have revealed an important role for CTLA-4 as a negative regulator of T cell activation. In the present study, we evaluated the importance of CTLA-4 to the immune response against the intracellular protozoan, Trypanosoma cruzi, the causative agent of Chagas' disease. We observed that the expression of CTLA-4 in spleen cells from naive mice cultured in the presence of live trypomastigote forms of T. cruzi increases over time of exposure. Furthermore, spleen cells harvested from recently infected mice showed a significant increase in the expression of CTLA-4 when compared with spleen cells from noninfected mice. Blockage of CTLA-4 in vitro and/or in vivo did not restore the lymphoproliferative response decreased during the acute phase of infection, but it resulted in a significant increase of NO production in vivo and in vitro. Moreover, the production of IFN-gamma in response to parasite Ags was significantly increased in spleen cells from anti-CTLA-4-treated infected mice when compared with the production found in cells from IgG-treated infected mice. CTLA-4 blockade in vivo also resulted in increased resistance to infection with the Y and Colombian strains of T. cruzi. Taken together these results indicate that CTLA-4 engagement is implicated in the modulation of the immune response against T. cruzi by acting in the mechanisms that control IFN-gamma and NO production during the acute phase of the infection.