Wesley Nogueira Brandão

The Brazilian Zika virus strain causes birth defects in experimental models

Summary Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (Family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys1. Until the 20th century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the island of Yap in Micronesia2. Patients experienced fever, skin rash, arthralgia and conjunctivitis2. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia. In 2013, ZIKV reached Brazil, later spreading to other countries in South and Central America3. In Brazil, the virus has been linked to congenital malformations, including microcephaly and other severe neurological diseases, such as Guillain-Barré syndrome4,5. Despite clinical evidence, direct experimental proof showing that the Brazilian ZIKV (ZIKVBR) strain causes birth defects remains missing6. Here we demonstrate that the ZIKVBR infects fetuses, causing intra-uterine growth restriction (IUGR), including signs of microcephaly in mice. Moreover, the virus infects human cortical progenitor cells, leading to an increase in cell death. Finally, we observed that the infection of human brain organoids resulted in a reduction of proliferative zones and disrupted cortical layers. These results indicate that ZIKVBR crosses the placenta and causes microcephaly by targeting cortical progenitor cells, inducing cell death by apoptosis and autophagy, impairing neurodevelopment. Our data reinforce the growing body of evidence linking the ZIKVBR outbreak to the alarming number of cases of congenital brain malformations. Our model can be used to determine the efficiency of therapeutic approaches to counteracting the harmful impact of ZIKVBR in human neurodevelopment.

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. In its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6+TGF-β dependent manner. Thus, using the oral tolerance model, by which 200 μg of MOG 35-55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-γ in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1α, IL-6, IL-9, IL-12p70 and the chemokines MIP-1β, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response.

Human Endometrial-Derived Mesenchymal Stem Cells Suppress Inflammation in the Central Nervous System of EAE Mice

Mesenchymal stromal cells (MSCs) are undifferentiated multipotent cells endowed with the capacity for self-renewal and the potential to differentiate into several distinct cell lineages [1]. It is well established that adult MSCs constitute a reservoir found within connective tissues of most organs, and whose biological function is involved in the maintenance and repair of tissues throughout the postnatal life of an individual. Over the past years we and others have shown that menstrual blood, the endometrium and fallopian tubes are very rich sources of MSCs and able to differentiate into different cell lineages in vitro and/or in vivo [2, 3]. The unique regenerative capacity of the human endometrium following menstruation, postpartum, surgical procedures (uterine curettage, endometrial ablation) and in postmenopausal women undergoing hormonal replacement therapy suggests that MSCs niches present in this tissue are responsible, at least in part, for this process. This makes these cells a very interesting approach to studies in regenerative medicine, especially in autoimmune disorders. Multiple sclerosis (MS) is a debilitating and neurodegenerative autoimmune disease with a significant social burden. It is mainly characterized by central nervous system (CNS) inflammation, gliosis, neuronal death and demyelination [4, 5]. Its murine model, experimental autoimmune encephalomyelitis (EAE), has generated many important data concerning MS pathology and treatment [6–9]. In EAE, mice are subcutaneously immunized with myelin-derived antigens such as myelin oligodendrocyte glycoprotein (MOG35-55), myelin basic protein (MBP) and also proteolipoprotein (PLP) [6]. In the periphery T CD4 cells differentiate into Th1 and Th17 cells and have been implicated in the pathogenesis of EAE [10–12]. Although IFN-γ and ILStem Cell Rev and Rep (2012) 8:940–952 DOI 10.1007/s12015-011-9338-3

Human Tubal-Derived Mesenchymal Stromal Cells Associated with Low Level Laser Therapy Significantly Reduces Cigarette Smoke–Induced COPD in C57BL/6 mice

Cigarette smoke-induced chronic obstructive pulmonary disease is a very debilitating disease, with a very high prevalence worldwide, which results in a expressive economic and social burden. Therefore, new therapeutic approaches to treat these patients are of unquestionable relevance. The use of mesenchymal stromal cells (MSCs) is an innovative and yet accessible approach for pulmonary acute and chronic diseases, mainly due to its important immunoregulatory, anti-fibrogenic, anti-apoptotic and pro-angiogenic. Besides, the use of adjuvant therapies, whose aim is to boost or synergize with their function should be tested. Low level laser (LLL) therapy is a relatively new and promising approach, with very low cost, no invasiveness and no side effects. Here, we aimed to study the effectiveness of human tube derived MSCs (htMSCs) cell therapy associated with a 30mW/3J—660 nm LLL irradiation in experimental cigarette smoke-induced chronic obstructive pulmonary disease. Thus, C57BL/6 mice were exposed to cigarette smoke for 75 days (twice a day) and all experiments were performed on day 76. Experimental groups receive htMSCS either intraperitoneally or intranasally and/or LLL irradiation either alone or in association. We show that co-therapy greatly reduces lung inflammation, lowering the cellular infiltrate and pro-inflammatory cytokine secretion (IL-1β, IL-6, TNF-α and KC), which were followed by decreased mucus production, collagen accumulation and tissue damage. These findings seemed to be secondary to the reduction of both NF-κB and NF-AT activation in lung tissues with a concomitant increase in IL-10. In summary, our data suggests that the concomitant use of MSCs + LLLT may be a promising therapeutic approach for lung inflammatory diseases as COPD.

Distinct courses of infection with Leishmania ( L. ) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

The wake-promoting drug Modafinil prevents motor impairment in sickness behavior induced by LPS in mice: Role for dopaminergic D1 receptor

&NA; The wake‐promoting drug Modafinil has been used for many years for treatment of Narcolepsy and Excessive Daytime Sleepiness, due to a dopamine‐related psychostimulant action. Recent studies have indicated that Modafinil prevents neuroinflammation in animal models. Thus, the aim of the present study was to evaluate the effect of Modafinil pretreatment in the Lipopolysaccharide (LPS)‐induced sickness and depressive‐like behaviors. Adult male C57BL/6J mice were pretreated with Vehicle or Modafinil (90 mg/Kg) and, 30 min later, received a single saline or LPS (2 mg/Kg) administration, and were submitted to the open field and elevated plus maze test 2 h later. After 24 h, mice were subjected to tail suspension test, followed by either flow cytometry with whole brain for CD11b+CD45+ cells or qPCR in brain areas for cytokine gene expression. Modafinil treatment prevented the LPS‐induced motor impairment, anxiety‐like and depressive‐like behaviors, as well as the increase in brain CD11b+CD45high cells induced by LPS. Our results indicate that Modafinil pretreatment also decreased the IL‐1&bgr; gene upregulation caused by LPS in brain areas, which is possibly correlated with the preventive behavioral effects. The pharmacological blockage of the dopaminergic D1R by the drug SCH‐23390 counteracted the effect of Modafinil on locomotion and anxiety‐like behavior, but not on depressive‐like behavior and brain immune cells. The dopaminergic D1 receptor signaling is essential to the Modafinil effects on LPS‐induced alterations in locomotion and anxiety, but not on depression and brain macrophages. This evidence suggests that Modafinil treatment might be useful to prevent inflammation‐related behavioral alterations, possibly due to a neuroimmune mechanism. HighlightsModafinil prevents sickness and depressive‐like behavior induced by LPS in mice.This effect is correlated with infiltration of macrophages in the brain.D1R blockage prevents Modafinil effects on sickness, but not on depressive behavior.

Thymic and Postthymic Regulation of Naïve CD4(+) T-Cell Lineage Fates in Humans and Mice Models.

Our understanding of how thymocytes differentiate into many subtypes has been increased progressively in its complexity. At early life, the thymus provides a suitable microenvironment with specific combination of stromal cells, growth factors, cytokines, and chemokines to induce the bone marrow lymphoid progenitor T-cell precursors into single-positive CD4+ and CD8+ T effectors and CD4+CD25+ T-regulatory cells (Tregs). At postthymic compartments, the CD4+ T-cells acquire distinct phenotypes which include the classical T-helper 1 (Th1), T-helper 2 (Th2), T-helper 9 (Th9), T-helper 17 (Th17), follicular helper T-cell (Tfh), and induced T-regulatory cells (iTregs), such as the regulatory type 1 cells (Tr1) and transforming growth factor-β- (TGF-β-) producing CD4+ T-cells (Th3). Tregs represent only a small fraction, 5–10% in mice and 1-2% in humans, of the overall CD4+ T-cells in lymphoid tissues but are essential for immunoregulatory circuits mediating the inhibition and expansion of all lineages of T-cells. In this paper, we first provide an overview of the major cell-intrinsic developmental programs that regulate T-cell lineage fates in thymus and periphery. Next, we introduce the SV40 immortomouse as a relevant mice model for implementation of new approaches to investigate thymus organogenesis, CD4 and CD8 development, and thymus cells tumorogenesis.

The Role of M. leprae Hsp65 Protein and Peptides in the Pathogenesis of Uveitis

Experimental autoimmune uveitis (EAU) is a well established model for immune-mediated organ-specific disease. Our group has recently shown that the M. leprae Hsp65 aggravated the uveitis in mice; in the present study, we evaluated the action of M. leprae  K409A mutant protein and the synthetic peptides Leader pep and K409A pep (covering amino acids residues 352–371 of WT and K409A proteins of M. leprae Hsp65, resp.) on the pathogenesis of EAU. Mice received the 161–180 IRBP peptide and B. pertussis toxin followed by the intraperitoneal inoculation of K409A protein or the Leader pep or K409A pep. The Leader pep aggravated the disease, but mice receiving the K409A pep did not develop the disease and presented an increase in IL-10 levels by spleen cells and a decrease in the percentage of CD4+ IFN-γ+ T cells. Moreover, animals receiving the Leader pep presented the highest scores of the disease associated with increase percentage of CD4+ IFN-γ+ T cells. These results would contribute to understanding of the pathogenesis of EAU and support the concept that immune responses to Hsp are of potential importance in exacerbating, perpetuating, or even controlling organ-restricted autoimmune diseases, and it is discussed the irreversibility of autoimmune syndromes.

Increased interferon-mediated immunity following in vitro and in vivo Modafinil treatment on peripheral immune cells

&NA; The wake‐promoting drug Modafinil has been used for treatment of sleep disorders, such as Narcolepsy, excessive daytime sleepiness and sleep apnea, due to its stimulant action. Despite the known effect of Modafinil on brain neurochemistry, particularly on brain dopamine system, recent evidence support an immunomodulatory role for Modafinil treatment in neuroinflammatory models. Here, we aimed to study the effects of in vitro and in vivo Modafinil treatment on activation, proliferation, cell viability, and cytokine production by immune cells in splenocytes culture from mice. The results show that in vitro treatment with Modafinil increased Interferon (IFN)‐&ggr;, Interleukin (IL)‐2 and IL‐17 production and CD25 expression by T cells. In turn, in vivo Modafinil treatment enhanced splenocyte production of IFN‐&ggr;, IL‐6 and tumor necrosis factor (TNF), and increased the number of IFN‐&ggr; producing cells. Next, we addressed the translational value of the observed effects by testing PBMCs from Narcolepsy type 1 patients that underwent Modafinil treatment. We reported increased number of IFN‐&ggr; producing cells in PBMCs from Narcolepsy type 1 patients following continuous Modafinil treatment, corroborating our animal data. Taken together, our results show, for the first time, a pro‐inflammatory action of Modafinil, particularly on IFN‐mediated immunity, in mice and in patients with Narcolepsy type 1. The study suggests a novel effect of this drug treatment, which should be taken into consideration when given concomitantly with an ongoing inflammatory or autoimmune process. HighlightsRecent studies indicated immunomodulatory role for the psychostimulant Modafinil.Our data showed increased splenic IL‐2, IFN‐&ggr; and IL‐17 after in vitro Modafinil treatment.In vivo Modafinil increased splenic production of IFN‐&ggr;, IL‐6 and TNF and number of IFN‐producing cells.Modafinil increased IFN‐producing cells in humans after continuous treatment.Data supports the pro‐inflammatory effect of Modafinil treatment in peripheral immune cells.

A Zika virus-associated microcephaly case with background exposure to STORCH agents

We present a case of microcephaly associated with Zika virus (ZIKV) in a chronological, multimodal imaging approach, illustrating the hallmarks of this disease on intrauterine morphological ultrasound, transfontanelar ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). We also determined the serological e immunological status of the mother and newborn. Noticeably, there was evidence for maternal infection by ZIKV, cytomegalovirus (CMV), herpes simplex virus (HSV), dengue virus (DENV) and Toxoplasma gondii, which indicates a possible role of previous exposures to STORCH agents and possibly comorbidities in the severe fetal congenital manifestation. Author Summary Zika virus (ZIKV) is an emerging mosquito-borne arbovirus causing dengue-like symptoms. In humans the illness is characterized by malaise and cutaneous rash and absent or short-termed fever. Recently, the Brazilian Ministry of Health reported an outbreak of microcephaly in Brazil as a delayed effect of the 2014-2015 outbreak of ZIKV in the Northeast of Brazil. A 20-fold increase in the notifications of newborns with microcephaly was documented during the second semester of 2015. This increase was almost immediately found to be associated with ZIKV infections, both in Brazil and, retrospectively, in French Polynesia. Herein we report a case of microcephaly associated with ZIKV and we also present evidence of other maternal infections. Our results indicated that, both mother and microcephaly infant had immunologic status compatible with previous exposure (in the mother) by STORCH agents. These indicate a possible role of previous exposures and possibly comorbidities in the severe fetal congenital manifestation. □