An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

Complement component 3 and its activation split-products in saliva associate with periodontitis.

BACKGROUND: Periodontitis (PD) is classified by Grades A through C according to the risk of further progression, PD Grade C (PD-C) being the most severe progressing form. It is a matter of controversy, whether the disease activity observed in PD-C is due to impaired immune reactivity toward bacteria embedded in biofilms or a hyper-reactive immune response causing tissue damage as a bystander phenomenon. Little is known about the role of complement in this respect. METHODS: Plasma and unstimulated saliva samples were collected from patients with PD-B (n = 34) or -C (n = 27) and healthy controls (HCs) (n = 28). Salivary and plasma levels of total C3, C3c, and C3dg were quantified using sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Salivary levels of total C3 and C3dg were elevated in PD-B and PD-C patients compared to HCs (both P < 0.05), while the levels of C3c were elevated in PD-C compared to HCs. Plasma levels of C3c were higher in PD-B patients than in HCs (P < 0.05). CONCLUSION: PD-B and PD-C patients show increased complement activation compared to HCs, but no difference was found between the two disease grades. PD-B, but not PD-C, is associated with increased systemic complement activation as assessed by C3c in plasma.

Complement Components C3 and C4 Indicate Vasculitis Manifestations to Distinct Renal Compartments in ANCA-Associated Glomerulonephritis.

Acute kidney injury (AKI) is a common and severe complication of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) causing progressive chronic kidney disease (CKD), end-stage renal disease (ESRD) or death. Pathogenic ANCAs, in particular proteinase 3 (PR3) and myeloperoxidase (MPO), trigger a deleterious immune response resulting in pauci-immune necrotizing and crescentic glomerulonephritis (GN), a common manifestation of glomerular injury in AAV. However, there is growing evidence that activation of the complement pathway contributes to the pathogenesis and progression of AAV. We here aimed to compare glomerular and tubulointerstitial lesions in ANCA GN and extrarenal manifestation of AAV in association with levels of circulating complement components C3c and C4. METHODS: Plasma levels of C3c and C4 in a total number of 53 kidney biopsies with ANCA GN were retrospectively included between 2015 and 2020. Glomerular and tubulointerstitial lesions were evaluated according to established scoring systems for ANCA GN and analogous to the Banff classification. RESULTS: We here show that circulating levels of C3c and C4 in ANCA GN were comparable to the majority of other renal pathologies. Furthermore, hypocomplementemia was only detectable in a minor subset of ANCA GN and not correlated with renal or extrarenal AAV manifestations. However, low levels of circulating C3c correlated with AKI severity in ANCA GN independent of systemic disease activity or extrarenal AAV manifestation. By systematic scoring of glomerular and tubulointerstitial lesions, we provide evidence that low levels of circulating C3c and C4 correlated with vasculitis manifestations to distinct renal compartments in ANCA GN. CONCLUSIONS: We here expand our current knowledge about distinct complement components in association with vasculitis manifestations to different renal compartments in ANCA GN. While low levels of C4 correlated with glomerulitis, our observation that low levels of circulating complement component C3c is associated with interstitial vasculitis manifestation reflected by intimal arteritis implicates that C3c contributes to tubulointerstitial injury in ANCA GN.

The Spectrum of C4d Deposition in Renal Biopsies of Lupus Nephritis Patients.

Objectives: This study aimed to determine the prevalence and localization of complement factor C4d in renal biopsies from patients with lupus nephritis (LN), as well as its associations with the disease's clinico-pathological features. The correlation between arteriolar C4d deposition and renal microvascular lesions (RVLs) was further analyzed. Methods: A total of 325 biopsy-proven LN patients were enrolled, and their clinico-pathological data were collected. C4d staining of renal biopsies was performed by immunohistochemistry. The associations between C4d deposition and the clinico-pathological features were further analyzed. Results: C4d deposition was present in most (98.8%) renal specimens in our cohort. These deposits were localized in the glomeruli (98.2%), tubular basement membrane (TBM) (43.7%), arterioles (31.4%), and peritubular capillary (33.8%). Patients with TBM C4d staining had higher disease activity (measured with the Systemic Lupus Erythematous Disease Activity Index) and higher National Institutes of Health pathological activity and chronicity indices (all P < 0.01). Patients with arteriolar C4d deposition were more likely to develop RVLs (91.2%) compared to those with no arteriolar C4d deposition (78.0%; P = 0.004), especially with two or more types of RVLs (P < 0.001). During the mean follow-up of 55.8 months, arteriolar C4d was related to worse renal outcomes [hazard ration (HR): 2.074, 95% confidence interval (CI) 1.056-4.075, P = 0.034]. Multivariate Cox hazard analysis showed that co-deposition of arteriolar C4d and C3c was an independent risk factor (HR: 3.681, 95% CI 1.519-8.921, P = 0.004) for predicting renal outcomes. Conclusions: C4d deposition was common in renal tissues from LN patients. TBM C4d deposition was related to the disease activity, and arteriolar C4d deposition was associated with RVLs and worse renal outcomes.

Complement in Renal Disease as a Potential Contributor to Arterial Hypertension.

OBJECTIVE: Complement deposition is prevalent in kidney biopsies of patients with arterial hypertension and hypertensive nephropathy, but an association of hypertension and complement deposition or involvement of complement in the pathogenesis of hypertensive nephropathy has not been shown to date. METHODS: In this study, we analyzed complement C1q and C3c deposition in a rat model of overload and hypertension by subtotal nephrectomy (SNX) and in archival human renal biopsies from 217 patients with known hypertension and 91 control patients with no history of hypertension using semiquantitative scoring of C1q and C3c immunohistochemistry and correlation with parameters of renal function. To address whether complement was only passively deposited or actively expressed by renal cells, C1q and C3 mRNA expression were additionally analyzed. RESULTS: Glomerular C1q and C3c complement deposition were significantly higher in kidneys of hypertensive SNX rats and hypertensive compared to nonhypertensive patients. Mean arterial blood pressure (BP) in SNX rats correlated well with the amount of glomerular C1q and C3c deposition and with left ventricular weight, as an indirect parameter of high BP. Quantitative mRNA analysis showed that C3 was not only deposited but also actively produced by glomerular cells of hypertensive SNX rats and in human renal biopsies. Of note, in patients CKD-stage correlated significantly with the intensity of glomerular C3c staining, but not with that of C1q. CONCLUSION: Renal complement deposition correlated with experimental hypertension as well as the presence of hypertension in a variety of renal diseases. To answer the question, if and how exactly renal complement is causative for the pathogenesis of arterial hypertension in men, further studies are needed.

Diagnostic Utility of C4d by Direct Immunofluorescence in Bullous Pemphigoid.

ABSTRACT: Bullous pemphigoid (BP) is an autoimmune blistering disease that commonly affects elderly patients. Direct immunofluorescence (DIF) for immunoglobulin G (IgG) and C3c on frozen skin biopsies is the gold standard for the diagnosis of BP. In a minority of cases, IgG and/or C3c are found negative, and in these situations, there is a need for a more stable diagnostic marker of BP. C4d is biologically inactive, but has a long half-life, rendering it a long-lived marker for antibody-mediated complement activation. Previous studies already demonstrated that C4d was diagnostically useful in formalin-fixed paraffin-embedded skin biopsies of patients with BP. We hypothesized that C4d detected by DIF could also be a promising diagnostic marker for BP, particularly in IgG and/or C3c DIF-negative cases. In this single-center retrospective study, 69 cases of BP were analyzed for linear deposition of C4d; of the 69 cases, n = 26 were IgG+/C3c-, n = 10 IgG+/C3c+, and n = 33 IgG-/C3c-. Results were compared with n = 39 negative controls. Seven of the 26 (27%) IgG+/C3c- and 3 of the 33 (9%) IgG-/C3c- BP cases were positive for C4d. All 10 IgG+/C3c+ cases were also C4d positive. In the negative control group, 2 of the 39 (5%) were found positive for C4d. In conclusion, the current study shows that C4d is a more sensitive but not a 100% specific marker of BP. We conclude that C4d by DIF could be an interesting diagnostic adjunct for BP, particularly in IgG-/C3c- double negative cases.

Complement Activation in the Disease Course of Coronavirus Disease 2019 and Its Effects on Clinical Outcomes.

BACKGROUND: Excessive activation of immune responses in coronavirus disease 2019 (COVID-19) is considered to be related to disease severity, complications, and mortality rate. The complement system is an important component of innate immunity and can stimulate inflammation, but its role in COVID-19 is unknown. METHODS: A prospective, longitudinal, single center study was performed in hospitalized patients with COVID-19. Plasma concentrations of complement factors C3a, C3c, and terminal complement complex (TCC) were assessed at baseline and during hospital admission. In parallel, routine laboratory and clinical parameters were collected from medical files and analyzed. RESULTS: Complement factors C3a, C3c, and TCC were significantly increased in plasma of patients with COVID-19 compared with healthy controls (P < .05). These complement factors were especially elevated in intensive care unit patients during the entire disease course (P < .005 for C3a and TCC). More intense complement activation was observed in patients who died and in those with thromboembolic events. CONCLUSIONS: Patients with COVID-19 demonstrate activation of the complement system, which is related to disease severity. This pathway may be involved in the dysregulated proinflammatory response associated with increased mortality rate and thromboembolic complications. Components of the complement system might have potential as prognostic markers for disease severity and as therapeutic targets in COVID-19.

Ex vivo assays to detect complement activation in complementopathies.

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB++ or GVB0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Complement component 3c and tumor necrosis factor-α systemic assessment after Candida antigen immunotherapy in cutaneous warts.

BACKGROUND: Cutaneous warts are the commonest benign lesion produced by human papillomavirus. Lesions often regress spontaneously yet have a high rate of recurrence. They impair patients' quality of life and carry the potential risk of cancer. Nowadays, Candida antigen immunotherapy has become an encouraging therapeutic modality for warts. We tried to assess the role of the complement pathway and T helper 1 immune response in clinical response to Candida antigen immunotherapy via complement component 3c (C3c) and tumor necrosis factor (TNF)-α, respectively. METHODS: A total of 44 patients with cutaneous warts were enrolled in the study. Patients were injected with Candida antigen at 2-week interval until complete clearance of the lesion or for a maximum of 5 sessions. Blood samples were collected before initiation and after completion of immunotherapy. C3 and C4 were measured using an automated turbidimetric method. Mannose-binding lectin (MBL), C3c, and TNF-α were measured using enzyme-linked immune sorbent assay. RESULTS: A total of 56.4%, 17.9%, and 25.7% of the patients showed complete, partial, and no response to immunotherapy, respectively. Lesions on the dorsum of the foot and sole showed significant clearance (p value = 0.037). All patients had no deficient C3, C4, and MBL serum levels. C3c and TNF-α serum levels were significantly higher in non-responder group (p value < 0.001 and < 0.001, respectively). C3c and TNF-α serum levels were strongly correlated in all the studied patients (r = 0.8, p value < 0.001). CONCLUSIONS: Candida antigen immunotherapy is an effective therapeutic modality for cutaneous warts. C3c and TNF-α serum levels were higher in patients who failed to respond to immunotherapy. CLINICAL TRIAL REGISTRY NUMBER: NCT04399577 , May 2020 "retrospectively registered".

Classical pathway activity C3c, C4 and C1-inhibitor protein reference intervals determination in EDTA plasma.

INTRODUCTION: Reference intervals (RIs) for complement assays in EDTA plasma samples have not previously been published. The objectives of the present study were to validate and/or determine RIs for classical pathway (CP50) activity and C3c, C4 and C1 inhibitor protein (C1INH) assays and to assess the need for age-specific RIs in EDTA plasma. MATERIALS AND METHODS: We retrospectively evaluated a cohort of 387 patients attending our university hospital and known to be free of complement-modifying diseases. The need for age partitioning was assessed and RIs were calculated according to the CLSI protocol. RESULTS: No need for age partitioning was evidenced for CP50 activity, C3c and C4 concentrations and RIs (90% CI) were calculated from the pooled data: 35.4 (33.1-37.2) to 76.3 (73.7-83.6) U/mL for CP50 activity, 0.80 (0.75-0.87) to 1.64 (1.59-1.72) g/L for C3c, and 0.12 (0.10-0.14) to 0.38 (0.36-0.40) g/L for C4. Our results highlight a positive association between age and C1INH concentrations. We derived 3 age partitions (6 months to 30 years, 30-50 and > 50 years) and the related RIs: 0.20 (0.18-0.21) to 0.38 (0.36-0.40) g/L, 0.22 (0.20-0.24) to 0.39 (0.36-0.41) g/L and 0.25 (0.22-0.27) to 0.41 (0.40-0.43) g/L, respectively). CONCLUSIONS: The newly determined RIs for CP50 activity were higher than those provided by the manufacturer for EDTA plasma samples, whereas those for C3c and C4 RIs were similar to the values provided for serum samples. The C1INH concentration and activity were found to be associated with age and age-specific RIs are mandatory for this analyte.

[Monoclonal gammopathy of renal significance and membranoproliferative glomerulonephritis: a complex relationship with promising therapeutic opportunities].

In the last few years, the increasing awareness of the complex interaction between monoclonal component and renal damage has determined not only a new classification of the associated disorders, called Monoclonal Gammopathy of Renal Significance (MGRS), but has also contributed to emphasize the importance of an early diagnosis of the renal involvement, which is often hard to detect but can evolve towards terminal uraemia; it has also pointed at the need to treat these disorders with aggressive regimens, even if they are not strictly neoplastic. The case described here presented urinary abnormalities and renal failure secondary to a membranoproliferative glomerulonephritis (MPGN), with intensively positive immunofluorescence (IF) for monoclonal k light chain and C3, and in the absence of a neoplastic lympho-proliferative disorder documented on bone marrow biopsy. After the final diagnosis of MGRS, the patient was treated with several cycles of a therapy including dexamethasone, cyclophosphamide and bortezomib, showing a good functional and clinical response.

A study of detection and comparison of immunofluorescence on formalin-fixed paraffin-embedded tissue with fresh frozen renal biopsy specimen.

Direct immunofluorescence on the fresh frozen tissue is established way of demonstrating of immunoglobulins and complement deposition in renal biopsies. IF studies can be done on paraffin-fixed tissue (IF-P) and give comparable results to those obtained on frozen tissue for most pathogenic immunoglobulins and immunoglobulin fragments; although, the detection of C3c may be more problematic. In our study, we used proteinase-K method for antigen retrieval. We aimed to detect immunoglobulins and complements in formalin-fixed paraffin-embedded (FFPE) tissue sections from renal biopsies and have comparison of IF staining intensity on FFPE sections with conventional IF on fresh frozen tissue. Based on our results, we conclude that IF-P can serve as salvage technique and has significant diagnostic utility.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway.

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Immunophenotypes and clinical features of lymphocytes in the labial gland of primary Sjogren's syndrome patients.

OBJECTIVE: To investigate consistency of lymphocyte immunophenotype between labial gland and peripheral blood in patients with primary Sjogren's syndrome (pSS). METHODS: Seventy-one pSS patients and 35 patients with maxillofacial trauma were included in the study. Based on the ratio of CD20 to CD3 in labial gland from 71 pSS patients, they were divided into the high and (n = 48) and low CD20 expression group (n = 23). Lymphocyte immunophenotypes in labial glands, course of disease, erythrocyte sedimentation rate (ESR), C-reactive protein, immunoglobulin, and complement levels were analyzed. RESULTS: In the labial gland, the levels of IgG, IgA, IgM, and C3c were higher, but C1q was lower in the pSS group than in the control group (all P < .05). CD20 was detected in labial gland samples of all pSS patients, in which CD3 was positive in 66 (93.0%) patients, and negative in 5 (7.0%). The plasma levels of IgG, IgA, IgM, and CRP, and ESR were higher, but serum C4 level was lower in pSS patients than in the control group (all P < .01). Serum IgG level, ESR, and labial gland CD20 were higher in the high CD20 expression group than the low expression group (all P < .05). CONCLUSION: Primary Sjogren's syndrome patients had a higher expression of CD20 positive infiltrating lymphocytes of the labial gland, accompanied with the changes of immunoglobulins, and complements in both the labial gland and peripheral blood.

Effect of Repeated Freeze-Thaw on Serum Biomarkers Associated with Eye Disease.

BACKGROUND Serum biomarkers are associated with eye diseases, which results in the need for cryopreservation of serum samples. However, the effect on serum biomarker levels of repeatedly freezing and thawing remains poorly understood. The aim of this study was to evaluate the effects of repeated freeze-thaw on the serum levels of the protein, complement C3c (C3c), the micromolecule, uric acid (UA), and the enzyme, angiotensin-converting enzyme (ACE). MATERIAL AND METHODS Serum samples were obtained from 50 patients who attended an ophthalmic outpatient department. Following baseline measurements, the serum samples from each subject were divided into aliquots and stored at -80°C for further analysis, following between one to six freeze-thaw cycles. The serum levels of C3c, UA, and ACE were measured immediately after the stored serum samples were thawed. RESULTS The serum level of C3c was significantly changed after the first freeze-thaw cycle (p<0.05), and a significant alteration in serum ACE levels occurred after the third freeze-thaw cycle (p<0.05). The serum UA level remained unchanged after all freeze-thaw cycles. Repeated freeze-thaw cycles significantly increased the serum levels of C3c and decreased the serum levels of ACE. The serum levels of C3c, UA, and ACE, respectively were significantly correlated (p<0.001), while the correlation coefficient for C3c and UA were improved when compared with ACE. CONCLUSIONS Repeated freeze-thaw can have variable effects on the serum levels of biomarkers, C3c, UA and ACE, which supports the need for quality control of cryopreserved serum for biomarker evaluation.

Putative salivary protein biomarkers for the diagnosis of oral lichen planus: a case-control study.

BACKGROUND: Salivary protein biomarkers for screening and diagnosis of oral lichen planus (OLP) are not well-defined. The objective of this study was to identify putative protein biomarkers for OLP using proteomic approaches. METHODS: Pooled unstimulated whole saliva was collected from five OLP patients and five healthy control participants. Saliva samples were then subjected to two-dimensional gel electrophoresis, followed by mass spectrometry to identify putative protein biomarkers. Subsequently, a subset of these putative biomarkers were validated in 24 OLP patients and 24 age-matched healthy control subjects, using an enzyme-linked immunosorbent assay (ELISA). Immunoblotting analyses were then performed in 3 pairs of age- and sex-matched OLP patients and healthy controls to confirm results from the ELISA study. RESULTS: Thirty-one protein spots were identified, corresponding to 20 unique proteins. Notably, fibrinogen fragment D and complement component C3c exhibited increased expression in OLP patients, while cystatin SA exhibited decreased expression in OLP patients, compared with healthy control subjects. ELISA analyses indicated increased expression of fibrinogen fragment D and complement component C3c, and decreased expression of cystatin SA, in the saliva of OLP patients. Statistical differences in the expression of salivary complement C3c were observed between OLP patients and healthy control subjects. Immunoblotting analyses confirmed the results of our ELISA study. CONCLUSION: Complement C3c, fibrinogen fragment D and cystatin SA may serve as salivary biomarkers for screening and/or diagnosis of OLP.

Altered glycan accessibility on native immunoglobulin G complexes in early rheumatoid arthritis and its changes during therapy.

The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment.

Dexrazoxane Shows No Protective Effect in the Acute Phase of Reperfusion during Myocardial Infarction in Pigs.

Calcium and iron overload participate in the mechanisms of ischemia/reperfusion (I/R) injury during myocardial infarction (MI). Calcium overload induces cardiomyocyte death by hypercontraction, while iron catalyses generation of reactive oxygen species (ROS). We therefore hypothesized that dexrazoxane, an intracellular metal chelator, would attenuate I/R injury. MI was induced in pigs by occlusion of the left anterior descending artery for 1 hour followed by 2 hours reperfusion. Thirty minutes before reperfusion either 5 mg/ml dexrazoxane (n = 5) or saline (n = 5) was infused intravenously. Myocardial necrosis as percentage of the area at ischemic risk was found to be similar in both groups (77.2 ± 18% for dexrazoxane and 76.4 ± 14% for saline group) as determined by triphenyl tetrazolium chloride staining of the ischemic myocardium. Also, serum levels of troponin-I were similar in both groups. A conductance catheter was used to measure left ventricular pressure and volume at all times. Markers for tissue damage due to ROS (HNE), endothelial cell activation (CD31) and inflammation (IgG, C3b/c, C5b9, MCP-1) were assessed on tissue and/or in serum. No significant differences were observed between the groups for the parameters analyzed. To conclude, in this clinically relevant model of early reperfusion after acute myocardial ischemia, dexrazoxane lacked attenuating effects on I/R injury as shown by the measured parameters.

Humoral immune response and coated or uncoated oxygenators during cardiopulmonary bypass surgery.

AIM: To investigate and compare uncoated and phosphorylcholine-coated oxygenators in terms of induction of humoral immune response during coronary artery bypass surgery. METHODS: A total of 20 consecutive patients who underwent coronary artery bypass surgery were randomly distributed into two groups according to the type of oxygenator used during surgery. Group 1 consisted of 10 patients who were operated on using phosphorylcholine-coated oxygenators. Group 2 contained 10 patients who underwent surgery using uncoated oxygenators. Blood and oxygenator fibre samples were obtained and compared in terms of immunoglobulins (IgG, IgM), complements (C3c, C4), serum total protein and albumin levels using electron microscopy and flow cytometry. RESULTS: In group 1, levels of IgM, IgG, total protein and serum albumin were significantly increased at the end of cardiopulmonary bypass (CPB) compared to those at the beginning of CPB. In group 2, C3c and C4 levels at the beginning of CPB were found to be significantly higher than at the end. Electron microscopic examination of oxygenator fibres demonstrated that phosphorylcholine-coated fibres were less likely to be adsorbed by serum proteins and complements than the uncoated fibres. CONCLUSION: Our results indicate that phosphorylcholine-coated oxygenators seemed to induce humoral immune response to a lesser extent than uncoated oxygenators during coronary artery bypass procedures.

Eupatorium lindleyanum DC. flavonoids fraction attenuates lipopolysaccharide-induced acute lung injury in mice.

Eupatorium lindleyanum DC., "Ye-Ma-Zhui" called by local residents in China, showed anti-inflammatory activity and is used to treat tracheitis. We had isolated and identified the flavonoids, diterpenoids and sesquiterpenes compounds from the herb. In the present study, we evaluated the protective effects of the flavonoids fraction of E. lindleyanum (EUP-FLA) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and the possible underlying mechanisms of action. EUP-FLA could significantly decrease lung wet-to-dry weight (W/D) ratio, nitric oxide (NO) and protein concentration in BALF, lower myeloperoxidase (MPO) activity, increase superoxide dismutase (SOD) activity and down-regulate the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß). Additionally, EUP-FLA attenuated lung histopathological changes and significantly reduced complement deposition with decreasing the levels of Complement 3 (C3) and Complement 3c (C3c) in serum. These results demonstrated that EUP-FLA may attenuate LPS-induced ALI via reducing productions of pro-inflammatory mediators, decreasing the level of complement and affecting the NO, SOD and MPO activity.