Papel da insulina na vigência da peritonite decorrente da infecção por Staphylococcus aureus em animais diabéticos e sadios / Insulin role in peritonitis presence due to Staphylococcus aureus infection in diabetic and healthy animals

A nefropatia diabética é uma doença crônica caracterizada por falência renal, que torna necessária a hemodiálise. A diálise peritoneal é uma alternativa para a hemodiálise, porém causa peritonite e morte, principalmente devido à infecção com Staphylococcus aureus, especialmente em pacientes imunodeprimidos, como pacientes diabéticos. Nossa hipótese é que a insulina possa modular a peritonite causada por S. aureus. Para tanto, investigamos sua intervenção, após a indução de diabetes mellitus, na infecção peritoneal por cepas diferentes de S. aureus, analisando os mecanismos moleculares (produção/liberação de citocinas, expressão de moléculas de adesão) e a atividade microbicida dos macrófagos peritoneais envolvidos. Ratos Wistar, machos, diabéticos (aloxana, 42 mg/kg, i.v., 10 dias) e respectivos controles (salina, i.v.) foram submetidos à injeção intraperitoneal de uma suspensão de S. aureus (5x109 CFU/mL) ou volume equivalente de PBS estéril. Os animais foram submetidos a dois tratamentos com insulina NPH: 1) dose única (1UI e 4UI respectivamente, controle e diabético), administrada por via subcutânea; ou, 2) com 4 doses sendo a primeira administrada 2 horas antes da infecção, seguida de metade desta dose às 17 horas e no mesmo horário pelos próximos 2 dias (dose inicial 4UI e 1UI, grupo diabético e grupo controle, respectivamente), passadas 16 horas da última dose de insulina, a glicemia foi determinada e, em seguida, foi realizada eutanásia e coleta de amostras. Avaliamos: a) número de células no lavado peritoneal (LPe), leucograma e glicemia (monitor de glicose); b) níveis séricos de corticosterona e insulina (ELISA); c) concentrações de citocinas (IL-1ß, TNF-α, IL-6, IFN-γ, IL-4, IL-10, IL-12) e quimiocinas (CINC-1, CINC-2, CINC-3) no sobrenadante do LPe (ELISA); d) expressão de moléculas de adesão (P-selectina, PECAM-1, ICAM-1) no endotélio vascular (imunoistoquímica); e) atividade microbicida. Após a infecção com a cepa ATCC 25923, comparados aos não infectados, ratos diabéticos apresentaram aumento no número de leucócitos (350%) e nas concentrações de CINC-1 (1900%), IL-1ß (1300%), IFN-γ (280%), IL-4 (800%). O tratamento destes animais com dose única de insulina diminuiu as concentrações de CINC-1 (17%) e IFN-γ (30%) e o leucócitos (55%); e aumentou a concentração de IL-4 (260%); enquanto o tratamento com 4 doses diminuiu o número de leucócitos (82%) e as concentrações de CINC-1 (96%) e CINC-2 (45%); e, aumentou as concentrações de TNF-α (270%), IFN-γ (220%), IL-1ß (42%), IL-6 (760%) e a expressão de ICAM-1 (1360%); enquanto as concentrações de CINC-3, IL-10 e IL-12 não foram alteradas pelos tratamentos com insulina. Após a infecção com a cepa N315 HLA+, comparados aos não infectados, ratos diabéticos apresentaram aumento do número de leucócitos (200%), nas concentrações de CINC-1 (1000%), IL-4 (860%), IFN-γ (200%) e na expressão de PECAM-1 (800%) e diminuição de CINC-2 (92%). O tratamento destes animais com dose única de insulina diminuiu a concentração de CINC-1 (85%); e aumentou a concentração de CINC-2 (2030%), IL-1ß (370%) e IL-4 (250%); enquanto o tratamento com 4 doses diminuiu a concentração de CINC-1 (92%); e, aumentou número de leucócitos (66%), as concentrações de CINC-2 (100%), IL-1ß (490%), IL-6 (1870%) e IFN-γ (330%), e os outros parâmetros não foram modificados pelos diferentes tratamentos com insulina. Estes dados sugerem que a insulina possa modular a peritonite induzida por cepas diferentes de S. aureus, controlando pelo menos em parte, o infiltrado inflamatório, a produção das citocinas CINC-1, CINC-2, IL-4, IL-6, IFN-γ, TNF-α e IL-1ß; e, consequentemente a expressão P-selectina e PECAM-1 no endotélio vascular do mesentério

Diabetic nephropathy is a chronic disease characterized by kidney failure, so hemodialysis is necessary. Peritoneal dialysis is an alternative to hemodialysis, but causes peritonitis and death primarily due to infection by Staphylococcus aureus, especially in immunocompromised patients, such as diabetics. Our hypothesis is that insulin can modulate peritonitis caused by S. aureus, therefore, we investigated its action, after diabetes induction, in peritoneal infection with different S. aureus strains, analyzing the molecular mechanisms (cytokines production/release, adhesion molecules expression) and the microbicidal activity of peritoneal macrophages involved. Wistar male diabetic (alloxan, 42 mg/kg, iv, 10 days) and their respective controls (saline,iv) were subjected to intraperitoneal injection of S. aureus suspension (5x109CFU/mL) or an equivalent volume of PBS sterile. Animals were submitted to two treatments with NPH insulin administered subcutaneously : single dose (1IU and 4IU respectively , control and diabetic) 8 hours prior to euthanasia ; or 4 doses : first dose 2 hours before infection (4IU and 1IU , diabetic and control group, respectively), then half this dose of 17 pm and in the same time for the next 2 days, after 16 hours of the last dose, blood glucose was determined, and then it was carried out euthanasia and sampling. We evaluated: a) number of cells in peritoneal wash (PW), white blood cell count and blood sugar (glucose monitor) ; b) serum insulin and corticosterone (ELISA); c) concentrations of cytokines (IL-1ß, TNF-α, IL-6, IFN-γ, IL-4, IL-10, IL-12) and chemokines (CINC -1, CINC-2, CINC-3 ) in supernatant of the SBA assay (ELISA); d) expression of adhesion molecules (P- selectin, ICAM-1, PECAM-1) in vascular endothelium ( immunohistochemistry); e) microbicidal activity. After infection with ATCC 25923 strain, compared to uninfected, diabetic rats showed an increase in leukocytes number (350%) and in concentrations of CINC-1 (1900%), IL-1ß (1300%), IFN-γ (280%), IL-4 (800%). Treatment of these animals with a single dose of insulin decreased concentrations of CINC-1 (17%) and IFN-γ (30%) and leukocytes number (55%); and increased IL-4 concentrations (260%); while treatment with 4 doses decreased leukocytes number (82%) and concentrations of CINC-1 (96%) and CINC-2 (45%); and increased concentrations of TNF-α (270%), IFN-γ (220%), IL-1ß (42%), IL-6 (760%) and expression of ICAM-1 (1360%); while the concentrations of CINC-3, IL-10 and IL-12 were not affected by treatments with insulin. After infection with N315 HLA+ strain, compared to uninfected, diabetic rats showed an increase in leukocytes (200%), concentrations of CINC-1 (1000%), IL-4 (860%), IFN-γ (200%) and in the expression of PECAM-1 (800%); and CINC-2 decrease (92%). Treatment of these animals with a single dose of insulin decreased the concentration of CINC-1 (85%); and increased concentrations of CINC-2 (2030%), IL-1ß (370%) and IL-4 (250%); while treatment with 4 doses decreased the concentration of CINC-1 (92%); and increased leukocytes number (66%), concentrations of CINC-2 (100%), IL-1ß (490%), IL-6 (1870%) and IFN-γ (330%), and other parameters were not modified by treatments with insulin. These results suggest that both S. aureus strains activate differently the innate response during peritonitis, and insulin was not always able to modulate this response

Avaliação clínica e hematológica de equinos submetidos ao modelo de laminite por oligofructose, tratados ou não com um agente antagonista de receptores CXCR1/2 / Clinical and hematological evaluation of horses subjected to oligofructose-induced laminitis treated or not with a CXCR1/2 antagonist

O recrutamento de leucócitos aos tecidos é uma parte essencial da resposta imune inata e esse processo de forma desregulada pode resultar em lesões aos tecidos. Assim, a infiltração de leucócitos tem sido implicada na patogênese de laminite aguda em equinos. Os objetivos dessa pesquisa foram verificar a ação da ICXCR1/2 sobre os sinais clínicos e parâmetros hematológicos de cavalos com laminite induzida por oligofrutose. Doze equinos receberam oligofrutose (10g/kg de peso vivo PO) no tempo 0 e foram divididos em 2 grupos: tratados (30mg/kg p.v. ICXCR1/2 IV, nos tempos 6, 12, 18 e 24 h) e não tratados. As frequências cardíaca e respiratória, temperatura retal, coloração de membranas mucosas, presença e intensidade de pulso digital, sensibilidade ao exame com pinça de casco e grau de claudicação segundo Obel, bem como parâmetros hematológicos e bioquímicos (hemograma e as concentrações sanguíneas de glicose, uréia, creatinina, ALT, AST, FA, GGT, bilirrubina total e proteína total) foram aferidos nos tempos 0, 6, 12, 18, 24, 36, 48, 60 e 72 horas . O modelo usando oligofructose foi adequado para induzir sinais de laminite e de sinais de endotoxemia, como diarreia, febre e leucocitose em cavalos sem raça definida de origem nacional. Também, não foram observadas quaisquer reações adversas clínicas ou hematológicas relacionadas ao uso intravenoso do antagonista de CXCR1/2, contudo essa substância, quando administrada na dose de 30mg/kg de peso vivo, 4 vezes ao dia, por 4 aplicações, não foi capaz de prevenir os sinais clínicos e as alterações hematológicas causadas pela administração de oligofructose nos equinos deste estudo.

Leucocytes recruitment to tissues is an essential part of the innate immune response and an unregulated process can result in tissue damage. Thus, leucocytes infiltration has been implicated in the pathogenesis of acute laminitis. The objectives of this stud were to determine the effect of an antagonist for CXCR1/2, a chemokine receptor for neutrophils attraction on clinical signs and hematological parameters in horses given oligofructose to induce laminitis. Twelve horses were given oligofructose (10g/kg bw PO) in time 0 and divided into two groups: one treated (30mg/kg bw. ICXCR1/2 IV, times 6, 12, 18 e 24 h) and the other not treated. Cardiac and respiratory frequency, rectal temperature, mucous membrane colour, digital pulse, hoof sensitivity and Obel's grade of lameness were recorded. Values for RBC, WBC and blood glucose, BUN, creatinin, ALT, AST, alkaline phosphatase, GGT, total bilirubin and serum protein concentrations were measured on times 0, 6, 12, 18, 24, 36, 48, 60 e 72 h. All the horses given oligofructose developed signs of endotoxemia like diarrhea, fever and leukocytosis and laminitis. Also, CXCR1/2 antagonist treatment did not cause any adverse effects. However, this substance when injected intravenously (30mg/kg) 6/6 hours in 4 applications, did not ameliorate clinical and hematological signs of endotoxemia.

Inhibition of Janus activated kinase-3 protects against myocardial ischemia and reperfusion injury in mice

Recent studies have documented that Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can modulate the apoptotic program in a myocardial ischemia/reperfusion (I/R) model. To date, however, limited studies have examined the role of JAK3 on myocardial I/R injury. Here, we investigated the potential effects of pharmacological JAK3 inhibition with JANEX-1 in a myocardial I/R model. Mice were subjected to 45 min of ischemia followed by varying periods of reperfusion. JANEX-1 was injected 1 h before ischemia by intraperitoneal injection. Treatment with JANEX-1 significantly decreased plasma creatine kinase and lactate dehydrogenase activities, reduced infarct size, reversed I/R-induced functional deterioration of the myocardium and reduced myocardial apoptosis. Histological analysis revealed an increase in neutrophil and macrophage infiltration within the infarcted area, which was markedly reduced by JANEX-1 treatment. In parallel, in in vitro studies where neutrophils and macrophages were treated with JANEX-1 or isolated from JAK3 knockout mice, there was an impairment in the migration potential toward interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), respectively. Of note, however, JANEX-1 did not affect the expression of IL-8 and MCP-1 in the myocardium. The pharmacological inhibition of JAK3 might represent an effective approach to reduce inflammation-mediated apoptotic damage initiated by myocardial I/R injury.

p38 MAPK and ERK activation by 9-cis-retinoic acid induces chemokine receptors CCR1 and CCR2 expression in human monocytic THP-1 cells

9-cis-retinoic acid (9CRA) plays an important role in the immune response; this includes cytokine production and cell migration. We have previously demonstrated that 9CRA increases expression of chemokine receptors CCR1 and CCR2 in human monocytes. To better understand how 9CRA induces CCR1 and CCR2 expression, we examined the contribution of signaling proteins in human monocytic THP-1 cells. The mRNA and surface protein up-regulation of CCR1 and CCR2 in 9CRA-stimulated cells were weakly blocked by the pretreatment of SB202190, a p38 MAPK inhibitor, and PD98059, an upstream ERK inhibitor. Activation of p38 MAPK and ERK1/2 was induced in both a time and dose-dependent manner after 9CRA stimulation. Both p38 MAPK and ERK1/2 phosphorylation peaked at 2 h after a 100 nM 9CRA treatment. 9CRA increased calcium influx and chemotactic activity in response to CCR1-dependent chemokines, Lkn-1/CCL15, MIP-1alpha/CCL3, and RANTES/CCL5, and the CCR2-specific chemokine, MCP-1/CCL2. Both SB202190 and PD98059 pretreatment diminished the increased calcium mobilization and chemotactic ability due to 9CRA. SB202190 inhibited the expression and functional activities of CCR1 and CCR2 more effectively than did PD98059. Therefore, our results demonstrate that 9CRA transduces the signal through p38 MAPK and ERK1/2 for CCR1 and CCR2 up-regulation, and may regulate the pro-inflammatory process through the p38 MAPK and ERK-dependent signaling pathways.

Immunology of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease caused predominantly by the ubiquitous fungus Aspergillus fumigatus. ABPA is characterized by eosinophilia, fleeting pulmonary infiltrates, central bronchiectasis, elevated serum IgE and Aspergillus specific IgG and IgE. The pathogenetic mechanism implicated in ABPA is not completely understood. The cytokine response detected in ABPA patients is of a CD4+ Th2 type as evidenced by the production of IL-4, IL-5, and very little or no IFN-g on stimulation of T-lymphocytes with Aspergillus antigens. Animal model studies using wild type and gene knockout mice indicate a more precise mechanism of lung injury in antigen exposed animals. IL-4 knockout mice invariably showed a predominant Th1 response. B-cell deficient and IgE knockout mice exposed to A. fumigatus antigens showed airway response similar to wild type mice indicating a lesser role for IgE and other antibodies in the pathogenesis of murine experimental ABPA. RAG negative mice failed to show airway hyperreactivity response, although airway hyperreactivity was induced in naive RAG negative animals when T-cells from wild type Aspergillus immunized mice were transferred. The results of these studies indicate a multi-factorial immunopathogenesis in ABPA, which include T-cells, IgE, eosinophils, mast cells, and various cytokines and chemokines.