Characteristics of cerebrospinal fluid in neonatal purulent meningitis / 中国小儿急救医学

Objective To analyze the changes of WBC classification,sugar and protein in cerebro-spinal fluid(CSF)and pathogenic bacteria of neonatal purulent meningitis.Methods Thirty-one neonates with bacterial meningitis in our department of neonatology from June 201 1 to June 2013 were enrolled,and the clinical features,pathogenic bacteria,laboratory examination of CSF were analyzed.Results Fever (90.3%),convulsions(67.7%)and changed consciousness(58.1 %)were common clinical symptoms.The incidences of other nervous system abnormal signs such as gastrointestinal dysfunction(25.8%),abnormal breathing(16.1 %),cervical resistance(16.1 %),bulging fontanel(9.7%)were lower.The Gram -negative bacteria was more than Gram -positive in both blood and CSF culture.The escherichia coli was the most common bacteria,with positive rate of 38.1 %(8 /21 )in blood culture and 55.5%(5 /9)in CSF culture.The germiculture positive rate in CSF was lower than in blood culture (29.0% vs.67.7%).Polymorphonuclear leukocyte(PMN)[(79.61 ±12.06)%]was the most predominant cell of the leukocyte classification in CSF within 1 week in all cases,PMN was still predominant in 1 to 2 weeks in 7 cases,while only 2 cases in 2 to 3 weeks still dominated by PMN,PMN was not the predominant cell 3 weeks later.Conclusion In the typi-cal neonatal purulent meningitis,PMN was the predominant cell in CSF within the first week,but the propor-tion of monocyte gradually increased and was dominant later.Escherichia coli was a common bacteria caused by this disease.

Correlação entre a contagem automática de células somáticas e a porcentagem de neutrófilos pela citometria de fluxo e pela técnica de citocentrifugação / Correlation between automatic somatic cell count and the percentage of neutrophils through flow cytometry and cytocentrifugation technique

O objetivo do presente estudo foi avaliar a correlação entre a contagem automática de células somáticas (CCS) com a porcentagem de neutrófilos pela técnica de citocentrifugação e pela citometria de fluxo. Para tal, 102 amostras de leite proveniente de 28 vacas da raça Holandesa foram coletadas e submetidas ao isolamento de células do leite e posterior identificação da população de neutrófilos. Após citocentrifugação, os neutrófilos foram identificados por microscopia óptica utilizando-se o corante de Rosenfeld. Os neutrófilos lácteos foram identificados por citometria de fluxo utilizando anticorpo monoclonal específico (CH138A) e anticorpo monoclonal secundário conjugado à ficoeritrina. O presente estudo demonstrou correlação positiva entre a CCS e a porcentagem de neutrófilos por citometria de fluxo (r= 0,625) e pela técnica de citocentrifugação (r= 0,267). Observou-se também correlação positiva entre a porcentagem de neutrófilos pela citometria de fluxo e pela técnica de citocentrifugação (r = 0,496), embora a porcentagem de neutrófilos no leite tenha sido maior pela técnica de citocentrifugação quando comparada com a citometria de fluxo. Deste modo, o presente estudo indica que a citometria de fluxo pode ser uma ferramenta útil no diagnóstico e controle da mastite bovina.

The purpose of the present study was to assess the correlation between the automatic somatic cell count (SCC) and the percentage of neutrophils through cytocentrifugation technique and flow cytometry. Thus, 102 milk samples from 28 Holstein dairy cows were collected and submitted to milk cell isolation procedures, and afterwards, the neutrophils were identified. After cytocentrigugation, the neutrophils were microscopically identified using the rosenfeld dye. The milk neutrophils were recognized by flow cytometry using primary mouse IgM monoclonal antibody (CH138A) and phycoerytrin (PE) goat anti-mouse IgM antibody. This study found a positive correlation between SCC and the percentage of neutrophils through cytocentrifugation (r= 0.267) and flow cytometry (r= 0.625). A positive correlation was also encountered between the percentage of neutrophils through cytocentrifugation and flow cytometry (r= 0.496), although the percentage of neutrophils was higher in samples submitted to cytocentrifugation. In conclusion, flow cytometry can be a useful tool in the diagnosis and control of mastitis.

Muerte leucocitaria por toxicidad del propóleo / Leukocyte death incited by propolis toxicity

Objetivo: El propósito de este estudio fue evaluar el grado de citotoxicidad in vitro que presenta el propóleo sobre leucocitos polimorfonucleares. Material y métodos: Estudio experimental, controlado, in vitro. Procedimiento estadístico de: análisis de varianza de repetidas medidas y post-hoc de Scheffé, estableciendo una significancia estadística de p < 0.05. Se tomaron 10 mL de sangre venosa periférica de seis individuos sanos, ambos sexos entre 20 y 30 años de edad escogidos al azar para obtener los leucocitos. Resultados: Análisis de Scheffé con 95% de confiabilidad para comparación entre grupo control y grupo experimental: significativo con p 0.0001 para propóleo 1, control 1 y propóleo 2, control 2, para propóleo 1, propóleo 2, p 0.5002/control 1, control 2, p 0.9621. Conclusiones: El propóleo en dilución 1:4 aplicado a leucocitos polimorfonucleares durante1-2 horas en el experimento provocó muerte celular en un 70%; dando significancia estadística.

Objective: The aim of the present study was to assess the extent of propolis' in vitro cytotoxicity on polymorphonuclear leukocytes. Materials and methods: The present study was an in vitro, controlled, experimental endeavor. Statistical procedure entailed variance analysis of repeated measures as well as Scheffe's post-hoc. A p < 0.05 statistical significance was established. To obtain leukocytes, 10 mL of peripheral venous blood was harvested from six randomly selected, healthy, 20-30 year old subjects, of both genders. Results: Scheffe's analysis with 95% reliability for comparison between control and experimental groups. Significant with p 0.0001 for propolis 1 control 1 and propolis 2 control 2. For propolis 1 propolis 2 p 0.5002/control 1 control 2 p 0.9621. Conclusions: In the present experiment propolis at a 1:4 dilution applied for 1-2 hours to polymorphonuclear leukocytes caused 70% cellular death. This resulted in statistical significance.

Effects of in vitro selenium supplementation on blood and milk neutrophils from dairy cows / Efeito da suplementaçãoin vitro de selenio sobre neutrófilos do leite e sanguineos em vacas leiteiras

The study was designed to assess the effects of in vitro selenium addition on intracellular hydrogen peroxide production by neutrophils from the milk and blood of dairy cows. Blood from 10 dairy cows and 20 milk samples from five dairy cows were incubated with 0 mg (control) or 10μM of sodium selenite. Then, milk and blood neutrophils were submitted for evaluation of intracellular hydrogen peroxide production by flow cytometry using 2',7'-dichlorofluorescein diacetate as a probe. The selenium status of the animals was evaluated by determination of the blood glutathione peroxidase activity. The results of the present work showed that in vitro selenium supplementation leads to an enhancement in intracellular hydrogen peroxide production, which indicates an improvement in the bactericidal effects of blood and milk neutrophils even in cows with a selenium-adequate status. Thus, the present study showed that in vitro Se supplementation leads to an enhancement in intracellular hydrogen peroxide production, indicating an improvement in the bactericidal effects of blood and milk neutrophils in cows with Se-adequate status.

O presente estudo avaliou o efeito da suplementação in vitro de selênio sobre a produção intracelular de perόxido de hidrogênio (H2O2) por leucόcitos polimorfonucleares do leite e do sangue em bovinos. Assim, 10 e 20 amostras de sangue e leite, respectivamente, foram incubadas com 0 mg (controle) ou 10μM de selenito de sόdio. A determinação da produção intracelular de peróxido de hidrogênio se deu por citometria de fluxo através da utilização do 2´,7´ diclorodihidrofluoresceína diacetato como sonda. A mensuração do conteúdo de selênio foi avaliada pela atividade da glutationa peroxidase eritrocitária. Os leucócitos polimorfonucleares tanto sanguíneos quanto do leite apresentaram significativo aumento na produção intracelular de H2O2 com a suplementação in vitro de selênio. Desta forma, o presente estudo apontou para aumento da produção intracelular de H2O2, indicando aumento da capacidade microbicida dos leucócitos polimorfonucleares sanguíneos e lácteos mesmo em animais com níveis adequados de selênio.

Leukocytes and lactobacilli of vaginal discharge as a predictor of pelvic inflammatory disease / 대한산부인과학회지

OBJECTIVE: We performed this study to evaluate whether the vaginal polymorphonuclear leukocytes and few lactobacilli on microscopic evaluation of a saline wet preparations would be associated with pelvic inflammatory disease (PID) in Korean women. MATERIALS AND METHODS: We performed a cross-sectional study of 556 women between 16 and 78yrs of age from May 2001 to May 2003. Wet-mount microscopic examination of vaginal discharge was done on all patients. Positive vaginal polymorphonuclear leukocytes was defined as more than 10 white blood cells per high-power field on microscopic examination, and few lactobacilli was defined as less than 30 per high-power field on microscopic examination. The diagnosis of PID was relied on the minimal criteria delineated by the Centers for Disease Contral and Prevention, elevated CRP or ESR and positive anaerobic cultures. RESULTS: On univariate analysis, positive vaginal polymorphonuclear leukocytes and few lactobacilli were associated with PID, but age (less than 25) and marital status were not. On multivariate analysis using multiple logistic regression, odds ratios of positive vaginal polymorphonuclear leukocytes and few lactobacilli for PID were 5.995 (95%CI: 3.056-11.761) and 24.39 (95%CI: 10.989-55.556) respectively. The sensitivity and negative predictive value of positive vaginal polymorphonuclear leukocytes or few bacilli for predicting PID were 90.6% and 94.42% respectively. CONCLUSION: Positive vaginal polymorphonuclear leukocytes and few lactobacilli were strongly associated with PID. Positive vaginal polymorphonuclear leukocytes and few lactobacilli have a high sensitivity and negative predictive value for predicting PID. Therefore the existence of vaginal polymorphonuclear leukocytes and few lactobacilli are thought hereafter to be useful marker to diagnose PID.

Influence of Sex and Age on the Activity of Antioxidant Enzymes of Polymorphonuclear Leukocytes in Healthy subjects

In this study, the main antioxidant enzymes (AOE) of glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) were identified, and the influence of sex and age in healthy human polymorphonuclear leukocytes (PMNL) was determined. The SOD, GPX, CAT and MPO activities were investigated in intestinal parasite negative human PMNL from 109 healthy subjects aged from 6 to 70 years (55 males and 54 females) using simple and sensitive enzyme assays. Blood cells, such as eosinophils, platelets, neutrophils, monocytes, and macrophages also synthesize antioxidant enzymes (AOE). They constitute an important proportion and are also the major participants in a number of pathological conditions that suggest the involvement of AOE. A linear effect of age on SOD activity (p < 0.05) both in males and females was found. A similar effect with GPX activity (p < 0.05) was observed in males only. This showed that the activities of all these enzymes increase with age. In addition, SOD activity was significantly higher in females than males between the age of 19 and 70 years (p < 0.001). This analysis also showed that there is a negative correlation between the CAT-GPX (p < 0.05) activities and positive correlations between MPO-GPX (p < 0.05) activities only in females. No correlation among the other enzyme activities was found in either sex group. This study showed the activities of antioxidant enzyme activities and the correlations of these enyzmes activities with each other in healthy human PMNLs were age- and sex-dependent. This information may assisit in understanding the importance of antioxidant enzymes in the physiological and pathological conditions associated with PMNL.

Lack of IL-10 Release from Human Polymorphonuclear Leukocytes

PURPOSE: Polymorphonuclear leukocytes (PMNs) are the first line of cellular response for host defense during acute inflammation. The ability of PMNs to produce and release numerous pro-inflammatory cytokines is now estabilished and plays an important role in triggering and maintaining the inflammatory response. We studied the autocrine downregulation of this process by invesgating the potential production by human PMNs of the major anti-inflammatory cytokine, interleukine 10 (IL-10). METHODS: Human PMNs were isolated from the peripheral blood of health volunteers by using the modified boyum method. Human PMNs were incubated at 37 degrees Cwith and without formyl Met-Leu-Phe (fMLP) for 30 minute, 2 hours, 4 hours, and 20 hours. The level of IL-10 was determined in each of the cell-free supernatants by using the enzyme linked immunosorbent assay (ELISA) method. RESULTS: Non-stimulated PMNs generated 1.40 +/- 1.791pg/mL to 3.46 +/- 1.607 pg/mL of IL-10 over the time course. Stimulation with fMLP resulted in an increase in the constitutive PMN-derived IL-10 by 2.74 +/- 0.762 pg/mL, 1.27 +/- 0.262 pg/mL, 1.19 +/- 0.364 pg/mL, and 2.44 +/- 1.317 pg/mL at 30 min, 2 hr, 4 hr, and 20 hr after stimulation, respectively, but these increases were not statiscally significant. CONCLUSION: Human PMNs seem unable to induce release of the most potent anti-inflammatory cytokine, IL-10, and down-regulate inflammatory response due to the autocrine mechanism. This could partly account for the persistence of local inflammation, when PMNs are the main infiltrating cells.

IL-1 and TNF-alpha release in human polymorphonuclear leukocytes after exposure to calcium hydroxide treated Porphyromonas endodontalis lipopolysaccharide / 대한치과보존학회지

Bacterial lipopolysaccharide (LPS) plays a major role in stimulating the synthesis and release of the principal osteoclast-activating cytokines, namely, interleukin 1 and tumor necrosis factor-alpha from immune cells. Although monocytes/macrophages are the main producers of these cytokines, recent evidence has indicated that polymorphonuclear leukocytes (PMN) have the ability to release IL-1 and TNF-alpha. Calcium hydroxide has been shown to be an effective medicament in root canal infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The purpose of this study was to investigate whether treatment of Porphyromonas endodontalis LPS with calcium hydroxide alters its biological action as measured by human PMN secretion of IL-1 and TNF-alpha, and it was compared with Escherichia coli LPS. P. endodontalis ATCC 35406 was cultured in anaerobic condition, and LPS was extracted using the hot-phenol water extraction method and purified. Purchased E. coli LPS was also purified. 100 microg/ml of each LPS in pyrogen free water were incubated with 25mg/ml Ca(OH)2 at 37degrees C for 7 days. The supernatants were subjected to ultrafiltration, and the isolates were lyophilized and weighed. PMNs were obtained from peripheral blood by centrifugation layered over Lymphoprep. The cells were resuspended (4x106 cells/ml) in RPMI 1640 followed by treatment with various concentrations of LPS (0, 0.1, 1, 10microg/ml) for 24 hours at 37degrees C in 5% CO2 incubator. The supernatants of cells were collected and the levels of IL-1alpha, IL-1beta and TNF-alpha were measured by enzyme-linked immunosorbent assay. The results were as follows; 1. The levels of IL-1alpha, IL-1beta, TNF-alpha from PMN treated with each LPS were significantly higher than those released from unstimulated PMN of the control group (p0.05). 3. The levels of secretion for all three cytokines were affected in a dose-dependent manner in PMN stimulated with each LPS (p0.05). 4. The levels of all three cytokines released from PMN stimulated with P. endodontalis LPS were significantly lower than those released from PMN stimulated with E. coli LPS (p<0.05).

Apoptosis of Peripheral Blood Cells Stimulated by Lipopolysaccharide, Cytokines, or Escherichia coli / 감염

INTRODUCTION AND METHODS: Apoptosis is a natural suicidal mechanism of eukaryotic cells to maintain the homeostasis of hosts. It comprises important pathogenesis of autoimmune diseases, cancers, and some infectious diseases. To access the role of apoptosis in the pathogenesis of infectious diseases, we studied the difference in apoptotic patterns of polymorphonuclear leukocytes and of monocytes to various stimuli, such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)-alpa, interleukin (IL)-6, granulocyte, macrophage-colony stimulating factor (GM-CSF), or Escherichia coli (E. coli). The cell survival and expression of Fas and Fas ligand (FasL), bcl-2, and IL-1beta converting enzyme (ICE) were evaluated by flow cytometry, northern blotting or western blotting analysis. RESULTS: The survival of polymorphonuclear leukocytes and monocytes was prolonged after stimulation with LPS and cytokines. Stimulation of Fas with Fas monoclonal antibody induced apoptosis in both polymorphonuclear leukocytes and monocytes. Expressions of Fas and FasL were more dominant in polymorphonuclear leukocytes than in monocytes. bcl-2 was expressed onlyin monocytes and was associated with the de-lay of apoptosis. ICE is a common pathway to apoptosis and thus pretreatment with ICE inhibitor could partially inhibit the apoptosis in both cells. Interestingly, E. coli prolonged the life-span of polymorphonuclear leukocytes but accelerated apoptosis in monocytes in spite of the over-expression of bcl-2. CONCLUSION: Polymorphonuclear leukocytes and monocytes might have different apoptotic mechanisms to maintain their homeostasis. Polymorphonuclear leukocytes have only one mechanism to apoptosis, namely via Fas- FasL, whereas, monocytes have both Fas-FasL and bcl-2 mechanisms. These differences may be associated with their primary functions and natural life-span. Direct stimulation by live E. coli accelerated apoptosis in monocytes in spite of the over-expression of inhibitor of apoptosis, i.e. bcl-2. It could be due to activation of other apoptotic mechanisms by E. coli, which may detour the anti-apoptotic action of bcl-2 or direct inhibition of bcl-2 function.

Selective inhibition of polymorphonuclear leukocytes by immunosuppressive concentration of prostaglandin E2

Prostaglandin E2(PGE2) has been implicated as an immunosuppressive agent and plasma levels of PGE2 are elevated in patients sustaining thermal injury. We examined the effect of 10(-7) M prostaglandin E2(PGE2) on human polymorphonuclear leukocytes (PMN) to determine whether it directly inhibits stimulated responses of these cells. At this concentration, PGE2 alone was incapable of stimulating PMN intracellular hydrogen peroxide production (indirectly assayed by fluorescence of 2',7'ichlorofluorescin) or expression of the PMN CD11b/CD16 surface glycoproteins. PMN incubated in the presence of the soluble stimul phorbol myristate acetate(PMA, 100 ng/ml) or recombinant human C5a(rHC5a, 10(-8) M) generated significant amounts of hydrogen peroxide, increased their CD11b expression and decreased their CD16 expression. Pre-incubation of cells with PGE2 caused significant inhibition of all the observed changes stimulated by rHC5a. In contrast, events stimulated by PMA were not affected by preincubation of cells with PGE2. We conclude that PGE2, in concentrations identical to those found in the plasma of patients with burn injuries, is capable of selectively inhibiting some stimulated events and phenotypic expression of PMN in vitro study.

SCAVENGING EFFECT OF TOTAL FLAVONOIDS OF HIPPOPHAE ON ACTIVE OXYGEN RADICALS / 中国药理学通报

The scavenging effects of Total Flavonoids of Hippophae ( TFH ) were studied by using ESR-spin trapping technique in different systems, and the inhibitery effects on chemiluminescence ( CL ) of poly-morphonuclear leukocytes ( PMN) were measured by using luminol-de-pendent CL. TFH ( 1.7mg/L ) significantly reduced the active oxygen radicals level of PMA-stimulated PMN; TFH ( 0.03-3mg/L ) could remarkably scavenged O2 in xanthine/xanthine oxidase (Xan/XO)system and the effect was concentration-dependent. The scavenging effect of TFH on O2 in irradiation riboflavin system was not so strong as in Xan/XO system. TFH ( 3 mg/L ) could scavenged OH produced in Fenton's reaction; TFH ( 1 mg/L ) effectively inhibited PMN CL stimulated by PMA.The effects of TFH on active oxygen radicals produced by PMN and O2 in Xan/XO system were stronger than Vit E.

Effect of procyanidins on the release of H_2O_2 induced by croton oil and on lipid peroxidation / 中国药理学通报

AIM To study the anti oxidation and cancer chemoprevention mechanism of procyanidins extracted from Vitis vinifera. METHODS A simple colorimetric method is used for the measurement of H 2O 2 produced by PMNs induced by croton oil. RESULTS It was shown that procyanidins could inhibit PMNs releasing H 2O 2 induced by croton oil. Procyanidins serum of rats also had the same effect. The effect was time and dose dependent. Inhibitory effect of procyanidins on hepatic mitochondria lipid peroxidation induced by croton oil was also observed. Procyanidins could raise the activity of SOD and decrease the level of MDA. CONCLUSION It was concluded that anti oxidation effect of procyanidins was one important mechanism of cancer chemoprevention.