The atypical chemokine receptor ACKR2 is protective against sepsis

Sepsis is a systemic inflammatory response as a result of uncontrolled infections. Neutrophils are the first cells to reach the primary sites of infection and chemokines play a key role in recruiting neutrophils. However, in sepsis chemokines could also contribute to neutrophil infiltration to vital organs leading to multiple organ failure. ACKR2 is an atypical chemokine receptor, which can remove and degrade inflammatory CC chemokines. The role of ACK2 in sepsis is unknown. Using a model of cecal ligation and puncture (CLP), we demonstrate here that ACKR2 deficient (−/−) mice exhibited a significant reduction in the survival rate compared to similarly treated wild type (WT) mice. However, neutrophil migration to the peritoneal cavity and bacterial load were similar between WT and ACKR2−/− mice during CLP. In contrast, ACKR2−/− mice showed increased neutrophil infiltration and elevated CC chemokine levels in the lung, kidney and heart compared to the WT mice. In addition, ACKR2−/− mice also showed more severe lesions in the lung and kidney than those in the WT mice. Consistent with these results, WT mice under non-severe sepsis (90% survival) had higher expression of ACKR2 in these organs than mice under severe sepsis (no survival). Finally, the lungs from septic patients showed increased number of ACKR2+ cells compared to those of non-septic patients. Our data indicate that ACKR2 may have a protective role during sepsis, and the absence of ACKR2 leads to exacerbated chemokine accumulation, neutrophil infiltration and damage to vital organs

PPAR-γ/IL-10 axis inhibits MyD88 expression and ameliorates murine polymicrobial sepsis

Polymicrobial sepsis induces organ failure and is accompanied by overwhelming inflammatory response and impairment of microbial killing. Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. The insulin-sensitizing drugs thiazolidinediones (TZDs) are specific PPAR-γ agonists. TZDs exert anti-inflammatory actions in different disease models, including polymicrobial sepsis. The TZD pioglitazone, which has been approved by the U.S. Food and Drug Administration, improves sepsis outcome; however, the molecular programs that mediate its effect have not been determined. In a murine model of sepsis, we now show that pioglitazone treatment improves microbial clearance and enhances neutrophil recruitment to the site of infection. We also observed reduced proinflammatory cytokine production and high IL-10 levels in pioglitazone-treated mice. These effects were associated with a decrease in STAT-1-dependent expression of MyD88 in vivo and in vitro. IL-10R blockage abolished PPAR-γ-mediated inhibition of MyD88 expression. These data demonstrate that the primary mechanism by which pioglitazone protects against polymicrobial sepsis is through the impairment of MyD88 responses. This appears to represent a novel regulatory program. In this regard, pioglitazone provides advantages as a therapeutic tool, because it improves different aspects of host defense during sepsis, ultimately enhancing survival

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice

PREVENÇÃO DE MALOCLUSÃO: CONHECIMENTO DE MÃES DE CRIANÇAS COM 3 E 4 ANOS

a maloclusão é classificada pela Organização Mundial da Saúde (OMS)como a terceira maior prioridade da Odontologia, referente a problemas de saúdebucal, sendo superada apenas pelas doenças cárie e periodontal. Tal patologiaassumiu aspectos tão significativos que se tornou um problema de saúde pública em nível mundial (DIAS; GLEISER, 2008). Acredita-se que o desconhecimento dasmães sobre os cuidados necessários para a prevenção da maloclusão, favorece agrande prevalência de mordida aberta anterior e mordida cruzada entre osportadores de hábitos bucais deletérios como a sucção não nutritiva e a utilização de chupeta. Nesse contexto o presente trabalho tem por objetivo analisar acompreensão das mães de crianças com 3 e 4 anos sobre os cuidados com a saúdebucal de seus filhos para evitar a maloclusão

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the “don’t eat me” ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)–specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies

Desenvolvimento e validação de método analítico para nistatina creme vaginal por cromatografia líquida de alta eficiência

A nistatina é um antibiótico poliênico, com características fungistáticas e fungicidas, que age desestruturando a membrana celular de fungos e leveduras. O creme vaginal de nistatina é usado para o tratamento da candidíase vaginal. Até recentemente, os compêndios oficiais preconizavam o ensaio microbiológico para doseamento deste antibiótico, método este considerado inviável na rotina de centros de controle de qualidade, devido ao tempo excessivo para liberação dos resultados. Visando obter um método alternativo para o doseamento do creme vaginal de nistatina, procurou-se desenvolver e validar um método cromatográfico (CLAE). O método desenvolvido utilizou como fase estacionária uma coluna de fase reversa, C18, 3,9 x 150 mm, 4 mm, à temperatura de 30 ºC. A fase móvel foi constituída por tampão fosfato de sódio 0,25 mM e EDTA 0,025 mM, pH 6,00, metanol e acetonitrila (40:30:30), vazão de 1,0 mL/minuto e comprimento de onda 305 nm. O método validado revelou-se exato, preciso, robusto, linear e específico, além de rápido e prático, podendo ser utilizado para o doseamento analítico de creme vaginal de nistatina.Nystatin is a polyenic antibiotic with fungistatic and fungicide characteristics that acts by de-structuring the cellular membrane of fungi and yeast. The nystatin vaginal cream is used for the treatment of vaginal candidiasis. Until recently, the official compendia professed the microbiological trial for dosing this antibiotic, method considered as non-feasible in the routine of quality control centers due to the excessive time for release of results. Aiming at obtaining an alternative method for dosing nystatin vaginal cream, a chromatographic method (HPLC) was developed and validated. The method developed used a reversible phase column of C18, 3.9 x 150 mm, 4 mm, at 30 ºC. The mobile phase was made up of a 0.25 mM sodium phosphate buffer and 0.025 mM EDTA, pH 6.00, methanol and acetonitrile (40:30:30), rate of 1.0 mL/minute and wavelength of 305 nm. The validated method showed to be accurate, precise, robust, linear and specific, in addition to being fast and practical, able to be used for analytic dosing of nystatin vaginal cream

Desenvolvimento e validação de método analítico para nistatina creme vaginal por cromatografia líquida de alta eficiência Development and validation of an analytical method for Nystatin vaginal cream by high performance liquid chromatography

A nistatina é um antibiótico poliênico, com características fungistáticas e fungicidas, que age desestruturando a membrana celular de fungos e leveduras. O creme vaginal de nistatina é usado para o tratamento da candidíase vaginal. Até recentemente, os compêndios oficiais preconizavam o ensaio microbiológico para doseamento deste antibiótico, método este considerado inviável na rotina de centros de controle de qualidade, devido ao tempo excessivo para liberação dos resultados. Visando obter um método alternativo para o doseamento do creme vaginal de nistatina, procurou-se desenvolver e validar um método cromatográfico (CLAE). O método desenvolvido utilizou como fase estacionária uma coluna de fase reversa, C18, 3,9 x 150 mm, 4 mm, à temperatura de 30 ºC. A fase móvel foi constituída por tampão fosfato de sódio 0,25 mM e EDTA 0,025 mM, pH 6,00, metanol e acetonitrila (40:30:30), vazão de 1,0 mL/minuto e comprimento de onda 305 nm. O método validado revelou-se exato, preciso, robusto, linear e específico, além de rápido e prático, podendo ser utilizado para o doseamento analítico de creme vaginal de nistatina.<br>Nystatin is a polyenic antibiotic with fungistatic and fungicide characteristics that acts by de-structuring the cellular membrane of fungi and yeast. The nystatin vaginal cream is used for the treatment of vaginal candidiasis. Until recently, the official compendia professed the microbiological trial for dosing this antibiotic, method considered as non-feasible in the routine of quality control centers due to the excessive time for release of results. Aiming at obtaining an alternative method for dosing nystatin vaginal cream, a chromatographic method (HPLC) was developed and validated. The method developed used a reversible phase column of C18, 3.9 x 150 mm, 4 mm, at 30 ºC. The mobile phase was made up of a 0.25 mM sodium phosphate buffer and 0.025 mM EDTA, pH 6.00, methanol and acetonitrile (40:30:30), rate of 1.0 mL/minute and wavelength of 305 nm. The validated method showed to be accurate, precise, robust, linear and specific, in addition to being fast and practical, able to be used for analytic dosing of nystatin vaginal cream

PPAR-γ/IL-10 axis inhibits MyD88 expression and ameliorates murine polymicrobial sepsis

Polymicrobial sepsis induces organ failure and is accompanied by overwhelming inflammatory response and impairment of microbial killing. Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. The insulin-sensitizing drugs thiazolidinediones (TZDs) are specific PPAR-γ agonists. TZDs exert anti-inflammatory actions in different disease models, including polymicrobial sepsis. The TZD pioglitazone, which has been approved by the U.S. Food and Drug Administration, improves sepsis outcome; however, the molecular programs that mediate its effect have not been determined. In a murine model of sepsis, we now show that pioglitazone treatment improves microbial clearance and enhances neutrophil recruitment to the site of infection. We also observed reduced proinflammatory cytokine production and high IL-10 levels in pioglitazone-treated mice. These effects were associated with a decrease in STAT-1-dependent expression of MyD88 in vivo and in vitro. IL-10R blockage abolished PPAR-γ-mediated inhibition of MyD88 expression. These data demonstrate that the primary mechanism by which pioglitazone protects against polymicrobial sepsis is through the impairment of MyD88 responses. This appears to represent a novel regulatory program. In this regard, pioglitazone provides advantages as a therapeutic tool, because it improves different aspects of host defense during sepsis, ultimately enhancing survival

Bi-specific autoantigen-T cell engagers as targeted immunotherapy for autoreactive B cell depletion in autoimmune diseases

IntroductionIn autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need.MethodsHere, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A2 receptor (PLA2R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA2R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker.ResultsBiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig+ and T cells. Ex vivo, the BiAATE successfully induced T cell-dependent depletion of PLA2R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA2R antibody levels following active immunization with PLA2R.DiscussionShould this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases