Comparison of oxidative stress & leukocyte activation in patients with severe sepsis & burn injury.

Background & objectives: We evaluated pro- and anti-oxidant disturbances in sepsis and non-sepsis burn patients with systemic inflammatory response syndrome (SIRS). Adhesion molecules and inflammation markers on leukocytes were also analyzed. We hypothesized that oxidative stress and leukocyte activation markers can lead to the severity of sepsis. Methods: In 28 severe sepsis and 27 acute burn injury patients blood samples were collected at admission and 4 days consecutively. Oxidative stress markers: production of reactive oxygen species (ROS), myeloperoxidase, malondialdehyde and endogenous antioxidants: plasma protein sulphydryl groups, reduced glutathione, superoxide dismutase and catalase were measured. Flow cytometry was used to determine CD11a, CD14, CD18, CD49d and CD97 adhesion molecules on leukocytes. Procalcitonin, C-reactive protein, fibrinogen, platelet count and lactate were also analyzed. Results: Pro-oxidant parameters were significantly elevated in sepsis patients at admission, ROS intensity increased in burn patients until the 5th day. Endogenous antioxidant levels except catalase showed increased levels after burn trauma compared to sepsis. Elevated granulocyte activation and suppressed lymphocyte function were found at admission and early activation of granulocytes caused by increasing activation/migration markers in sepsis. Leukocyte adhesion molecule expression confirmed the suppressed lymphocyte and monocyte function in sepsis. Interpretation & conclusions: Severe sepsis is accompanied by oxidative stress and pathological leukocyte endothelial cell interactions. The laboratory parameters used for the evaluation of sepsis and several markers of pro- and antioxidant status were different between sepsis and non-sepsis burn patients. The tendency of changes in these parameters may refer to major oxidative stress in sepsis and developing SIRS in burns.

Possible involvement of granulocyte oxidative burst in Nrf2 signaling in cancer.

The activation process of granulocytes is accompanied by the intense production of reactive oxygen species (ROS). Overproduction of ROS is cytotoxic, damages macromolecules and can lead to the occurrence of lipid peroxidation. Cellular defense against the toxicity of ROS is enhancement of detoxifying enzymes activation. Regulation of many detoxifying enzymes is mediated by the antioxidant response element (ARE) that is located in the promoter region of related genes. In eukaryotes, there are only few transcription factors known to be activated by ROS. One of them is NF-E2-related factor 2 (Nrf2). Normally, Nrf2 is present in the cytoplasm as an inactive Keap1-Nrf2 complex. However, after direct attack by ROS, Nrf2 is released from Keap1 repression and translocated into nucleus where it binds with ARE sequence to initiate gene expression. ROS may also influence nuclear factor-κB (NF-κB) intracellular signaling repressing the Nrf2-ARE pathway at transcriptional level. Since ROS are crucial in granulocyte-mediated tumor cell lysis the induction of NF-κB signaling pathway may be an important mechanism in suppressing the tumor growth.

Assessment of inflammation based on the release of oxygen radicals by granulocytes in chronic uncontrolled asthma / Avaliação da inflamação com base na liberação de radicais oxidantes por granulócitos na asma crônica não-controlada

Objetivo: Avaliar a liberação espontânea de ânion superóxidopor granulócitos de sangue periférico de pacientescomasma crônica não-controlada antes e após corticoterapia e de indivíduos sadios. Métodos: Foram estudados 32 pacientes entre 6 e 18 anos (média 12,04 anos) e 29 indivíduos sadios como grupo decomparação.Os pacientes oramagrupados de acordocomo volumeexpiratório forçado no primeiro segundo: grupo I, volume expiratórioforçado no primeiro segundo entre 60 e 80%, 19 pacientes; e grupo II, volume expiratório forçado no primeiro segundo = 60%, 13 pacientes. A liberação espontânea de superóxido por granulócitos, medida por espectrofotometria utilizando superóxido dismutase, foi avaliada nos pacientes antes e após o tratamento com prednisona por via oral e beclometasona, budesonida ou fluticasona administradas por via inalatória. Na análise estatística foramutilizados os testes de análise de variância, Tukey e de Wilcoxon.Resultados: Comparando-se a liberação de ânion superóxido por granulócitos dos pacientes asmáticos e indivíduos sadios observamosque a liberação foi maior nos asmáticos não-controladosdo grupo II (p < 0,05). Avaliando-se a liberação de superóxido pelas células dos pacientes antes e após a terapiacomcorticosteroideuma diminuição significativa foi observada apenas no grupo I. Conclusão: O impacto dos glicocorticoides sobre a modulação da inflamação ocorreu nos indivíduos asmáticos não-controlados com volume expiratório forçado no primeiro segundo entre 60 e 80%.Naqueles com volume expiratório forçado no primeiro segundo = 60não foi observada essa modulação, havendo necessidade de mais estudos para avaliar o impacto de tal achado nos pacientes asmáticos.

Objective: To evaluate spontaneous release of superoxide anion by peripheral blood granulocytes of atopic patients with uncontrolled asthma undergoing glucocorticoid therapy and of healthy subjects. Methods: We studied 32 patients, aged 6 to 18 (mean 12.04), and 29 healthy subjects as a comparative group. Patients weregrouped according to the forced expiratory vital capacity in the firstsecond. Group I, forced expiratory vital capacity in the first second of between60and80%,had 19 patients, and group II, forced expiratoryvital capacity in the first second=60%,had 13 patients. Spontaneous superoxide release by granulocytes was measured by aspectrophotometer method based on superoxide dismutase, before and after oral prednisone and beclomethasone, budesonide or fluticasone inhaled therapy. Statistical analyses were performed using ANOVA, Wilcoxon and Tukey tests. Results: Comparing the superoxide anion release bygranulocytes of asthmatic patients and healthy subjects,weobserved a higher release by cells of the uncontrolled patient group II (p < 0.05). Evaluating the superoxide release by cells of asthmatic patients before and after steroid therapy, a significant decrease was found only in patient group I.Conclusion: The impact of corticosteroids on inflammatorymodulation occurred in the uncontrolled asthmatics with forced expiratory vital capacity in the first second between 60 and 80%. In those with forced expiratory vital capacity in the first second of = 60%, this findingwas not observed. Further studies are necessary to evaluate the effect of this finding on asthmatic patients.

Dorfman-Chanarin syndrome.

A four-year-old girl was brought to the dermatology outpatient department with scaling all over the body since birth. She had history of episodic vomiting and abdominal distension. A dermatological diagnosis of lamellar ichthyosis was made. Abdominal examination revealed a nontender hepatomegaly, fatty liver on ultrasonography and deranged liver function tests. Peripheral blood smear showed lipid vacuoles in the granulocytes consistent with Jordans' anomaly. Similar lipid vacuoles were seen in the basal layer in skin biopsy. An inflammatory infiltrate, moderate fibrosis in the portal tract and diffuse severe fatty change in hepatocytes were seen in liver biopsy. The patient was diagnosed as a case of Dorfman-Chanarin syndrome.

Resposta terapêutica e inflamatória de ratos com peritonite secundária submetidos ao uso tópico de ampicilina/sulbactam / Therapeutical and inflammatory response in rats with secondary peritonitis and topical use of ampicilin/sulbactam

OBJETIVOS: A peritonite aguda representa uma importante causa de sepsis e óbito nas unidades de terapia intensiva e cirurgia. Classicamente o seu tratamento deve incluir: a administração sistêmica de antibióticos, a remoção mecânica dos contaminantes e a restauração da integridade gastrintestinal. A utilização de antibióticos diretamente na cavidade peritoneal é controversa. Estudo com o objetivo de avaliar o uso terapêutico, intraperitoneal da ampicilina associada ao sulbactam. MÉTODOS: foram mensurados os níveis plasmáticos do óxido nítrico, bem como a contagem de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal, utilizando-se modelo de peritonite em ratos (ligadura-transfixação cecal). Vinte quatro ratos Wistar, machos, foram divididos em quatro grupos de seis animais, assim distribuídos: grupo A: método de indução de peritonite - soltura da ligadura + tratamento com soro fisiológico; grupo B: método de indução de peritonite + soltura da ligadura + tratamento com soro fisiológico acrescido de ampicilina / sulbactam; grupo C: método de indução de peritonite + soltura da ligadura-transfixação cecal; e grupo D: laparatomia para realização de lavado peritoneal mais coleta de sangue. A ligadura-transfixação do cecum permaneceu por 24 horas, antes do tratamento instaurado. Foi realizada uma relaparotomia nos 18 ratos com coleta de líquido de lavado peritoneal e sangue. Foram dosados os níveis plasmáticos de óxido nítrico e determinado o número de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal. RESULTADOS: Não ocorreu diferença estatisticamente significante (p > 0,05) nos níveis de óxido nítrico, bem como no número de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal, entre os grupos. CONCLUSÃO: Neste estudo, concluiu-se que: a utilização de ampicilina associada a sulbactam por via intraperitoneal nos ratos com peritonite fecal: não modificou a sobrevida; não alterou os níveis plasmáticos de óxido nítrico; não alterou a contagem de eosinófilos, linfócitos, monócitos e neutrófilos tanto no sangue como no lavado peritoneal.

Genotypic and phenotypic implications in paroxysmal nocturnal hemoglobinuria (PNH): a preliminary investigation.

The genetic and biochemical defects underlying paroxysmal nocturnal hemoglobinuria (PNH) have recently been elucidated. The deficiency of the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins caused by a somatic mutation of the PIG-A gene, an X-chromosomal gene that participates in the first step of the GPI anchor synthesis, has been shown to be responsible for PNH in all patients. The mutations of PIG-A studied to date are highly heterogeneous. They are however mainly of the frameshift type (61.5%). The characteristic abnormalities of PNH phenotypes has also been shown especially by DAF- and/or CD59-based fluorescent immunocytometry. A great degree of heterogeneity in the patterns and levels of expression of GPI-anchored proteins in various cell types was demonstrated indicating a discrepancy of lineage involvement. In this investigation, major blood cell populations, i.e erythrocytes and granulocytes were analyzed immunophenotypically, the mutations of PIG-A were identified by heteroduplex analysis and nucleotide sequencing and the consequences of PIG-A mutations were observed. All the mutations identified in 9 patients with PNH resulted in complete loss of function as clones of affected granulocytes completely negative for CD59 expression were shown in all patients. Interestingly, granulocytes in these patients contained variable proportions of affected cells varied from 50% to 100% and four of the patients had erythrocytes with diminished expression of GPI-anchored DAF and CD59 coexisting with normal and completely negative cells. Immunophenotypic analysis of reticulocytes in peripheral blood of patients with PNH demonstrated the conserved patterns of DAF and CD59 expression in circulating erythroid cells and the discrepancies between granulocytic and erythroid lineages. These findings suggested that the characteristics of abnormal phenotypes which appear to be highly variable between different hematopoietic lineages are not solely caused by mutation of PIG-A but are influenced by other factor(s).

PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria.

Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.

Control y alteraciones de la granulopoyesis: neutropenias-neutrofilias / Control and changes in granulopoiesis: neutropenia-neutrophylias

Se analiza la cinética de los granulocitos en la médula ósea, sangre periférica y tejidos, y los mecanismos fisiológicos de control de la granulopoyesis con su variación en la enfermedad. Se describen los cuadros clínicos que producen una neutrofilia y una neutropenia, con los mecanismos fisiopatológicos involucrados. Hay una especial referencia a las drogas inductoras de neutropenia y su posible vía de acción