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Background: Alzheimer's disease (AD) is a common cause of dementia. Serum complement factor 5a (C5a) is exceedingly implicated in AD. We explored the role of C5a levels in AD patients of different severity., Methods: Mild, moderate, and severe AD patients, and healthy controls were included. C5a and pro-inflammatory factor (TNF-α, IL-1β, IL-6, CRP) levels were assessed by ELISA, and cognitive function was evaluated by Mini-Mental state examination (MMSE) score. The correlations between C5a, inflammatory factor levels, MMSE score, and plasma Aβ42/Aβ40 ratio were analyzed by Pearson tests. Independent risk factors for AD aggravation were assessed by logistic multivariate regression analysis. According to the cut-off value of receiver operating characteristic (ROC) curve analysis of C5a level, AD patients were assigned into low/high expression groups, and severe AD incidence was compared. Severe AD cumulative incidence was analyzed by Kaplan-Meier curve., Results: Serum C5a, TNF-α, IL-1β, IL-6 and CRP levels were raised, and MMSE score was lowered in AD. Serum C5a, TNF-α, IL-1β, IL-6 and CRP levels in severe AD patients were higher than those in mild/moderate AD patients, but there were no significant differences in these cytokines between moderate and mild AD groups. The MMSE score of severe AD patients was lower than that of mild/moderate AD patients. Serum C5a level was positively correlated with serum TNF-α, IL-1β, IL-6, and CRP levels, and negatively correlated with MMSE score, with no obvious correlation with plasma Aβ42/Aβ40 ratio. Serum C5a level was one of the independent risk factors for AD aggravation. The occurrence of severe AD might be related to an increase in serum C5a level., Conclusion: Serum C5a level increased with AD severity, and its expression was positively correlated with serum pro-inflammatory factor levels, and negatively correlated with cognitive function., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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.

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.

Background: Elizabethkingia miricola is a rare Gram-negative bacterium found in water and clinical specimens. Typical culturing methods often misidentify Elizabethkingia spp. as Flavobacterium or Chryseobacterium. Although diagnosis is based on culturing samples taken from sterile sites, such as blood, a proper identification of this bacterium requires an expertise that goes beyond the capabilities of a typical clinical laboratory., Case Presentation: A 35-year-old woman diagnosed with common variable immunodeficiency was admitted to our center. Previous treatment with antibiotics (amoxicillin plus clavulanate, first and third generation of cephalosporins, macrolides) and systemic corticosteroids (up to 120 mg/day of prednisolone) failed to arrest the spread of inflammation. Gingival recession was observed in her oral cavity, resulting in an apparent lengthening of her teeth. In addition to typical commensal bacteria, including streptococci and neisseriae, strains of Rothia mucilaginosa and Elizabethkingia miricola were identified upon a detailed microbiological examination using a MALDI-TOF MS Biotyper system. The presence of the latter strain correlated with severe periodontitis, lack of IgA in her saliva and serum, a very low IgG concentration (< 50 mg/dl), IgM-paraproteinemia, decreases in C3a and C5a and microvascular abnormality. High-dose immunoglobulin (to maintain IgG > 500 mg/dl) and targeted levofloxacin treatment resulted in immune system reconstitution, oral healing, and eradication of the Elizabethkingia infection., Conclusions: E. miricola rarely causes disease in healthy individuals. However, the overgrowth of commensal bacteria, lack of IgG/IgA, microvasculopathy and complement cascade activation in patients with humoral immunodeficiency may facilitate Elizabethkingia invasion. Overuse of antibiotics, particularly beta-lactams, may cause mucosal colonization by E. miricola, followed by its multiplication combined with periodontitis that prompts bacterial translocation. MALDI-TOF Biotyper analysis may become a method of choice for identification of Elizabethkingia infections.

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.

Introduction: The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a plays a central role. In this study, plasma and urinary levels of C5a as well as renal C5a receptors (CD88 and C5L2) expression were investigated in patients with AAV., Methods: Twenty-four patients with AAV in the active phase, 19 patients with AAV in the remission phase, and 20 patients with lupus nephritis (LN) were included. Plasma and urinary levels of C5a were measured with enzyme-linked immunosorbent assay (ELISA). The staining of CD88 and C5L2 in renal specimens was detected with immunohistochemistry., Results: The level of plasma C5a was significantly higher in patients with AAV in the active phase than that in patients in remission, that in patients with LN, and that in normal controls. The urinary C5a level was significantly higher in patients with AAV in the active phase than that in patients in remission and that in normal controls, but not significantly different between patients with active AAV and patients with LN. The mean optical density of CD88 staining in the tubulointerstitium was significantly lower in AAV patients than that in normal controls (0.0052 ± 0.0011 versus 0.029 ± 0.0042; P = 0.005). The mean optical density of C5L2 in glomeruli was significantly higher in AAV patients than that in normal controls (0.013 ± 0.0027 versus 0.0032 ± 0.0006; P < 0.001). The mean optical density of CD88 staining closely correlated with the initial eGFR (r = 0.835; P < 0.001) in AAV patients. Double-labeling immunofluorescence assay suggested that CD88 did not express on neutrophils, monocytes, or macrophages, but C5L2 expressed on neutrophils (or monocytes) and macrophages., Conclusion: The elevated plasma and urinary C5a levels indicated complement activation in human AAV. The level of renal CD88 expression could reflect the disease severity of ANCA-associated glomerulonephritis. CD88 expression was downregulated, and C5L2 was upregulated in ANCA-associated glomerulonephritis.

The response of the bovine mammary gland to lipoteichoic acid (LTA), which is a major pathogen-associated molecular pattern of Gram-positive bacteria, was investigated by infusing purified Staphylococcus aureus LTA in the lumen of the gland. LTA was able to induce clinical mastitis at the dose of 100 microg/quarter, and a subclinical inflammatory response at 10 microg/quarter. The induced inflammation was characterized by a prompt and massive influx of neutrophils in milk. LTA proved to induce strongly the secretion of the chemokines CXCL1, CXCL2, CXCL3 and CXCL8, which target mainly neutrophils. The complement-derived chemoattractant C5a was generated in milk only with the highest dose of LTA (100 microg). The pro-inflammatory cytokine IL-1beta was induced in milk, but there was very little if any TNF-alpha and no IFN-gamma. The re-assessment of CXCL8 concentrations in milk whey of quarters previously challenged with S. aureus, by using an ELISA designed for bovine CXCL8, showed that this chemokine was induced in milk, contradicting previous reports. Overall, S. aureus LTA elicited mammary inflammatory responses that shared several attributes with S. aureus mastitis. Purified LTA looks promising as a convenient tool to investigate the inflammatory and immune responses of the mammary gland to S. aureus.

Complement activation generates two potent inflammatory mediators from C5, C5a and its derivative C5a(desArg), which results from the removal of the C-terminal arginine by ubiquitous carboxypeptidases. In this paper we describe the purification of milligram amounts of bovine C5a(desArg) by a simplified procedure, and the preparation of mouse monoclonal antibodies (MAbs) to C5a/C5a(desArg) which do not recognize native C5. A MAb was used to develop a sandwich ELISA which made it possible to quantify levels of C5a/C5adesArg in bovine biological fluids. Small amounts (means +/- SEM) of C5a/C5a(desArg) were found in EDTA-plasma (0.58 +/- 0.06 ng.mL-1). The anticoagulant EDTA was more efficient than citrate or heparin in inhibiting in vitro activation of the complement system. Complement activation occurred during coagulation since the baseline concentration of C5a/C5a(desArg) (15.4 +/- 4.1 ng.mL-1) was higher than in plasma. Zymosan, a potent activator of the complement cascade, was used to generate C5a/C5a(desArg). The time-course of the reaction and the dose-effect of zymosan were investigated. Optimal conditions were incubation at 39 degrees C for 1 or 2 h with 2 mg of zymosan per mL of serum. The maximal concentration of C5a/C5a desArg attained in zymosan-activated serum was 4.28 +/- 0.14 micrograms.mL-1. Normal milk (from healthy, uninflamed mammary glands) contained on average 0.12 ng of C5a/C5a(desArg).mL-1 (range 0.02-0.19 ng.mL-1). The maximal amount of C5a/C5a(desArg) which was generated in milk with zymosan was 1.1 ng.mL-1 (range 0.68-2.17 ng.mL-1). In milk from quarters with subclinical infections by coagulase-negative staphylococci, values were 0.18 ng.mL-1 and 2.37 ng.mL-1 for spontaneous and zymosan-generated C5a/C5a(desArg) concentrations, respectively. In milk from Escherichia coli endotoxin-induced mastitis, C5a/C5a(desArg) concentrations (means of four cows) before and after zymosan activation reached 6.5 ng.mL-1 and 55 ng.mL-1, respectively. These results indicate that a C5-convertase can operate in normal milk, that only minute amounts of C5a/C5a(desArg) can be generated (less than 1/1,000 of plasma potential), but that much higher concentrations are reached in milk during endotoxin-induced inflammation. The ELISA made it possible to determine normal ranges of C5a/C5a(desArg) in bovine blood plasma and in milk, and is a valuable tool to define the variations of its concentrations in exudates during inflammatory reactions.