Cytokine responses differ by compartment and wasting status in patients with HIV infection and healthy controls

Inflammatory cytokines are implicated in the loss of lean tissue that occurs in patients with inflammatory and infectious diseases, including HIV infection. However, it is not known whether plasma levels or cellular production of cytokines, or their antagonists, are more closely related to lean tissue loss. We studied whether plasma cytokine analysis could substitute for PBMC production assays in studies of nutrition status and disease state, and if cytokine antagonists could offer an alternative in assessing cytokine status. We used a bout of moderately difficult exercise to perturb cytokine production in 12 adults with HIV without wasting, 10 adults with HIV wasting, and nine healthy controls. Plasma and peripheral blood mononuclear cell (PBMC) production of interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1ra) and soluble TNF receptor type II (sTNFrII) were measured at baseline and 2, 6, 24 and 168h following exercise. PBMC production of IL-1beta, TNF-alpha and IL-6 were all higher in the HIV-infected patients without wasting than in the controls (P<0.05) or the patients with AIDS wasting (P<0.05). Plasma concentrations of TNF-alpha and IL-6 were higher in the HIV wasted patients than in the controls (P<0.05). Both plasma and PBMC levels of sTNFrII were higher in HIV patients, regardless of wasting, than in controls. These data suggest that the PBMC cytokine compartment is more sensitive to nutritional and metabolic abnormalities than is the plasma compartment. PBMC production of IL-1beta, IL-6 and TNF-alpha best distinguish between HIV patients with and without wasting, while plasma concentrations of IL-6 and TNF-alpha are elevated in AIDS wasting, but do not reliably distinguish patients with wasting from HIV-infected patients without wasting.     

Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue

High circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are found in patients with hyperinsulinemia. Insulin stimulates release of IL-6 from adipocyte cultures, and it stimulates IL-6 gene expression in insulin-resistant, but not control, rat skeletal muscle. In addition, TNF-alpha may be involved in the pathogenesis of insulin resistance. Therefore, we studied the effect of insulin on IL-6 and TNF-alpha gene expression in human skeletal muscle and adipose tissue. Nine healthy young volunteers participated in the study. They underwent a 6-h hyperinsulinemic euglycemic clamp at a fixed insulin infusion rate, with blood glucose clamped at fasting level. Blood samples drawn at 0, 1, 2, 3, 4, 5, and 6 h were analyzed for IL-6 and TNF-alpha. Muscle and fat biopsies, obtained at 0, 2, 4, and 6 h, were analyzed for IL-6 and TNF-alpha mRNA with real-time PCR. IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, in adipose tissue (ANOVA P = 0.027), whereas there was no significant effect of insulin on skeletal muscles. Plasma IL-6 increased during insulin stimulation. TNF-alpha mRNA increased 2.4-, 1.4-, and 2.2-fold in adipose tissue (ANOVA P = 0.001) and decreased 0.74-, 0.64-, and 0.68-fold in muscle tissue (ANOVA P = 0.04). Plasma levels of TNF-alpha were constant. In conclusion, the finding that insulin stimulates IL-6 and TNF-alpha gene expression in adipose tissue only and inhibits the TNF-alpha production in skeletal muscles suggests a differential regulation of muscle- and adipose tissue-derived IL-6 and TNF-alpha.     

Phenotypic and genotypic alterations characterize patients bearing plasma cell dyscrasias with a high M-component

As at present only a long-term follow-up can fully determine whether monoclonal gammapathies of undetermined significance (MGUS) will evolve into multiple myeloma (MM), this study attempted to identify other variables connected with the amount of monoclonal component (MC), generally considered as the most reliable marker of malignant evolution. Thirty-four MGUS subjects showing a high MC (> or = 15.0 g/l) but without clinical evidence of MM (MGUS group b), were characterized for their phenotypic and genotypic profile by comparing them either with 40 MM patients or with 24 subjects affected by a benign form of monoclonal gammapathy (MGUS group a) according to the standard criteria. In addition to the usual laboratory markers, the levels of expression of a panel of CD membrane subsets were measured on B and T lymphocytes. Also, the serum level of the p53 mutant protein and the structural alterations of the c-myc oncogene were evaluated. The results show that for MGUS group b patients, an increased M-protein was accompanied by significantly increased levels of peripheral blood CD3+ T cells and oncogenetic aberrations in c-myc. Since a high serum MC level seems to indicate a greater likelihood of malignant transformation for MGUS patients, these findings suggest that this relationship may be a result of the concomitant alterations observed at a phenotypic and genotypic level. Such alterations may be potentially useful as surrogate markers for the transition of benign to malignant (MM) plasma cell dyscrasia.     

Honey stimulates inflammatory cytokine production from monocytes

Clinical observations indicate that honey may initiate or accelerate the healing of chronic wounds and has, therefore, been claimed to have anti-inflammatory properties. The aim of this study was to investigate the effects of honey on the activation state of immunocompetent cells, using the monocytic cell line, MonoMac-6 (MM6), as a model.We investigated the effect of each of the three honeys (manuka, pasture and jelly bush) on the release of important inflammatory cytokines from MM6 cells. These honeys, together with a sugar syrup control (artificial honey), were incubated with MM6 cells at a concentration of 1% (w/v) for 0-24h. Cell culture supernatants were tested using specific ELISA assays for tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta and IL-6. All honeys significantly increased the TNF-alpha, IL-1beta and IL-6 release from MM6 cells (and human monocytes) when compared with untreated and artificial-honey-treated cells (P<0.001). Jelly bush honey significantly induced the maximal release of each cytokine compared with manuka, pasture or artificial honeys (P<0.001).These results suggest that the effect of honey on wound healing may in part be related to the stimulation of inflammatory cytokines from monocytic cells. Such cell types are known to play an important role in healing and tissue repair.     

Muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase in human skeletal muscle

Physiological stress associated with muscle damage results in systemic insulin resistance. However, the mechanisms responsible for the insulin resistance are not known; therefore, the present study was conducted to elucidate the molecular mechanisms associated with insulin resistance after muscle damage. Muscle biopsies were obtained before (base) and at 1 h during a hyperinsulinemic-euglycemic clamp (40 mU x kg(-1) x min(-1)) in eight young (age 24+/-1 yr) healthy sedentary (maximal O(2) consumption, 49.7+/-2.4 ml x kg(-1) x min(-1)) males before and 24 h after eccentric exercise (ECC)-induced muscle damage. To determine the role of cytokines in ECC-induced insulin resistance, venous blood samples were obtained before (control) and 24 h after ECC to evaluate ex vivo endotoxin-induced mononuclear cell secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Glucose disposal was 19% lower after ECC (P<0.05). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was 45% lower after ECC (P<0.05). Insulin-stimulated phosphatidylinositol (PI) 3-kinase, Akt (protein kinase B) serine phosphorylation, and Akt activity were reduced 34, 65, and 20%, respectively, after ECC (P < 0.05). TNF-alpha, but not IL-6 or IL-1beta production, increased 2.4-fold 24 h after ECC (P<0.05). TNF-alpha production was positively correlated with reduced insulin action on PI 3-kinase (r = 0.77, P = 0.04). In summary, the physiological stress associated with muscle damage impairs insulin stimulation of IRS-1, PI 3-kinase, and Akt-kinase, presumably leading to decreased insulin-mediated glucose uptake. Although more research is needed on the potential role for TNF-alpha inhibition of insulin action, elevated TNF-alpha production after muscle damage may impair insulin signal transduction.     

Immune status evaluation of patients with chronic heart failure

Chronic heart failure (CHF) may be considered a state of immune activation and persistent inflammation expressed by increased circulating levels of pro- and anti-inflammatory cytokines. The purpose of the study was to investigate the immune status in patients with CHF compared to normal individuals. We measured serum cytokine levels as well as cytokine production after ex vivo LPS stimulation of whole blood taken from 14 patients with CHF and 14 healthy volunteers. We used 500 pg/ml of LPS for an incubation period of 4h to stimulate 100 microL of whole blood. Patients with CHF had significantly higher levels of TNF-RI, and TNF-RII in serum compared to normal individuals. TNF-alpha, IL-6, and IL-10 did not differ significantly. After LPS stimulation, patients with CHF had significantly higher levels of TNF-alpha and IL-10, and significantly lower IL-6 levels compared to normal individuals. TNF-alpha receptors did not differ significantly. Patients with CHF may be found in a pro- as well as an anti-inflammatory state. They also do not develop endotoxin tolerance in an ex vivo laboratory model using whole blood stimulated with LPS. They may have increased TNF-alpha and IL-10 production after LPS stimulation of whole blood, which may contribute to a worsening of heart function, more severe disease presentation and a worse outcome during infections.     

Detection of human P2X7 nucleotide receptor polymorphisms by a novel monocyte pore assay predictive of alterations in lipopolysaccharide-induced cytokine production

The nucleotide receptor P2X(7) is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X(7) gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X(7) pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 +/- 4; AC, 42 +/- 4; and CC, 6 +/- 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X(7) pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-alpha (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-alpha to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X(7) polymorphisms to regulate the LPS-induced TNF-alpha to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X(7) pore activity.     

Increased production of IL-1alpha and TNF-alpha in lipopolysaccharide-stimulated blood from obese patients with non-alcoholic fatty liver disease

Enhanced pro-inflammatory cytokine production is considered a pathogenic factor in non-alcoholic fatty liver disease (NAFLD). Peripheral blood production of interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) was studied in relation to the severity of histological changes of the liver in obese NAFLD patients. Basal levels in serum and production of IL-1alpha and TNF-alpha in peripheral blood cell cultures after stimulation with lipopolysaccharide (enzyme-linked immunoabsorbent assays) were measured in 11 patients with steatosis and 15 with steatohepatitis, who underwent gastrectomy with a gastro-jejunal anastomosis in roux and Y, and in 9 controls who underwent anti-reflux surgery. Production of IL-1alpha and TNF-alpha was 122 and 67% higher in patients with steatosis than control values, respectively. In patients with steatohepatitis, IL-1alpha production was 300 and 80% higher and that of TNF-alpha 110 and 26% higher, as compared with controls and steatosis patients, respectively. Production of IL-1alpha was positively correlated with that of TNF-alpha (r=0.78, p<0.0001). IL-1alpha and TNF-alpha production were both positively correlated with the degree of steatosis (r=0.68, p<0.001 and r=0.74, p<0.0001) and steatohepatitis (r=0.77 and r=0.75, p<0.0001) at liver biopsy, and with the homeostasis model assessment index (r=0.73, p<0.0001 and r=0.63, p<0.01), respectively. Basal serum IL-1alpha and TNF-alpha levels were comparable in the three groups studied. It is concluded that elevated production of IL-1alpha and TNF-alpha by in vitro stimulated whole blood cell cultures occurs in NAFLD obese patients, which might play a pathophysiological role upon inflammatory leukocyte infiltration of the liver.     

Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and ¹²⁵I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5–3.8 fold) and decrease in intact IGFBP-3 (0.6–0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.     

Cytokine secretion after cardiac surgery and its relationship to postoperative fever

A relationship between the inflammatory response to cardiopulmonary bypass (CPB) and fever after coronary artery bypass graft surgery (CABG) is assumed, but has not been studied. Therefore, we sought to assess the temporal pattern of cytokines' elevation and its association with post-CABG fever. In 355 primary elective CABG patients, serum cytokines (TNF-alpha, IL-1ra, IL-1beta, IL-6, and IL-8) were measured before surgery, at cessation of CPB and 2.5, 4.5, 24, and 48 h post-CPB. Fever was defined as a temperature >38 degrees C. TNF-alpha, IL-1beta and IL-8 peaked within the first 2.5 h after bypass, returning to near normal levels by 24h and increasing again by 48 h. IL-6 peaked early after bypass and remained elevated at 48 h. IL-1ra was elevated early, before returning to baseline by 24 h. Postoperative fever developed in 27% of patients. Increased IL-6 levels and male gender were significant predictors of fever (C-index=0.68; p=0.0003). No other cytokine showed a significant association with fever development. Of note is the previously undescribed bimodal pattern of cytokines' secretion after CABG. The association of fever with IL-6 levels suggests inflammatory mediation.     

Serum levels of interleukin-15 and interleukin-10 and their correlation with proliferating cell nuclear antigen in multiple myeloma

In order to determine prognostic factors characterizing multiple myeloma (MM) cell kinetics, bone marrow proliferative activity and serum Interleukin-10 (IL-10), and Interleukin-15 (IL-15) levels were measured in 40 newly diagnosed MM patients, compared with 10-age and sex-matched-healthy controls. Cell proliferation was evaluated by employing a monoclonal antibody directed against the proliferating cell nuclear antigen (PCNA), whereas IL-10 and IL-15 were measured with quantitative sandwich enzyme immunoassay methods. IL-15, IL-10 and PCNA were higher in the patient group than in controls (P<0.001). IL-10 levels, and PCNA increased significantly with increasing Durie-Salmon disease stage (I-III, P<0.002, and P=0.001, respectively). Serum IL-15 levels in MM stage III patients were elevated in comparison with stages I and II, the difference however, did not reach statistical significance. There was a significant positive correlation between serum IL-15 and IL-10 levels (r: 0.372, P<0.01), and between serum IL-10 and PCNA (r: 0.608, P<0.0001), as well as a positive correlation of serum IL-15 with PCNA, which marginally failed to reach statistical significance. Serum IL-15 levels are elevated in MM patients, increase with advancing stage, and correlate with Il-10 and PCNA. These proliferative factors may be useful in assessing disease progression in MM.     

Oxidative stress precedes peak systemic inflammatory response in pediatric patients undergoing cardiopulmonary bypass operation

Oxidative stress seems to contribute to cardiopulmonary bypass (CPB)-related postoperative complications. Pediatric patients are particularly prone to these complications. With this in mind, we measured oxidative stress markers in blood plasma of 20 children undergoing elective heart surgery before, during, and up to 48 h after cessation of CPB, along with inflammatory parameters and full analysis of iron status. Ascorbate levels were decreased by approximately 50% (P < 0.001) at the time of aorta cross-clamp removal (or pump switch-off in 4 patients with partial CPB), and associated with corresponding increases in dehydroascorbate (P < 0.001, r = -0.80) and malondialdehyde (P < 0.01, r = -0.59). In contrast to the immediate oxidative response, peak levels of IL-6 and IL-8 were not observed until 3-12 h after CPB cessation. The early loss of ascorbate correlated with duration of CPB (P < 0.002, r = 0.72), plasma hemoglobin after cross-clamp removal (P < 0.001, r = 0.70), and IL-6 and IL-8 levels at 24 and 48 h after CPB (P < 0.01), but not with postoperative lactate levels, strongly suggesting that hemolysis, and not inflammation or ischemia, was the main cause of early oxidative stress. The correlation of ventilation time with early changes in ascorbate (P < 0.02, r = 0.55), plasma hemoglobin (P < 0.01, r = 0.60), and malondialdehyde (P < 0.02, r = 0.54) suggests that hemolysis-induced oxidative stress may be an underlying cause of CPB-associated pulmonary dysfunction. Optimization of surgical procedures or therapeutic intervention that minimize hemolysis (e.g., off-pump surgery) or the resultant oxidative stress (e.g., antioxidant treatment) should be considered as possible strategies to lower the rate of postoperative complications in pediatric CPB.     

Tumour necrosis factor-alpha, interleukin-6, and fasting serum insulin correlate with clinical outcome in metastatic breast cancer patients treated with chemotherapy

INTRODUCTION: To assess the relationship of various growth factors and cytokines with the clinical outcome in metastatic breast cancer patients receiving chemotherapy. METHODS: Consecutive, metastatic breast cancer patients with measurable disease and receiving palliative chemotherapy were prospectively evaluated for the predictors of progression free survival (PFS) and overall survival (OAS) in relation to serum insulin, insulin resistance, interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). RESULTS: Estrogen receptor (ER) status, serum IL-6 and serum TNF- were the independent determinants of PFS, with RR=0.28 (0.13-0.60), P=0.001, RR=2.48 (1.24-5.61), P=0.012, and RR=0.48 (0.23-1.01), P=0.053, respectively. The factors related with OAS in the multivariate analysis were histological grade (RR=7.88 (2.33-26.62), P=0.001), ER status (RR=0.18 (0.06-0.57), P=0.003), serum insulin (RR=0.87 (0.77-0.97), P=0.016), and serum IL-6 (RR=5.99 (1.89-18.97), P=0.002). CONCLUSIONS: We show for the first time that fasting serum insulin and TNF-alpha levels are independent predictors for OAS and PFS, respectively, in metastatic breast cancer patients. In addition, we also confirm that IL-6 is a poor prognosticator in this group. These results suggest that insulin and TNF-alpha are important biomolecules that may be directly involved in vivo in the progression of metastatic breast cancer.     

Tumor necrosis factor is a survival and proliferation factor for human myeloma cells

Interleukin-6 (IL-6) is a major survival factor for malignant plasma cells. In patients with multiple myeloma (MM), cell lines whose survival and proliferation are dependent upon addition of exogenous IL-6 have been obtained. We show here that tumor necrosis factor-alpha (TNF-alpha) is also a survival factor for myeloma cell lines, although less potent than IL-6. The survival activity of TNF-alpha is not affected by anti-IL-6 or anti-gp130 monoclonal antibodies (mAbs). TNF-alpha also induces myeloma cells in the cell cycle and promotes the long-term growth of malignant plasma cell lines. As TNF-alpha is produced in patients with MM and associated with a poor prognosis, these results suggest that anti-TNF-alpha therapies could be useful in this disease.     

Rare Circulating Cells in Familial Waldenstr?m Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization

The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10⁻⁷). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.     

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.     

连续肾脏替代治疗对脓毒症患者血清中TNF-alpha、IL-6 和IL-8 表达的影响

目的：探讨连续肾脏替代治疗（CRRT）对脓毒症患者血清中肿瘤坏死因子alpha（TNF-alpha）、白介素-6（IL-6）和白介素-8（IL-8）的 影响。方法：将我院2013 年1 月-2014 年6 月间收治的80 例脓毒症患者随机分为观察组与对照组各40 例,两组患者均给予脓毒 症常规治疗,观察组另给予CRRT 治疗。观察比较两组患者治疗前1 天,治疗后24 h,72 h空腹静脉血TNF-alpha、IL-6、IL-8 水平。结 果：观察组治愈率为85.0%（34/40）,明显高于对照组的55.0%（22/40）,差异有统计学意义（P<0.05）;治疗24h、72h 后两组患者 TNF-alpha、IL-6和IL-8 水平均明显下降,其中观察组下降更显著,差异均有统计学意义（P<0.05）。结论：CRRT 能有效降低脓毒症患 者血清中TNF-alpha、IL-6 和IL-8 水平,有助于对炎症反应的正向调节。     

Effect of pro-inflammatory cytokines on spontaneous apoptosis in leukocyte sub-sets within a whole blood culture

Pro-inflammatory cytokines are known to affect apoptosis in human peripheral blood cells. Neutrophils, which are an essential component of the immune response and usually undergo apoptosis rapidly, are greatly affected by these cytokines. In this study, the effect of varying concentrations of TNF-alpha, IL-1beta and IL-6 on the apoptotic response of leukocytes and their sub-sets in cultured whole blood were studied over a 48 h culture period. At clinically relevant concentrations, it was found that these pro-inflammatory cytokines reduced the amount of spontaneous apoptosis in neutrophils in culture, but had little effect on the lymphocyte population. Distinct differences in the sensitivity of neutrophils to cytokine-mediated protection against spontaneous apoptosis were apparent when compared to previous studies conducted using purified or enriched neutrophil cultures. IL-1beta, at a dose of 0.01 pg/mL, was observed to significantly inhibit spontaneous neutrophil apoptosis by approximately 90% and 65% at 24 and 48 h of culturing, respectively. This concentration used in whole blood is dramatically lower than that required to elicit similar protection in neutrophil-enriched cell cultures. Higher concentrations of TNF-alpha (1.0 pg/mL) and IL-6 (125 pg/mL) were also found to significantly inhibit neutrophil apoptosis, at levels much lower than previously published using neutrophil-enriched cultures. Furthermore, each cytokine displayed a unique signature with respect to the optimal applied doses required to elicit maximal protection against spontaneous neutrophil apoptosis. These results demonstrate the dramatic differences in cellular responses that exist between neutrophil-enriched cultures and whole blood culture systems, where multiple blood cell types provide a much more complex environment.     

长效干扰素对CHB患者CD4^＋T细胞ICOS表达及血清IFN-γ、IL-4水平的影响

目的探讨长效干扰素对慢性乙型肝炎患者外周血CD4^＋T细胞上ICOS表达及血清IFN-γ、IL-4水平的影响。方法慢性乙型肝炎患者52例,其中聚乙二醇干扰素α2a治疗28例,常规治疗24例,另征集健康志愿者20例为正常对照组。采集治疗前及治疗后24、48周的患者外周血,以FCM检测ICOS^＋CD4^＋T细胞在PBMC中的频数变化;以ELISA检测治疗前后患者血清中IFN-γ、IL-4的水平变化;以Realtime—PCR检测患者HBV—DNA载量变化。结果慢性乙肝患者CD4^＋T细胞ICOS表达水平明显高于正常对照者（P〈0．001）,干扰素治疗者48周时ICOS表达水平低于常规治疗者,差异有统计学意义（P〈0．01）。经干扰素治疗后,患者Th1细胞因子IFN-γ水平升高,与常规治疗者相比有差异有统计学意义（P〈0．001）;而Th2细胞因子IL-4水平逐渐降低,与常规治疗者相比差异有统计学意义（P〈0．001）。干扰素治疗者ICOS、HBV-DNA载量变化值同IFN-γ水平的变化值呈负相关（P〈0．001）,而与IL4水平的变化值则为正相关（P〈0．001）。结论慢性乙肝患者存在着细胞免疫紊乱,干扰素治疗可以在一定程度纠正患者体内的Th2偏移,降低CD4^＋T细胞上ICOS的表达,促进IFN-γ表达,发挥抗病毒作用。     

Macrolide antibiotics modulate ERK phosphorylation and IL-8 and GM-CSF production by human bronchial epithelial cells

Macrolide antibiotics decrease proinflammatory cytokine production in airway cells from subjects with chronic airway inflammation. However, in subjects with chronic obstructive pulmonary disease, short-term azithromycin (AZM) therapy causes a transient early increase in the blood neutrophil oxidative burst followed by a decrease in inflammatory markers with longer administration. We studied the effects of clarithromycin (CAM) and AZM on proinflammatory cytokine production from normal human bronchial epithelial (NHBE) cells. CAM decreased IL-8 over the first 6 h and then significantly increased interleukin (IL)-8 at 12-72 h after exposure (P < 0.0001). AZM also increased IL-8 at 24 and 48 h, and CAM increased granulocyte-macrophage colony-stimulating factor at 48 h. In the presence of LPS, both CAM and AZM dose-dependently increased IL-8 secretion over 24 h, but after 5 days of exposure to 10 microg/ml CAM there is suppression of IL-8 (P < 0.001). PD-98059, an inhibitor of MAP kinase/ERK kinase, inhibited CAM-induced IL-8 (P < 0.0001) and GM-CSF (P < 0.01) release. The p38 MAP kinase inhibitor SB-203580 increased CAM-induced IL-8 release (P < 0.001), and the c-jun NH2-terminal kinase inhibitor SP-600125 had no effect on IL-8. At 120 min and 6 h, CAM increased phospho-ERK1/2 (pERK) but not phospho-p38 or phospho-JNK. Over the first 90 min, CAM at 10 microg/ml inhibited pERK and then increased pERK in parallel with measured IL-8 secretion. After daily CAM exposure for 5 days, both IL-8 and pERK returned to baseline. The p38 MAP kinase inhibitor, SB-203580 increased ERK phosphorylation and IL-8 secretion. These results suggest that macrolide antibiotics can differentially modulate proinflammatory cytokine secretion in NHBE cells, in part through ERK.