Airways hyperreactivity and inflammation produced by aerosolization of human C5A des arg

The premise of this study was that complement fragments would lead to granulocyte infiltration and to associated changes in airways reactivity. Inflammation was induced in large airways (greater than 1.0 mm) by aerosolization of the complement chemotactic factor, C5a des arg, which was isolated from activated human serum. Pulmonary function (resistance, compliance, and lung volume) and histamine reactivity were assessed at 4 and at 48 h after C5a des arg or a saline sham aerosol. Four hours after exposure to C5a des arg, the animals exhibited evidence of significant bronchoconstriction and hyperinflation. In addition, an increased responsiveness to histamine was demonstrated, which was not correlated with the degree of bronchospasm. By 48 h, these alterations were partially or completely resolved. Histologic examination and quantitation demonstrated significant accumulation of neutrophils, which was limited to airways 0.5 mm in or larger. Saline-treated animals also demonstrated a neutrophil infiltration, but significantly less than those treated with C5a des arg. However, these animals did not demonstrate hyperreactivity to histamine, and there was little change in baseline mechanical function. The mild inflammation associated in the saline control animals was demonstrated to be a result of intubation of the airways. Granulocyte dependence of the effects of C5a des arg was partially established by abrogation of these effects when the animals were rendered granulocytopenic with nitrogen mustard. We conclude that the physiologic responses of the airways to C5a des arg were largely granulocyte dependent. However, since granulocyte accumulation could occur without airways dysfunction, it is suggested that cell activation is an important step in producing neutrophil-associated airways hyperresponsiveness.     

The priming effects of the products of stimulated mononuclear cells on the response of neutrophils to C5a des arg

Certain recombinant human cytokines have been shown to enhance polymorphonuclear leucocyte (PMN) responses to subsequent stimulation. Mononuclear cells (MNC) from normal healthy individuals were stimulated for 5 h with 1 micrograms/ml bacterial lipopolysaccharide (LPS) in order to induce production and secretion of inflammatory cytokines into the surrounding medium. These mononuclear cell conditioned media (MNCM) were then used to prime PMN isolated from healthy volunteers. Preincubating the PMN with MNCM for 15 min at 4 degrees C followed by washing and warming to 37 degrees C caused a 344% increase (n = 26) in the rate of superoxide anion production in response to zymosan-activated serum (ZAS), a source of C5a des arg. This effect could not be reproduced with recombinant human forms of interleukin 1 beta (Il-1 beta) or granulocyte-macrophage-colony stimulating factor (GM-CSF), although, with the latter, there was some effect when the preincubation stage was carried out for 60 min at 37 degrees C. Only recombinant human tumour necrosis factor-alpha (rh-TNF-alpha) gave a similar PMN priming effect to that seen with MNCM. This effect could not be reversed by washing away either the MNCM or rh-TNF-alpha. The priming effect could be markedly reduced (74.8%, n = 6) by employing the use of polyclonal antibody to TNF-alpha in the preincubation step; assaying for TNF-alpha in these MNCMs showed that the degree of priming corresponded to the amount of TNF-alpha present. Rh-TNF-alpha alone appeared to have very little direct stimulatory effect on respiratory burst activity. The results show that TNF-alpha produced by LPS stimulated MNC after 5 h binds to a PMN surface receptor in the cold and warming of the cells to 37 degrees C allows for an immediate and dramatic response to ZAS stimulation. This suggests that TNF-alpha is the important cytokine upregulating PMN responses to other physiological mediators, including C5a des arg during the early phases of an inflammatory reaction.     

C5a对人中性粒细胞凋亡影响的实验观察

目的 探讨C5a影响人中性粒细胞(PMN)的凋亡.方法 采用流式细胞术,检测PMN被C5a作用后其凋亡率随浓度及时间的变化.结果 随着C5a浓度增加PMN凋亡率下降;随着C5a刺激时间的延长,其抑制PMN凋亡作用有所增强.结论 C5a抑制中性粒细胞凋亡存在浓度及时间依赖关系.C5a延长中性粒细胞的炎性反应的时间,可能由此导致炎症失控和消散延迟,从而参与了脓毒症及急性肺损伤的发生发展.     

C5a对人中性粒细胞凋亡的影响

目的探讨C5a影响人中性粒细胞的凋亡。方法采用流式细胞术，检测PMN被C5a作用后其凋亡率随浓度及时间的变化。结果随着C5a浓度增加PMN凋亡率下降，随着C5a和刺激时间延长，其抑制作用有所增强。结论C5a依浓度及时间抑制中性粒细胞的凋亡。     

Separation and functional characterization of human neutrophil subpopulations

Human neutrophils have been considered to be a functionally homogeneous population of cells. We have developed a density sedimentation technique for separation of neutrophils into two populations based on their ability to form rosettes with IgG-coated human erythrocytes (7SEA). Under the experimental conditions 80% +/- 4.3% of normal human peripheral blood neutrophilis form rosettes. Functionally rosette- forming neutrophils are more adherent to nylon wool, able to phagocytize more 14C-labeled Staphylococcus aureus, more efficient in killing S. aureus, and more responsive to endotoxin-activated human serum in a 51-cr chemotaxis assay that the non-rosette forming neutrophils. However, there is no difference among neutrophil subpopulations' ability to phagocytize latex particles. Paired samples of exudate neutrophils from cutaneous abscess fluid and peripheral neutrophils from three patients were investigated for their ability to form 7SEA rosettes. In each case exudate neutrophils contained greater than 96% rosette-forming neutrophils, whereas peripheral blood contained the normal 80% ( less than 0.01). Thus we show that peripheral blood contains at least two distinct populations of neutrophils. However, an essentially homogeneous neutrophil population is present in cutaneous exudate fluid.     

Impact of hepatitis C virus infection on neutrophil oxidative burst function in hemodialysis patients

Polymorphonuclear leukocyte (PMN) functions have been studied extensively in hemodialysis (HD) patients; however, results are contradictory and the mechanisms that modulate phagocytosis and oxidative burst are not completely understood. Hepatitis C virus (HCV) is a frequent complication of HD that may be associated with disturbed PMN function; however, the impact of HCV infection on neutrophil oxidative burst function in HD patients is unknown. We investigated Neutrophil oxidative burst function in 24 HD patients (15 HCV-positive and, 9 HCV-negative patients) before and after dialysis. HCV-RNA was detected by RT-nested PCR while, quantitative measurement of oxidative burst function was assessed by flowcytometry. Neutrophil Oxidised burst function was significantly diminished in HD patients as comapred to controls (P = 0.001, oxidised PMN (%); P = 0.02 mean flueresnce intensity, MFI), and in pre-dialysis as compared to post-dialysis samples (oxidised PMNs (%): 60.5 +/- 3.2 vs. 72.1 +/- 3.9, P = 0.02); (MFI: 352 +/- 42 vs. 500 +/- 50, P = 0.03). Alteration in Neutrophil oxidative burst function in the pre-dialysis samples was significant in HCV-positive patients as compared to HCV-negative patients (oxidized PMNs (%): 50 +/- 2.9 vs. 63 +/- 5.1, P = 0.02); (MFI: 291 +/- 31 vs. 438 +/- 64, P = 0.006). Marked reduction in E. coli induced burst in pre-dialysis samples compared to post-dialysis was found in HCV-positive when compared to HCV-negative patients (oxidized PMNs (%): 50 +/- 2.9 vs. 74.8 +/- 4.7, P = 0.001), (MFI: 291 +/- 31 vs. 493 +/- 63, P = 0.002). In conclusion, a possible role of concomitant HCV infection in alteration of Neutrophil oxidative burst function is highly suggested.     

Identification of neutrophil subpopulations with monoclonal antibodies

Two monoclonal antibodies have been produced by the hybridoma technique that recognize subpopulations of human neutrophils. The antibodies, termed 1B5 and 4D1, react with a mean percentage of 57% and 51% of peripheral blood granulocytes, respectively. The antigens recognized appear to be neutrophil specific in that these antibodies do not react with eosinophils, platelets, erythrocytes, monocytes, or nonadherent peripheral blood mononuclear cells. Although the neutrophil subpopulations recognized by these antibodies are nearly identical (coinclusive), the antigenic determinants recognized appear to be different. These monoclonal antibodies to neutrophil subpopulations may prove useful to studying functional heterogeneity among neutrophils as well as for investigations of normal and abnormal myeloid differentiation.     

The role of formylpeptide receptors, C5a receptors, and cytosolic-free calcium in neutrophil priming

Polymorphonuclear leukocytes (PMNL) exposed to chemoattractants or cytokines change their functional capacity. The effect of endotoxin-activated serum as a priming agent on human PMNL was tested. Pretreatment of PMNL with endotoxin-activated serum increased their oxidative burst in response to formylpeptide (FMLP) (P less than .02) and C5a (P less than .05). Priming for membrane depolarization was observed in PMNL preincubated with either endotoxin-activated serum, low concentrations of purified C5a, or endotoxin but not with decomplemented plasma. Primed PMNL had an increased number of FMLP but not C5a receptors as compared with control PMNL. The "resting" cytosolic free calcium was increased in primed PMNL (P less than .02). Intracellular calcium buffering abolished the priming effect of endotoxin-activated serum. Thus, endotoxin-activated serum can prime cellular responsiveness for membrane depolarization and superoxide production in response to FMLP and to C5a. Priming may be due to an increased resting cytosolic-free calcium.     

The effect of intravascular neutrophil chemotactic factors on blood neutrophil and platelet kinetics

Intravenous infusion of an analogue (f-met-leu-phe [FMLP]) of a bacterial-derived polymorphonuclear leukocyte (PMNL) chemotactic factor, or of the complement-derived chemotactic stimulus, zymosan-activated plasma (ZAP, containing C5ades Arg) into rabbits induces acute PMNL margination in the pulmonary vasculature. This process also occurs during hemodialysis and the adult respiratory distress syndrome. The pulmonary PMNL sequestration is accompanied by thrombocytopenia. Because of the role platelets and PMNLs play in hemostasis and defense against infection, we studied the fate of these blood elements following sequestration induced by chemotactic factors. By employing 111In-labelled platelets and external radioisotope scanning, platelets were found to sequester in the pulmonary vasculature during FMLP infusion. Simultaneous 51Cr PMNL and 111In-platelet studies showed that following sequestration, PMNLs returned to the circulation and disappeared with a normal half-life (T1/2) whereas the T1/2 of the platelets was markedly shortened (T1/2 of control = 49 +/- 3.0 hr; FMLP or ZAP infused T1/2 = 27 +/- 2.7 hr). Infusion of platelet-activating factor (PAF) induced PMN and platelet sequestration with similar abnormalities in platelet kinetics. Studies with 51Cr- and 14C-serotonin-labelled platelets showed that platelets did not release serotonin during FMLP, ZAP, or low dose PAF-induced sequestration. In contrast to platelet survival, platelet size, platelet aggregation responses, and platelet glycoproteins were not affected by transient sequestration. These results indicate that during PMNL margination induced by relatively "pure" PMNL stimuli such as FMLP, platelets may reversibly marginate and subsequently be cleared at an accelerated rate. The reason for accelerated platelet clearance is not a result of circulating platelet aggregates or detectable proteolytic modification of membrane glycoproteins. Such altered platelet kinetics may contribute to thrombocytopenia during sepsis, the adult respiratory distress syndrome, and other states in which excess PMNL margination occurs.     

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease

The absolute numbers of total leukocytes, lymphocytes. T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin-stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.     

Effect of low dose methotrexate on neutrophil chemotaxis induced by leukotriene B4 and complement C5a

When mediators of inflammation such as complement component C5a or leukotriene B4 are introduced into an air pouch created in mice, these mediators induce the migration of neutrophils into the air pouch. Pretreatment of mice with low doses of methotrexate inhibits leukotriene B4 or C5a induced neutrophil migration into the air pouch. Inhibition of neutrophil chemotaxis by methotrexate may, at least in part, account for the rapid onset of antiinflammatory activity that was observed in clinical trials with methotrexate in rheumatoid arthritis.     

C5 contents and neutrophil chemotactic activities in bronchiolar and alveolar regions

To clarify the localization and mechanism of neutrophil infiltration in the lower respiratory tract, we measured neutrophil number, neutrophil chemotactic factor (NCF) activity and content of C5 in bronchial lavage (BL) fluid and bronchoalveolar lavage (BAL) fluid. Numbers of neutrophils, NCF activity and C5 content were higher in the BL fluid from normal volunteers (NV) and control patients (CP) than those in the BAL fluid from the same subjects. The NCF activity in the BL fluid was inhibited approximately 40% by anti-C5 antiserum, and correlated with C5 content in the BL fluid. In the BAL fluids of patients with chronic airway diseases (CAD) and patients with idiopathic interstitial pneumonia (IIP), neutrophil number, NCF activity and C5 content were increased compared to those in BAL fluid from NV or CP. These results indicated that neutrophils are predominant in the bronchial region compared to the alveolar region, and that C5-derived NCF play important roles in the accumulation of neutrophils in the bronchial region. Also C5-derived NCF are thought to be related to, at least, a part of the neutrophil infiltration in the respiratory tract of patients with CAD and IIP.     

Tissue factor: a link between C5a and neutrophil activation in antiphospholipid antibody induced fetal injury

Fetal loss in patients with antiphospholipid (aPL) antibodies has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically activation of complement with generation of the anaphylotoxin C5a, is an essential trigger of fetal injury. In this study, we analyzed the role of the procoagulant molecule tissue factor (TF) in a mouse model of aPL antibody-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild-type mice or a genetic reduction of TF prevented aPL antibody-induced inflammation and pregnancy loss. In response to aPL antibody-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells but not fetal-derived cells (trophoblasts) was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL antibodies. The identification of TF as an important mediator of C5a-induced oxidative burst in neutrophils in aPL-induced fetal injury provides a new target for therapy to prevent pregnancy loss in the antiphospholipid syndrome.     

The effect of intravenous iron on oxidative stress in hemodialysis patients at various levels of vitamin C

BACKGROUND/AIMS: Vitamin C levels decrease during hemodialysis (HD), which deteriorates antioxidant defense. Vitamin C may also act pro-oxidatively, via reduction in Fe(III). We sought to determine whether intravenous iron (Fe(iv))-induced oxidative stress differs in HD patients with low and physiological vitamin C levels and whether intravenous vitamin C (C(iv)) administration during HD would change the response to Fe(iv). Patients and METHODS: Twenty patients with vitamin C deficiency (median 15.7 micromol/l, range 8.0-22.7) received Fe(iv) (100 mg iron sucrose between 150 and 180 min of HD). After 4 weeks of oral supplementation, the levels of vitamin C were comparable with those of controls (60.1 micromol/l, range 47.4-70.9). Patients were subsequently treated with (1) Fe(iv), (2) Fe(iv) and continuous 2 mg/min C(iv) throughout HD, (3) saline (S), and (4) S+C(iv). Plasma thiobarbituric acid reacting substances (TBARS) and vitamin C were assessed before, during and after FE(iv)(S), and 15, 30 and 60 min after infusion. RESULTS: Fe(iv) induced a comparable rise in TBARS in patients with vitamin C deficiency (before Fe(iv), 1.9 micromol/l, range 1.4-1.9; after Fe(iv), 2.6 micromol/l, range 2.3-2.9; p < 0.01) and in those with normal vitamin C (before Fe(iv), 1.9 micromol/l, range 1.7-2.1; after Fe(iv), 2.6 micromol/l, range 2.5-2.9; p < 0.01). Fe(iv)+C(iv) resulted in a greater increase in TBARS (after Fe(iv), 3.1 micromol/l, range 2.8-3.2) compared with Fe(iv) (p < 0.01). CONCLUSION: Iron sucrose-induced oxidative stress is comparable in HD patients with vitamin C deficiency and in those with normal vitamin C. We documented a pro-oxidative effect of vitamin C during Fe(iv)+C(iv) administration.     

The ability of uremic serum to induce neutrophil chemotaxis in relation to hemodialysis

The ability of serum to attract normal neutrophil granulocytes was investigated in 11 uremic patients before and after hemodialysis using a modified assay system for granulocyte migration under agarose. The chemotactic responses towards serum from the uremic patients were significantly decreased (p less than 0.01) prior to hemodialysis compared to control serum from a healthy individual. Hemodialysis acutely normalized the depressed chemotactic responses. It is concluded that hemodialysis improves the ability of uremic serum to attract neutrophils, and it is suggested that this improvement might be due to removal of granulocyte inhibitory factors from the sera.     

丙型肝炎病毒非结构蛋白5a对细胞周期的影响

丙型肝炎病毒(HCV)感染是慢性肝炎、肝硬化的主要病因之一，并与肝细胞癌的发生关系非常密切。但HCV导致原发性肝细胞癌的机制，特别是其基因产物(结构蛋白和非结构蛋白)在致癌中的作用仍不清楚。HCV非结构蛋白5a(NS5a)能激活多种转录因子，并与细胞内因子结合，影响其功能，导致细胞增殖分化异常，可能与原发性肝细胞癌的发生有关。为进     

运动对血液透析充分性的影响

目的 探讨运动对血液透析（HD）充分性的影响。方法 20例病情稳定的维持性HD的尿毒症患者,随机分为运动组和对照组。运动组在HD期间做蹬脚踏器的运动,对照组不运动。观察透析前、后及透析后1h的尿素氮（BUN）、肌酐（Scr)和尿酸（UA）的下降率及反弹率,并分别测定两组患者的尿素清除指数（KT／V）、溶质清除指数（SRI）、尿素气清除量（AUR）和标准化蛋白分解率（nPCR)。结果 运动组患者BUN、Scr的下降率较对照组明显增加（P＜0．05）,BUN、Scr、UA的反弹率较对照组明显降低（P＜0．05－0．01）,KT／V、SRI、AUR明显高于对照组（P＜0．05－0．01）,两组nPCR差异无显著性。结论 患者HD期间运动可以提高透析效果,是一个实用、可行的新方法。     

C5a-mediated neutrophil dysfunction is RhoA-dependent and predicts infection in critically ill patients

Sensitivity of chronic lymphocytic leukemia (CLL) cells to anti-CD20 mAbs is low and, therefore, the efficacy of monotherapy with current anti-CD20 mAbs is limited. At present, it is not known whether sensitivity of CLL cells to CD20 mAbs is modulated by microenvironmental stimuli. We have shown previously that in vitro CD40 stimulation of peripheral blood-derived CLL cells results in resistance to cytotoxic drugs. In the present study, we show that, in contrast, CD40 stimulation sensitizes CLL cells to the recently described novel type II anti-CD20 mAb GA101. Cell death occurred without cross-linking of GA101 and involved a lysosome-dependent mechanism. Combining GA101 with various cytotoxic drugs resulted in additive cell death, not only in CD40-stimulated CLL cells, but also in p53-dysfunctional CLL cells. Our findings indicate that GA101 has efficacy against chemoresistant CLL, and provide a rationale for combining cytotoxic drugs with anti-CD20 mAbs.     

Late hepatitis C virus seroconversion in a patient on hemodialysis

We present a case of an exceptionally late HCV seroconversion in a hemodialysis patient. The clinical case illustrates the difficulty of appropriately and quickly isolating a HCV infected patient, and it demonstrates the necessity to consider all the dialysis patients as potentially infectious patients. The standard precautions in hemodialysis must be applied routinely to all the patients, because the diagnosis of the HCV infection is very often late, and on some occasions it is difficult to interpret correctly the results of the available diagnostic tests.