CCR5-Positive Inflammatory Monocytes are Crucial for Control of Sepsis.

Neutrophils and inflammatory monocytes control sepsis by migration to the site of infection via their chemokine receptors. CCR5 is a chemokine receptor that is not expressed on neutrophils and inflammatory monocytes under homeostatic conditions. However, it has been demonstrated that CCR5 can become expressed on these cells during different models of inflammation. In the present study, we investigated if CCR5 is also expressed on neutrophil and inflammatory monocytes during sepsis, exerting an important role in the migration of these cells to the infectious focus. Using cecal ligation and puncture model to induce polymicrobial sepsis, we demonstrated that the expression of CCR5 is induced on CD11bLy6GLy6C inflammatory monocytes, but not on neutrophils (CD11bLy6GLy6C). Furthermore, CCR5 plays an important role for the migration of the inflammatory monocytes to infection focus during sepsis. CCR5-expressing inflammatory monocytes migrate from the bone marrow to the circulation and then into the site of infection, where they phagocytize and kill the bacteria. Consequently, CCR5 mice showed increased systemic inflammatory response and mortality compared to wild-type mice. These data therefore demonstrate a hitherto unrecognized protective role of CCR5 in sepsis.

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 with 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 with 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 nonsevere 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 with those of nonseptic 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.

The host control of a clinical isolate strain of P. aeruginosa infection is independent of Nod-1 but depends on MyD88.

OBJECTIVE AND DESIGN: The objective of this study was to investigate the role of Nod1 in the recruitment of neutrophils into the infection site and in the establishment of the inflammatory response elicited by a clinical isolate strain of P. aeruginosa in vivo, while comparing it to the well-established role of MyD88 in this process. SUBJECTS: Wild-type, Nod1-/- and MyD88-/- mice, all with a C57Bl/6 background. METHODS: Mice were intranasally infected with Pseudomonas aeruginosa DZ605. Bronchoalveolar lavage and blood were harvested 6 or 20 h post-infection for evaluating bacterial load, chemokine levels and neutrophil migration. Survival post-infection was also observed. RESULTS: We show here that wild-type and Nod1-/- mice induce similar lung chemokine levels, neutrophil recruitment, and bacterial load, thus leading to equal survival rates upon P. aeruginosa pulmonary infection. Furthermore, we confirmed the essential role of MyD88-dependent signalling in recruiting neutrophils and controlling P. aeruginosa-induced pulmonary infection. CONCLUSION: The results suggest that in contrast to MyD88, under our experimental conditions, the absence of Nod1 does not impair the recruitment of neutrophils in response to P. aeruginosa DZ605.

The aggravation of arthritis by periodontitis is dependent of IL-17 receptor A activation.

AIM: To evaluate whether Porphyromonas gingivalis-induced periodontitis aggravates the antigen-induced arthritis (AIA) model, and whether this effect is dependent on the Th17/IL-17 signalling pathway. MATERIALS AND METHODS: Antigen-induced arthritis was triggered by local injection of methylated bovine serum albumin into the knee joint of previously immunized C57BL/6 wild-type (WT) and IL-17 receptor A (IL-17RA)-knockout mice. Periodontal disease in naïve or arthritic mice was induced by oral infection with P. gingivalis. Animals were sacrificed 7, 15 and 30 days after infection. Alveolar bone loss, joint histopathology, articular hyperalgesia and joint cytokine production were assessed, in addition to the proportion of Th17 and Treg cells isolated from the inguinal lymph nodes. RESULTS: No influence of experimentally-induced arthritis was found on the alveolar bone resorption induced by P. gingivalis. However, mice with experimentally-induced arthritis that were exposed to P. gingivalis presented higher joint damage and Th17 frequencies when compared to non-infected mice. The aggravation of arthritis by periodontitis was accompanied by increased TNF and IL-17 production and articular neutrophil infiltration, whereas arthritis aggravation and changes in neutrophil infiltration were absent in IL-17RA-deficient mice. CONCLUSION: The effects of P. gingivalis-induced periodontitis on arthritis are dependent on Th17 expansion and IL-17RA signalling, which lead to increased neutrophil infiltration into the joints.

Paradoxical Roles of the Neutrophil in Sepsis: Protective and Deleterious.

Sepsis, an overwhelming inflammatory response syndrome secondary to infection, is one of the costliest and deadliest medical conditions worldwide. Neutrophils are classically considered to be essential players in the host defense against invading pathogens. However, several investigations have shown that impairment of neutrophil migration to the site of infection, also referred to as neutrophil paralysis, occurs during severe sepsis, resulting in an inability of the host to contain and eliminate the infection. On the other hand, the neutrophil antibacterial arsenal contributes to tissue damage and the development of organ dysfunction during sepsis. In this review, we provide an overview of the main events in which neutrophils play a beneficial or deleterious role in the outcome of sepsis.

Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis.

Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.

MyD88-, but not Nod1- and/or Nod2-deficient mice, show increased susceptibility to polymicrobial sepsis due to impaired local inflammatory response.

Pathogen recognition and triggering of the inflammatory response following infection in mammals depend mainly on Toll-like and Nod-like receptors. Here, we evaluated the role of Nod1, Nod2 and MyD88-dependent signaling in the chemokine production and neutrophil recruitment to the infectious site during sepsis induced by cecal ligation and puncture (CLP) in C57Bl/6 mice. We demonstrate that Nod1 and Nod2 are not involved in the release of chemokines and recruitment of neutrophils to the infectious site during CLP-induced septic peritonitis because these events were similar in wild-type, Nod1-, Nod2-, Nod1/Nod2- and Rip2-deficient mice. Consequently, the local and systemic bacterial loads were not altered. Accordingly, neither Nod1 nor Nod2 was involved in the production of the circulating cytokines and in the accumulation of leukocytes in the lungs. By contrast, we showed that MyD88-dependent signaling is crucial for the establishment of the local inflammatory response during CLP-induced sepsis. MyD88-deficient mice were susceptible to sepsis because of an impaired local production of chemokines and defective neutrophil recruitment to the infection site. Altogether, these data show that Nod1, Nod2 and Rip2 are not required for local chemokine production and neutrophil recruitment during CLP-induced sepsis, and they reinforce the importance of MyD88-dependent signaling for initiation of a protective host response.

Targeting neutrophils in sepsis.

Sepsis continues to have a high mortality rate worldwide. The multi-step effects of this syndrome make it difficult to develop a comprehensive understanding of its pathophysiology and to identify a direct treatment. Neutrophils play a major role in controlling infection. Interestingly, the recruitment of these cells to an infection site is markedly reduced in severe sepsis. The systemic activation of Toll-like receptors and high levels of TNF-α and nitric oxide are involved in the reduction of neutrophil recruitment due to down-regulation of CXCR2 in neutrophils. By contrast, CCR2 is expressed in neutrophils after sepsis induction and contributes to their recruitment to organs far from the infection site, which contributes to organ damage. This review provides an overview of the recent advances in the understanding of the role of neutrophils in sepsis, highlighting their potential as a therapeutic target.

Inibição da guanilato ciclase pelo azul de metileno no choque circulatório causado por pancreatite aguda necrosante: uma palavra de cuidado embasada em modelo suíno / Guanylate cyclase inhibition by methylene blue in circulatory shock caused by acute necrotizing pancreatitis: a word of caution based on a porcine model

OBJETIVO: estudar o uso terapêutico do bloqueio da guanilato ciclase pelo azul de metileno em um modelo experimental de pancreatite aguda grave em suínos. MÉTODOS: a pancreatite aguda necrotizante foi induzida em porcos anestesiados por infusão ductal pancreática retrógrada de 1ml/kg de taurocolato de sódio a 5% e 8U/kg de enteroquinase. Três grupos foram estudados (n=5): controle (C), pancreatite (PA), "bolus" de azul seguido por pancreatite (AM+PA). Os dados incluíram enzimas séricas e do líquido abdominal, variáveis hemodinâmicas, hemogasometria arterial, volume de líquido abdominal, marcadores inflamatórios plasmáticos, nitrito/nitrato e mieloperoxidase e malondialdeído plasmático. Aplicou-se a análise de variância seguida do pós-teste de Bonferroni (p<0,05). RESULTADOS: os valores de amilase e lipase foram três e dez vezes mais elevados no grupo PA. A atividade da mieloperoxidase foi 50% superior no grupo PA. Os dados hemodinâmicos indicaram choque hipovolêmico precoce seguido de choque cardiogênico. Observou-se grave translocação de líquidos para a cavidade peritoneal. A nitrito/nitrato plasmática permaneceu inalterada. O grupo AM+PA teve aumento de cinco vezes do mieloperoxidase em comparação com o grupo C. CONCLUSÕES: a utilização de azul de metileno em suínos com pancreatite não demonstrou efeitos significativos sobre variáveis hemodinâmicas e inflamatórias. Seu uso terapêutico na pancreatite necro-hemorrágica pode ser inadequado e extremo cuidado deve ser tomado dado o aumento da peroxidação lipídica evidenciado pelo aumento dos valores do malondialdeído.

OBJECTIVE: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs. METHODS: acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05). RESULTS: amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group. CONCLUSION: preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.

Heme oxygenase inhibition enhances neutrophil migration into the bronchoalveolar spaces and improves the outcome of murine pneumonia-induced sepsis.

A reduction of the neutrophil migration into the site of infection during cecal ligation and puncture-induced sepsis increases host mortality. Inhibition of heme oxygenase (HO) prevents this neutrophil paralysis and improves host survival in the cecal ligation and puncture model. Taking into account that almost 50% of all sepsis cases are a consequence of pneumonia, we designed the present study to determine the role of HO in an experimental model of pneumonia-induced sepsis. The objective of this study was to evaluate whether the inhibition of HO improves the outcome and pathophysiologic changes of sepsis induced by an intratracheal instillation of Klebsiella pneumoniae. The pretreatment of mice subjected to pneumonia-induced sepsis with ZnDPBG (zinc deuteroporphyrin 2,4-bis glycol), a nonspecific HO inhibitor, increased the number of neutrophils in the bronchoalveolar spaces, reduced the bacterial load at the site of infection, and prevented the upregulation of CD11b and the downregulation of CXCR2 on blood neutrophils. Moreover, the pretreatment with ZnDPBG decreased alveolar collapse, attenuating the deleterious changes in pulmonary mechanics and gas exchanges and, as a consequence, improved the survival rate of mice from 0% to ∼20%. These results show that heme oxygenase is involved in the pathophysiology of pneumonia-induced sepsis and suggest that HO inhibitors could be helpful for the management of this disease.

Guanylate cyclase inhibition by methylene blue in circulatory shock caused by acute necrotizing pancreatitis: a word of caution based on a porcine model.

OBJECTIVE: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs. METHODS: acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05). RESULTS: amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group. CONCLUSION: preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.

Toll-like receptor 9 activation in neutrophils impairs chemotaxis and reduces sepsis outcome.

OBJECTIVES: To investigate the role of toll-like receptor 9 on sepsis-induced failure of neutrophil recruitment to the site of infection. DESIGN: Prospective experimental study. SETTING: University research laboratory. INTERVENTIONS: Model of polymicrobial sepsis induced by cecal ligation and puncture in wild-type and toll-like receptor 9-deficient mice. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor 9-deficient mice with cecal ligation and puncture-induced severe sepsis did not demonstrate failure of neutrophil migration and consequently had a low systemic inflammatory response and a high survival rate. Upon investigating the mechanism by which toll-like receptor 9 deficiency prevents the failure of neutrophil migration, it was found that neutrophils derived from toll-like receptor 9--deficient mice with cecal ligation and puncture-induced severe sepsis expressed high levels of chemokine C-X-C motif receptor 2 (CXCR2) and had reduced induction of G-protein-coupled receptor kinase 2. CONCLUSIONS: These findings suggest that the poor outcome of severe sepsis is associated with toll-like receptor 9 activation in neutrophils, which triggers G-protein-coupled receptor kinase 2 expression and CXCR2 downregulation. These events account for the reduction of neutrophil migration to the site of infection, with consequent spreading of the infection, onset of the systemic inflammatory response, and a decrease in survival.

Divergent role of heme oxygenase inhibition in the pathogenesis of sepsis.

The reduction of neutrophil migration to an infectious focus is associated with a high mortality in severe sepsis. Previously, we showed that heme oxygenase (HO) products downregulate neutrophil recruitment in a noninfectious inflammatory model. The present study was designed to determine the role of HO in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pretreatment, but not the combination of pretreatment plus posttreatment with zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to the site of infection. Consequently, bacterial dissemination, systemic inflammatory response, and organ injury were prevented. In addition, pretreatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to severe CLP, the pretreatment, but not the combination of pretreatment plus posttreatment with ZnPP IX, prevented the increase of plasmatic free heme observed in nontreated severe CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pretreatment with the HO inhibitor prevents the pathological findings in severe CLP. However, the combination of pretreatment plus posttreatment with ZnPP IX enhances sepsis severity because of an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.

Metalloproteinase inhibition protects against cardiomyocyte injury during experimental acute pulmonary thromboembolism.

OBJECTIVES: Up-regulated matrix metalloproteinases may be involved in the development of cardiomyocyte injury and the degradation of troponin associated with acute pulmonary thromboembolism. We examined whether pretreatment with doxycycline (a nonspecific matrix metalloproteinase inhibitor) protects against cardiomyocyte injury associated with acute pulmonary thromboembolism. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Mongrel dogs. INTERVENTIONS: Anesthetized animals received doxycycline (10 mg/kg intravenously) or saline and acute pulmonary thromboembolism was induced with autologous blood clots injected into the right atrium. Control animals received doxycycline (or saline). MEASUREMENTS AND MAIN RESULTS: Hemodynamic measurements were performed, and acute pulmonary thromboembolism increased baseline mean pulmonary arterial pressure and pulmonary vascular resistance by approximately 160% and 362%, respectively (both p < .05), 120 mins after acute pulmonary thromboembolism. Pretreatment with doxycycline attenuated these increases (to 125% and 232%, respectively; both p < .05). Although acute pulmonary thromboembolism tended to increase the right ventricle maximum rate of isovolumic pressure development and the maximum rate of isovolumic pressure decay, doxycycline produced no effects on these parameters. Gelatin zymograms of right ventricle showed that acute pulmonary thromboembolism marginally increased matrix metalloproteinase-9 (but not matrix metalloproteinase-2) levels in the right ventricle. A fluorometric assay to assess net matrix metalloproteinase activities showed that acute pulmonary thromboembolism increased matrix metalloproteinase activities in the right ventricle by >100% (p < .05), and this finding was confirmed by in situ zymography of the right ventricle. Doxycycline attenuated acute pulmonary thromboembolism-induced increases in right ventricle matrix metalloproteinase activities. Acute pulmonary thromboembolism induced neutrophil accumulation in the right ventricle, as estimated by myeloperoxidase activity, and doxycycline blunted this effect (p < .05). Serum cardiac troponin I concentrations, which reflect cardiomyocyte injury, increased after acute pulmonary thromboembolism, and this increase was attenuated by pretreatment with doxycycline (p < .05). CONCLUSIONS: We found evidence supporting the idea that acute pulmonary thromboembolism is associated with increased matrix metalloproteinase activities in the right ventricle, which may lead to degradation of sarcomeric proteins, including cardiac troponin I. Inhibition of matrix metalloproteinases may be an effective therapeutic intervention in the management of acute pulmonary thromboembolism.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1ß maturation.

BACKGROUND: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1ß and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). RESULTS: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE2 and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1ß and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)α and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1ß and PGE2 did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-α and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1ß was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE2 production in the paw of WT mice, but was reduced in casp1-/- mice. CONCLUSION: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1ß maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE2 production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila.

The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases.

Role of regulatory T cells in long-term immune dysfunction associated with severe sepsis.

OBJECTIVE: To investigate the role of regulatory T cells in the modulation of long-term immune dysfunction during experimental sepsis. It is well established that sepsis predisposes to development of a pronounced immunosuppression. Nevertheless, the mechanisms underlying the immune dysfunction after sepsis are still not well understood. DESIGN: Prospective experimental study. SETTING: University research laboratory. INTERVENTIONS: Wild-type mice underwent cecal ligation and puncture and were treated with antibiotic during 3 days after surgery. On days 1, 7, or 15 after cecal ligation and puncture, the frequency of regulatory T cells, proliferation of CD4 T cells and bacterial counts were evaluated. Fifteen days after cecal ligation and puncture, surviving mice underwent secondary pulmonary infection by intranasal inoculation of nonlethal dose of Legionella pneumophila. Some mice received agonistic glucocorticoid-induced tumor necrosis factor receptor antibody (DTA-1) before induction of secondary infection. MEASUREMENTS AND MAIN RESULTS: Mice surviving cecal ligation and puncture showed a markedly increased frequency of regulatory T cells in thymus and spleen, which was associated with reduced proliferation of CD4 T cells. Fifteen days after cecal ligation and puncture, all sepsis-surviving mice succumbed to nonlethal injection of L. pneumophila. Treatment of mice with DTA-1 antibody reduced frequency of regulatory T cells, restored CD4 T cell proliferation, reduced the levels of bacteria in spleen, and markedly improved survival of L. pneumophila infection. CONCLUSION: These findings suggest that regulatory T cells play an important role in the progression and establishment of immune dysfunction observed in experimental sepsis.

IL-17 receptor signaling is required to control polymicrobial sepsis.

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis.

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.

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.