Pseudomonas aeruginosa adaptation in cystic fibrosis patients increases C5a levels and promotes neutrophil recruitment.

Cystic fibrosis (CF) disease is characterized by an intense airway inflammatory response mediated by neutrophils and chronic respiratory infections caused by P. aeruginosa. High levels of the complement component C5a, the strongest neutrophil chemoattractant molecule, are commonly found in the CF lung and have been associated with a worsening of the disease. In this study, we investigated how the isolates from CF patients modulate the levels of C5a and identified the bacterial factors involved. We demonstrated that most isolates from airway chronic infections induce the production and accumulation of C5a, an effect attributable to the loss of C5a cleavage by the exoproteases alkaline protease (AprA) and elastase B (LasB). Furthermore, we found that lack of the bacterial protease-dependent C5a degradation is due to mutations in the master regulator LasR. Thus, complementation of a non-C5a-cleaving CF isolate with a functional wild-type LasR restored its ability to express both proteases, cleave C5a and reduce neutrophil recruitment in vitro. These findings suggest that the non-cleaving C5a phenotype acquired by the LasR variants frequently isolated in CF patients may account for the strong neutrophilia and general neutrophil dysfunction predisposing toward increased inflammation and reduced bacterial clearance described in CF patients.

The Identification of Immune-Related Plasma Proteins Associated with Spontaneous Preterm Delivery and Intra-Amniotic Infection in Women with Premature Cervical Dilation or an Asymptomatic Short Cervix.

BACKGROUND: We aimed to investigate whether various immune-related plasma proteins, alone or in combination with conventional clinical risk factors, can predict spontaneous preterm delivery (SPTD) and intra-amniotic infection in women with premature cervical dilation or a short cervix (≤ 25 mm). METHODS: This retrospective study included 80 asymptomatic women with premature cervical dilation (n = 50) or a short cervix (n = 30), who underwent amniocentesis at 17-29 weeks. Amniotic fluid (AF) was cultured, and maternal plasma was assayed for interleukin (IL)-6, matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and complements C3a and C5a, using enzyme-linked immunosorbent assay (ELISA) kits. The primary outcome measures were SPTD at < 32 weeks and positive AF cultures. RESULTS: The plasma levels of IL-6, C3a, and C5a, but not of MMP-9 and TIMP-1, were significantly higher in women with SPTD at < 32 weeks than in those who delivered at ≥ 32 weeks. The women who delivered at < 32 weeks had more advanced cervical dilatation, and higher rates of antibiotic and tocolytic administration and were less likely to be given vaginal progesterone than those who delivered at ≥ 32 weeks. Using a stepwise regression analysis, a combined prediction model was developed, which included the plasma IL-6 and C3a levels, and cervical dilatation (area under the curve [AUC], 0.901). The AUC for this model was significantly greater than that for any single variable included in the predictive model. In the univariate analysis, plasma IL-6 level was the only significant predictor of intra-amniotic infection. CONCLUSION: In women with premature cervical dilation or a short cervix, maternal plasma IL-6, C3a, and C5a levels could be useful non-invasive predictors of SPTD at < 32 weeks. A combination of these biomarkers and conventional clinical factors may clearly improve the predictability for SPTD, as compared with the biomarkers alone. An increased plasma level of IL-6 predicted intra-amniotic infection.

Complement activation products in the circulation and urine of primary membranous nephropathy.

BACKGROUND: Complement activation plays a substantial role in the pathogenesis of primary membranous nephropathy (pMN). C5b-9, C3c, MBL, and factor B have been documented in the subepithelial immune deposits. However, the changing of complement activation products in circulation and urine is not clear. METHODS: We measured the circulating and urinary levels of C1q, MBL, C4d, Bb, properdin, C3a, C5a, and sC5b-9, in 134 patients with biopsy-proven pMN, by enzyme-linked immunosorbent assay. All the plasma values were corrected by eGFR and all the urinary values were corrected by urinary creatinine and urinary protein excretion. Anti-PLA2R antibodies were measured in all patients. RESULTS: The plasma complement activation products were elevated both in the patients with and without anti-PLA2R antibodies. C3a levels were remarkably increased in the circulation and urine, much higher than the elevated levels of C5a. C5b-9 was in normal range in plasma, but significantly higher in urine. The urinary C5a had a positive correlation with anti-PLA2R antibody levels and urinary protein. The plasma level of C4d was elevated, but C1q and MBL were comparable to healthy controls. Positive correlations were observed between plasma C4d/MBL and urinary protein, only in the patients with positive anti-PLA2R antibodies but not in those without. The plasma level of Bb was elevated and had positive correlation with urinary protein only in the patients without anti-PLA2R antibodies. CONCLUSION: Complement activation products were remarkable increased in pMN and may serve as sensitive biomarkers of disease activity. The complement may be activated through lectin pathway with the existence of anti-PLA2R antibodies, while through alternative pathway in the absence of antibody.

Effect of preoperative inhaled budesonide on pulmonary injury after cardiopulmonary bypass: A randomized pilot study.

BACKGROUND: Cardiopulmonary bypass can result in lung injury. This prospective, double-blinded, randomized trial aimed to evaluate the protective effect of inhaled budesonide on lung injury after cardiopulmonary bypass. METHODS: Sixty patients, aged 25 to 65 years, requiring cardiopulmonary bypass were randomized to groups treated with saline or budesonide inhalation preoperatively. The respiratory mechanics were recorded. Bronchoalveolar lavage fluid was collected before cardiopulmonary bypass and after sternal closure. Serum and bronchoalveolar lavage fluid levels of proinflammatory and anti-inflammatory factors were analyzed. The primary end point was the lowest ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen after cardiopulmonary bypass. The durations of ventilation and postoperative recovery time were noted. RESULTS: Budesonide significantly improved respiratory mechanics after cardiopulmonary bypass. Budesonide improved the partial pressure of arterial oxygen to the fraction of inspired oxygen ratio from 8 to 48 hours after the operation. Budesonide shortened the durations of mechanical ventilation and postoperative recovery time. Budesonide decreased the levels of proinflammatory factors while increasing the levels of anti-inflammatory factors in bronchoalveolar lavage fluid and serum (all P < .05). The macrophage and neutrophil counts, and protein and elastase concentrations were decreased by budesonide treatment. CONCLUSIONS: Budesonide treatment shortened the durations of mechanical ventilation, inhibited local and systemic inflammation, and improved respiratory function after cardiopulmonary bypass.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study.

BACKGROUND: We investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP). METHODS: This retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis. RESULTS: In bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery. CONCLUSIONS: Maternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

Complement activation in hidradenitis suppurativa: a new pathway of pathogenesis?

BACKGROUND: Despite the heavy purulence observed in hidradenitis suppurativa (HS), the kinetics of complement anaphylatoxins acting to prime chemotaxis of neutrophils has not been studied. OBJECTIVES: To explore complement activation in HS. METHODS: Circulating concentrations of complement factor C5a, as well as of membrane attack complex C5b-9, were determined in the plasma of 54 treatment-naïve patients and of 14 healthy controls, as well as in the pus of seven patients. Results were correlated with Hurley stage and International Hidradenitis Suppurativa Severity Score. Peripheral blood mononuclear cells (PBMCs) were isolated from seven patients with Hurley stage III HS and seven healthy volunteers and stimulated in the presence of 25% of plasma for the production of tumour necrosis factor-α (TNF-α). RESULTS: Circulating C5a and C5b-9 were significantly greater in patient than in control plasma; however, concentrations in pus were very low. Circulating C5a levels exceeding 28 ng mL-1 were associated with a specificity > 90% with the occurrence of HS. Circulating levels of C5a and C5b-9 were greater in patients with more severe HS. PBMCs of patients produced high concentrations of TNF-α only when growth medium was enriched with patient plasma; this was reversed with the addition of the C5a blocker IFX-1. CONCLUSIONS: Systemic complement activation occurs in HS and may be used as a surrogate biomarker of HS. C5a stimulates overproduction of TNF-α and may be a future therapeutic target.

Complement Alternative Pathway׳s Activation in Patients With Lupus Nephritis.

OBJECTIVE: The aim of this study was to detect the spectrum of complement activation pathways in circulation and to assess their correlations with clinical and pathologic features in a large lupus nephritis cohort from China. MATERIALS AND METHODS: Plasma levels of C1q, mannose-binding lectin, C4d, Bb, C3, C3a, C5a and soluble C5b-9 were detected by enzyme-linked immunosorbent assay in 222 patients with active biopsy-proven lupus nephritis, 34 patients with lupus nephritis at remission, 82 patients with active systemic lupus erythematosus without renal involvement and 39 normal controls. The correlations between levels of complement components and clinicopathological features of these patients were further analyzed. RESULTS: Plasma levels of C1q and C3 significantly decreased, and the levels of Bb, C3a, C5a and soluble C5b-9 were significantly elevated in patients with active lupus nephritis compared with those in remission, active systemic lupus erythematosus without renal involvement group and normal controls. In the lupus nephritis group, soluble C5b-9 levels were inversely correlated with C1q and C4d levels (r = -0.412, P < 0.001 and r = -0.221, P = 0.002, respectively), but more strongly correlated with the level of Bb (r = 0.546, P < 0.001). C3b, Bb and C5b-9 could colocalize on glomeruli in lupus nephritis. Plasma Bb level was significantly correlated with some renal disease activity indices and was a risk factor for renal outcomes (hazard ratio = 1.745; 95% CI: 1.106-2.754; P = 0.017) in the lupus nephritis group. CONCLUSIONS: Our findings suggested that the activation of the complement alternative pathway might play a more important role in the pathogenesis of lupus nephritis, and factor Bb might be a useful marker for evaluating renal disease activity and outcomes.

Complement 5a is an indicator of significant fibrosis and earlier cirrhosis in patients chronically infected with hepatitis B virus.

PURPOSE: To investigate the association between serum complement 5a (C5a) concentration and liver fibrosis and cirrhosis in a large cohort of patients chronically infected with hepatitis B virus (HBV). METHODS: Five hundred and eight patients with chronic HBV infection undergoing liver biopsy were included. Serum concentrations of C5a was measured by Luminex screening system. Ishak histological system was obtained. RESULTS: C5a levels were negatively associated with liver fibrosis stages and significantly declined in patients with severe fibrosis and cirrhosis (P < 0.001). Multiple analysis showed C5a, AST, laminin, Co-IV, platelet count, albumin, HBsAg associated with liver fibrosis independently. Based on the markers above, we created two scores, Fib-model for significant fibrosis and Cirrh-model for earlier cirrhosis. Fib-model was performing better to differentiate from significant fibrosis, with an AUROC of 0.82 (95 % CI 0.78, 0.86), in comparison to existed models APRI, FIB-4 and Forns' index with AUROCs of 0.71 (95 % CI 0.66, 0.76), 0.72 (95 % CI 0.67, 0.77), 0.77 (95 % CI 0.72, 0.81), respectively. Although, Cirrh-model showed AUROC of 0.85 (95 % CI 0.80, 0.91) for evaluation of earlier cirrhosis, superior to APRI, and Forns' index, C5a + FIB-4 performed best with an AUROC of 0.94 (95 % CI 0.90, 0.97). CONCLUSION: In patients with chronic HBV infection, serum C5a concentration significantly decreased in severe fibrosis stages and earlier cirrhosis. Fib-model and C5a + FIB-4 performed better than existed models for assessment of significant fibrosis and earlier cirrhosis, respectively.

Enrichment of C5a-C5aR axis predicts poor postoperative prognosis of patients with clear cell renal cell carcinoma.

Anaphylatoxin C5a and its receptor C5aR on cancer cells constitute a vital axis to cancer progression. In this study, we measured C5aR level by immunohistochemistry in the same cohort of our previous C5a research, and C5a-C5aR axis status was determined by synthesizing C5a and C5aR data. C5aR was an adverse independent prognostic factor for ccRCC patients. Kaplan-Meier analyses revealed the unique position of both C5a and C5aR high population in postoperative survival, based on which patients were then shunted into C5a-C5aR enriched and non-enriched groups. Obviously, C5a-C5aR enriched patients significantly had a poorer overall survival (OS) and recurrence free survival (RFS) compared with non-enriched ones, and the independence of C5a-C5aR axis was verified by multivariable analyses (HR 2.118, P = 0.001 for OS, HR 1.715, P = 0.035 for RFS). Established nomograms based on our findings reflected much better predicting accuracy in contrast with most common used TNM and Fuhrman systems. Meanwhile, consistent with HR, C5a-C5aR axis in this study held its advantages over C5a and C5aR for OS prediction by c-index analyses, rather than RFS prediction.

C5aR inhibition in the early inflammatory phase does not affect bone regeneration in a model of uneventful fracture healing.

BACKGROUND: Recent studies were able to demonstrate involvement of the complement cascade in bone biology. Further studies analyzed the role of complement in traumatic injuries and demonstrated negative effects after excessive systemic activation of the inflammatory response with early abrogation of complement activation after application of a C5aR-antagonist exerting beneficial effects upon bone regeneration. In contrast, own fracture healing experiments with complement-deficient animals implied a crucial role of the complement cascade for sufficient fracture healing. METHODS: To analyze the effect of a short abrogation of the complement system in the local process of fracture healing, a fracture healing experiment with wild-type mice (C57BL6), femoral osteotomy, consecutive external fixation for 21 days and blockade of the early complement activation (C5aRA) directly after trauma and after 12 h was performed. Control animals received a peptide without any biological effects. After 1-3 days, the inflammatory response was monitored with IL-6 immunostaining, serum analyses of C5a and after 3 days with histological evaluation of PMN. Fracture healing was examined with biomechanical, radiological and histological methods after 21 days. RESULTS: While a decrease of the early inflammatory response was seen on day 1 of the C5aRA-treated group regarding immunostaining for IL-6 and after 3 days in the histological evaluation of PMN, no significant differences were demonstrated between both experimental groups after 21 days in the biomechanical, radiological and histological evaluation. CONCLUSIONS: The present results demonstrate that the short-term inhibition of complement activation immediately after fracture does not significantly affect bone regeneration in an experimental model of regular fracture healing. Whereas other studies demonstrated that the early posttraumatic blockade of the C5aR improves fracture healing in a scenario of combined trauma, the present findings implicate that the same treatment has no effect in uneventful bone healing.

Correlations between complement system's activation factors and anti-angiogenesis factors in plasma of patients with early/late-onset severe preeclampsia.

OBJECTIVE: To investigate the expression of complement system's activation factors in plasma of patients with severe preeclampsia and their correlations with anti-angiogenesis factors. METHODS: A case-control study was performed. The study group consisted of 30 cases of early-onset severe preeclampsia (EOSPE) and 30 cases of late-onset severe preeclampsia (LOSPE). Thirty cases were selected as the early-onset control group (E-control) and 30 as the late-onset control group (L-control), with the weeks of gestation matched. Enzyme-linked immunosorbent assay (ELISA) was used to test C3a, C5a, MAC, sEng, and sflt-1 in the maternal peripheral circulation. RESULTS: The complement system's activation factors C3a, C5a, and MAC were increased significantly in EOSPE and LOSPE (all P < 0.01) compared with E/L-control. Plasma levels of C3a correlated inversely with plasma sEng (r = -0.454, P < 0.001) and sflt-1 (r = -0.326, P = 0.011) in preeclampsia patients, while MAC correlated with soluble endoglin (sEng; r = 0.343, P = 0.007) and soluble fms-like tyrosine kinase-1 (sflt-1; r = 0.318, P = 0.013). There were no significant correlations between complement system's activation-related factors and the anti-angiogenesis factors in healthy control group. CONCLUSIONS: Abnormal activation of the complement system exists in the maternal circulation of patients with E/L-onset severe preeclampsia. There were correlations between the abnormal activation of the complement system and the abnormal expression of anti-angiogenesis factors in patients with severe preeclampsia, but the correlation was not strong.

[Association between protective effect of Liuwei Wuling tablets against acute liver injury and its inhibitory effect on cytoplasmic translocation of high-mobility group box-1 in hepatocytes in mice].

OBJECTIVE: To investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). METHODS: A Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes. RESULTS: At 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1ß, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01). CONCLUSION: Liuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Complement mediates a primed inflammatory response after traumatic lung injury.

BACKGROUND: Pulmonary contusion (PC) is a common, potentially lethal injury that results in the priming for exaggerated responses to subsequent immune challenge such as an infection (second hit). We hypothesize a PC-induced complement (C) activation participates in the priming effect for a second hit. METHODS: Male, 8 weeks to 9 weeks, C57BL/6 mice (wild-type, C5) underwent blunt chest trauma resulting in PC. At 3 hours/24 hours after injury, the inflammatory response was measured in tissue, serum, and bronchoalveolar lavage (BAL). The thrombin inhibitor, hirudin, was used to determine if injury-induced thrombin participated in the activation of C. Injury-primed responses were tested by challenging injured mice with bacterial endotoxin (lipopolysaccharide, LPS) as a second hit. Inflammatory responses were assessed at 4 hours after LPS challenge. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest (significance, p ≤ 0.05). Protocols were approved by the Institutional Animal Care and Use Committee. RESULTS: We found significantly increased levels of C5a in the BAL of injured animals as early as 24 hours, persisting for up to 72 hours after injury. Hirudin-treated injured mice had significantly decreased levels of thrombin in the BAL that correlated with reduced C5a levels. Injured mice challenged with intratracheal (IT) LPS had increased C5a and inflammatory response. Conversely, inhibition of C5a or its receptor, C5aR, before LPS challenge correlated with decreased inflammatory responses; C5a-deficient mice showed a similar loss of primed response to LPS challenge. CONCLUSION: Complement C5a levels in the BAL are increased over several days after PC. Premorbid inhibition of thrombin markedly decreases C5a levels after PC, suggesting that thrombin-induced C activation is the major pathway of activation after PC. Similarly, inhibition of C5a after PC will decrease injury-primed responses to LPS stimulation. Our findings suggest cross-talk between the coagulation and complement systems that induce immune priming after PC.

C5a receptor-dependent cell activation by physiological concentrations of desarginated C5a: insights from a novel label-free cellular assay.

The complement anaphylatoxins C3a, C5a, and desarginated C5a (C5a(desArg)) play critical roles in the induction of inflammation and the modulation of innate and acquired immune responses after binding to their G protein-coupled receptors, C3a receptor and C5a receptor (C5aR). The role of C5a(desArg) in inducing cell activation has been often neglected, because the affinity of C5a(desArg) for C5aR has been reported to be much lower than that of C5a. We have used a novel label-free cellular assay to reassess the potential of C5a(desArg) to induce activation of transfected and primary immune cells. Our results indicate that physiological levels of C5a(desArg) induce significant levels of cell activation that are even higher than those achieved by stimulating cells with analogous concentrations of C5a. Such activation was strictly dependent on C5aR, because it was completely abrogated by PMX-53, a C5aR antagonist. Pharmacological inhibition of specific G proteins located downstream of C5aR indicated differential involvement of G(α) proteins upon C5aR engagement by C5a or C5a(desArg). Further, mass spectrometric characterization of plasma-derived C5a and C5a(desArg) provided important insight into the posttranslational modification pattern of these anaphylatoxins, which includes glycosylation at Asn(64) and partial cysteinylation at Cys(27). Although the context-specific physiological contribution of C5a(desArg) has to be further explored, our data suggest that C5a(desArg) acts as a key molecule in the triggering of local inflammation as well as the maintenance of blood surveillance and homeostatic status.

Complement factor C5a in acute exacerbation of Chronic Obstructive Pulmonary Disease.

The complement component C5a is a potent inflammatory peptide, which may be involved in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). We analysed the induced sputum and plasma of 28 patients with stable COPD, 12 healthy smokers and 7 non-smokers. In 13 of the patients with COPD, we also observed paired samples during acute exacerbation. The concentrations of C5a/C5a desArg and C3a/C3a desArg were measured using cytometric bead array. Both C5a and C3a concentrations in induced sputum of stable patients with COPD were significantly increased compared to the control groups of healthy smokers and non-smokers. In addition, there was a significant elevation in C5a values in exacerbation of COPD that was independent from the airway C3a levels. Airway C5a levels were negatively correlated with forced expiratory volume in first second (FEV1)% predicted and diffusing capacity of the lung (TLCO). Plasma C5a concentrations in patients with COPD were significantly higher than in healthy smokers, but no further significant systemic C5a elevation was detected with acute exacerbation of COPD. There was no important difference in local or systemic C5a concentrations between healthy smokers and non-smokers. These in vivo results clearly show that local and systemic C5a concentrations in COPD are elevated, and that the local, in contrast to systemic, C5a concentrations additionally increase in the acute exacerbation of COPD. It seems that the cigarette smoke is not related to C5a increase. The elevated local and systemic C5a levels, and additional individual local C5a increase during the exacerbation support the importance of C5a in COPD.

The ScpC protease of Streptococcus pyogenes affects the outcome of sepsis in a murine model.

The ScpC protease of Streptococcus pyogenes degrades interleukin-8 (IL-8), a chemokine that mediates neutrophil transmigration and activation. The ability to degrade IL-8 differs dramatically among clinical isolates of S. pyogenes. Bacteria expressing ScpC overcome immune clearance by preventing the recruitment of neutrophils in soft tissue infection of mice. To study the role of ScpC in streptococcal sepsis, we generated an ScpC mutant that did not degrade IL-8 and thus failed to prevent the recruitment of immune cells as well as to cause disease after soft tissue infection. In a murine model of sepsis, challenge with the ScpC mutant resulted in more severe systemic disease with higher bacteremia levels and mortality than did challenge with the wild-type strain. As expected, the blood level of KC, the murine IL-8 homologue, increased in mice infected with the ScpC mutant. However, the elevated KC levels did not influence neutrophil numbers in blood, as it did in soft tissue, indicating that additional factors contributed to neutrophil transmigration in blood. In addition, the absence of ScpC increased tumor necrosis factor, IL-6, and C5a levels in blood, which contributed to disease severity. Thus, the ScpC mutant triggers high neutrophil infiltration but not lethal outcome after soft tissue infection, whereas intravenous infection leads to highly aggressive systemic disease.

Innate immune response to intramammary Mycoplasma bovis infection.

The objective of the current study was to characterize the systemic and local innate immune response of dairy cows to IMI with Mycoplasma bovis, a pathogen of growing concern to the dairy industry. Ten Holstein cows were each infused in 1 quarter with M. bovis and studied for a 10-d period. Acute phase protein synthesis, which reflects 1 parameter of the systemic response to infection, was induced within 108 h of infection, as evidenced by increased circulating concentrations of lipopolysaccharide binding protein and serum amyloid A. Transient neutropenia was observed from 84 to 168 h postinfection, whereas a constant state of lymphopenia and thrombocytopenia was observed from 84 h until the end of the study. Milk somatic cell counts initially increased within 66 h of M. bovis infusion and remained elevated, relative to control (time 0) concentrations, for the remainder of study. Increased milk concentrations of BSA, which reflect increased permeability of the mammary epithelial-endothelial barrier, were evident within 78 h of infection and were sustained from 90 h until the end of the study. Milk concentrations of several cytokines, including IFN-gamma, IL-1beta, IL-10, IL-12, tumor growth factor-alpha, and tumor necrosis factor-alpha, were elevated in response to infection over a period of several days, whereas increases in milk IL-8 were of a more limited duration. Complement activation, reflected by increased milk concentrations of complement factor 5a, was also observed over several days. Despite the indication by these observed changes that the cows mounted a prolonged inflammatory response to M. bovis intramammary infection, all quarters remained infected throughout the study with persistently high concentrations of this bacterium. Thus, a sustained inflammatory response is not sufficient to eradicate M. bovis from the mammary gland and may reflect the ongoing struggle of the host to clear this persistent pathogen.

Comparison of in vitro and in vivo complement activation by metal and bioabsorbable screws used in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of the study was to evaluate the biocompatibility of polylactide (PLLA) screws in comparison with standard metal screws for fixation of the patellar tendon graft in human anterior cruciate ligament (ACL) reconstruction. METHODS: A total of 41 patients (22 women and 19 men) were prospectively randomized for the use of metal interference screws (20 patients) or biologically resorbable PLLA screws from Linvatec, Largo, FL (21 patients). Average age at the time of surgery was 26 years (15 to 51 y). Synovial fluid and plasma were collected preoperatively and after 6 weeks in both groups. Plasma was analyzed for C5a and synovial fluid, as well as for terminal SC5b-9 complement complex (TCC) and interleukin (IL)-8. At 1 year after surgery, serum was incubated with metal, PLLA, and no screws; this was followed by analysis of C5a after 1 and 6 hours of incubation. Inflammatory mediators were measured through enzyme-linked immunosorbent assay (ELISA). RESULTS: In the BioScrew group, 4 patient samples showed high C5a concentration in synovial fluid after 6 weeks, but no statistically significant difference was observed between the 2 groups (P = .11). One patient in the BioScrew group had a high TCC value after 6 weeks, but no statistically significant difference was seen between the 2 groups (P = .20). In the in vitro study, no increased C5a generation was observed in sera incubated with a BioScrew or a metal screw compared with controls. CONCLUSIONS: No statistically significant difference was observed between the BioScrew and metal screw groups concerning C5a, TCC, and IL-8 formation. However, some patients in the BioScrew group showed elevated values. LEVEL OF EVIDENCE: Level II, prospective randomized trial.

Drusen complement components C3a and C5a promote choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.