Modelling biological age based on plasma peptides in Han Chinese adults.

Age-related disease burdens increased over time, and whether plasma peptides can be used to accurately predict age in order to explain the variation in biological indicators remains inadequately understood. Here we first developed a biological age model based on plasma peptides in 1890 Chinese Han adults. Based on mass spectrometry, 84 peptides were detected with masses in the range of 0.6-10.0 kDa, and 13 of these peptides were identified as known amino acid sequences. Five of these thirteen plasma peptides, including fragments of apolipoprotein A-I (m/z 2883.99), fibrinogen alpha chain (m/z 3060.13), complement C3 (m/z 2190.59), complement C4-A (m/z 1898.21), and breast cancer type 2 susceptibility protein (m/z 1607.84) were finally included in the final model by performing a multivariate linear regression with stepwise selection. This biological age model accounted for 72.3% of the variation in chronological age. Furthermore, the linear correlation between the actual age and biological age was 0.851 (95% confidence interval: 0.836-0.864) and 0.842 (95% confidence interval: 0.810-0.869) in the training and validation sets, respectively. The biological age based on plasma peptides has potential positive effects on primary prevention, and its biological meaning warrants further investigation.

Impact of C4, C4A and C4B gene copy number variation in the susceptibility, phenotype and progression of systemic lupus erythematosus

Abstract Background Complement component 4 (C4) gene copy number (GCN) affects the susceptibility to systemic lupus erythematosus (SLE) in different populations, however the possible phenotype significance remains to be determined. This study aimed to associate C4A , C4B and total C4 GCN and SLE, focusing on the clinical phenotype and disease progression. Methods C4 , C4A and C4B GCN were determined by real-time PCR in 427 SLE patients and 301 healthy controls, which underwent a detailed clinical evaluation according to a pre-established protocol. Results The risk of developing SLE was 2.62 times higher in subjects with low total C4 GCN (< 4 copies, OR = 2.62, CI = 1.77 to 3.87, p < 0.001) and 3.59 times higher in subjects with low C4A GCN (< 2 copies; OR = 3.59, CI = 2.15 to 5.99, p < 0.001) compared to those subjects with normal or high GCN of total C4 (≥4) and C4A (≥2), respectively. An increased risk was also observed regarding low C4B GCN, albeit to a lesser degree (OR = 1.46, CI = 1.03 to 2.08, p = 0.03). Furthermore, subjects with low C4A GCN had higher permanent disease damage as assessed by the Systemic Lupus International Collaborating Clinics - Damage Index (SLICC-DI; median = 1.5, 95% CI = 1.2-1.9) than patients with normal or high copy number of C4A (median = 1.0, 95% CI = 0.8-1.1; p = 0.004). There was a negative association between low C4A GCN and serositis ( p = 0.02) as well as between low C4B GCN and arthritis ( p = 0.02). Conclusions This study confirms the association between low C4 GCN and SLE susceptibility, and originally demonstrates an association between low C4A GCN and disease severity.

Difference in serum complement component C4a levels between hepatitis C virus carriers with persistently normal alanine aminotransferase levels or chronic hepatitis C.

Certain hepatitis C virus (HCV) carriers exhibit persistently normal alanine aminotransferase (ALT) levels (PNALT) (≤ 30 IU/l) accompanied by normal platelet counts (≥ 15 x 10(4)/µl); these individuals show milder disease activity and slower progression to cirrhosis. This study aimed to elucidate the characteristics of HCV carriers with PNALT using serum proteomics. The first group of subjects, who underwent clinical evaluation in the hospital, consisted of 19 HCV carriers with PNALT (PNALT-1) and 20 chronic hepatitis C (CHC-1) patients. The second group of subjects was part of a cohort study on the natural history of liver disease, and included 37 PNALT (PNALT-2) and 30 CHC (CHC-2) patients. Affinity bead-purified serum protein was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. Serum proteomics showed that 6 protein peaks with mass-to-charge ratios ranging from 1,000 to 3,000 differed significantly between the PNALT-1 and CHC-1 groups. Among these peaks, a 1738-m/z peak protein was identified as a fragment of complement component 4 (C4) and correlated significantly with serum C4a concentrations as determined by enzyme immunoassay. Serum C4a levels were also significantly higher in the PNALT-2 group compared to the CHC-2 group and healthy volunteers. Furthermore, in the PNALT-2 group, serum C4a levels negatively correlated with transaminase levels, but not with other biochemical tests, HCV core antigen levels, peripheral blood cell counts or serum hepatic fibrosis markers. This study indicates that host factors such as C4a not only differ between HCV carriers with PNALT and CHC, but that proteomic approaches could also contribute to the elucidation of factors in PNALT as more differences are discovered.

Selective enrichment and sensitive detection of peptide and protein biomarkers in human serum using polymeric reverse micelles and MALDI-MS.

Reverse-micelle forming amphiphilic homopolymers with carboxylic acid and quaternary amine substituents are used to selectively enrich biomarker peptides and protein fragments from human serum prior to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. After depletion of human serum albumin (HSA) and immunoglobulin G (IgG), low abundance peptide biomarkers can be selectively enriched and detected by MALDI-MS at clinically relevant concentrations by using the appropriate homopolymer(s) and extraction pH value(s). Three breast cancer peptide biomarkers, bradykinin, C4a, and ITIH(4), were chosen to test this new approach, and detection limits of 0.5 ng mL(-1), 0.08 ng mL(-1), and 0.2 ng mL(-1), respectively, were obtained. In addition, the amphiphilic homopolymers were used to detect prostate specific antigen (PSA) at concentrations as low as 0.5 ng mL(-1) by targeting a surrogate peptide fragment of this protein biomarker. Selective enrichment and sensitive MS detection of low abundance peptide/protein biomarkers by these polymeric reverse micelles should be a sensitive and straightforward approach for biomarker screening in human serum.

Downregulation of C3 and C4A/B complement factor fragments in plasma from patients with squamous cell carcinoma of the penis.

PURPOSE: To investigate the use of ClinProt technique to identify cancer markers in plasma of patients suffering from squamous cell carcinoma of the penis (SCCP). MATERIALS AND METHODS: Plasma of 36 healthy subjects and 25 patients with penile carcinoma who underwent surgical treatment between June 2010 and June 2011 was collected and analyzed by the ClinProt/MALDI/ToF technique. Then the peptides were identified from the C8 MB eluted fraction of patients' and control subjects' plasma by LIFT MS/MS. RESULTS: A cluster of 2 peptides (A=m/z 1897.22 ± 9 Da and B=m/z 2021.99 ± 9 Da) was able to discriminate patients from control subjects. Cross validation analysis using the whole casuistic showed 62.5 % and 86.76 % sensitivity and specificity, respectively. The cluster also showed very high sensitivity (100 %) and specificity (97%) for SCCP patients that died due to the disease. Furthermore, patients with lymph node involvement presented sensitivity and specificity of 80 % and 97 %, respectively. These two peptides were identified by the proteomic approach based on a MALDI-TOF/TOF as fragments of C3 (m/z 1896.17) and C4a/b (m/z 2021.26) complement proteins. CONCLUSIONS: The results showed that as the disease progresses, the fragments C3 and C4 A/B are less expressed in comparison with healthy subjects. These results may be useful as prognostic tools.

Serum proteomic profiling reveals that pretreatment complement protein levels are predictive of esophageal cancer patient response to neoadjuvant chemoradiation.

OBJECTIVE: To identify serum-based biomarkers predicting response to neoadjuvant chemoradiotherapy (neo-CRT) in esophageal cancer. PURPOSE: Increasingly, the standard of care for esophageal cancer involves neo-CRT followed by surgery. The identification of biomarkers predicting response to therapy may represent a major advance, enabling clinical trials and improved outcomes. BACKGROUND DATA: Patients with esophageal cancer (n = 31) received a standard neo-CRT regimen. Histopathologic response to therapy was assessed by using the Mandard tumor regression grade (TRG) classification. Serum was collected pretreatment and at 24-hour and 48-hour time points into treatment. Serum samples were analyzed by using Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and enzyme-linked immunosorbent assay. A leave-one-out cross-validation predictive algorithm assessed the ability of validated biomarkers to correctly predict therapeutic outcome. RESULTS: Fifty-one percent (16) of patients were poor responders (TRG 3-5), whereas 49% (15) responded well (TRG 1-2). On CM10 biochips, serum expression of 9 protein peaks was significantly different between the response groups. Two differential spectrum peaks were identified as complement C4a and C3a and were subsequently analyzed by enzyme-linked immunosorbent assay. Pretreatment serum C4a and C3a levels were significantly higher in poor responders versus good responders. Subdivision of the response groups by TRG indicated an inverse correlation between levels of C4a and C3a and pathological response to neo-CRT. The leave-one-out cross-validation analysis revealed that these serum proteins could predict response to neo-CRT with a sensitivity and specificity of 78.6% and 83.3%, respectively. CONCLUSIONS: This translational application of proteomics technology identifies pretreatment serum levels of C4a and C3a as predictive biomarkers of response. Large validation studies in an independent cohort are merited.

[Enzyme immunoassay of masked complement component C4 deficiency in patients with urogenital Chlamydia infection].

AIM: Development of new method of C4B isotype functional activity evaluation in enzyme immunoassay by using pharmaceutical preparation derinat as a classical pathway complement activator and its use for blood sera isotyping in confirmed urogenital tract chlamydia infection. MATERIALS AND METHODS: Enzyme immunoassay was used to detect C4A and C4B isotype functional deficiency in blood sera of patients. Chlamydia etiology urogenital infection diagnosis was based on results of standard clinical-instrumental examination methods: vaginal clinical smear analysis, scrape sample light microscopy with consequent treatment by fluorescent monoclonal antibodies against Chlamydia trachomatis and PCR. RESULTS: In acute form of the disease C4A deficiency frequency of occurrence was 0.36, and C4B deficiency - 0.55. In chronic form of the disease deficiency frequency of occurrence was 0.38 for both isotypes. In the group of healthy people isotype deficiency was 0.08 and 0.25, respectively. CONCLUSION: Innate masked C4 deficiency interfere with the normal immune defense of organism against chlamydia infection, and antigen carbohydrate pathogenicity may possibly be more significant for the development of immune response to which C4B isotype activity is necessary.

Complement component C4A and apolipoprotein A-I in plasmas as biomarkers of the severe, early-onset preeclampsia.

Preeclampsia is a common pregnancy complication that is associated with maternal perinatal morbidity and mortality. Because of its early onset (before 34 weeks) and the potential for serious outcomes, severe, early-onset preeclampsia (sePE) should be regarded as a different form of preeclampsia. It is an important cause of preterm birth and fetal growth restriction and adverse maternal and neonatal outcomes. As there is no diagnostic test yet available for this disease, we used a proteomic approach to identify novel plasma biomarkers for developing severe, early-onset preeclampsia. We conducted case-control studies comparing nulliparous women with severe preeclampsia requiring delivery prior to 34 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Plasma was depleted of albumin and IgG and analyzed by two-dimensional gel electrophoresis (2DE). Seven specific plasma proteins for early-onset preeclampsia were detected by mass spectrometry had statistically significant expression differences when compared to controls. The expression of complement component C4A and apolipoprotein A-I were validated by immunoblotting. The complement component C4A in the plasmas of sePE women is lower than the severe, late-onset PE (slPE) women [mean ± SD; 3.05 ± 0.14 times reference level (normal/sePE) in sePE women vs. 2.73 ± 0.10 times reference level (normal/slPE) in slPE women, P < 0.05]. Apolipoprotein A-I is higher in sePE women than slPE women [mean ± SD; 1.58 ± 0.14 times reference level (sePE/normal) in sePE women vs. 1.04 ± 0.16 times reference level (slPE/normal) in slPE women, P < 0.05]. Furthermore, C4A can accurately distinguish severe PE (sePE and slPE) from mild PE (mePE and mlPE) and was proved by the results of ELISA. Further studies have been done to determine the relation between PE and hypoxia. JAR cells were cultured under hypoxia for 72 h. Total cellular proteins were gathered and lysed. Lower C4A and higher apolipoprotein A-I had been observed in JAR of hypoxia conditions than normoxia conditions through western blotting. The result proved that PE is correlated with hypoxia. In summary, C4A and apolipoprotein A-I are able to function as markers to distinguish ePE women from lPE women, and severe PE from mild PE, or perhaps even as disease predictors that might become relevant for diagnostics.

Serum degradome markers for the detection of breast cancer.

Many proteins have been proposed as potential biomarkers for breast cancer. Yet, validation of their discriminative value using quantitative methods has scarcely been performed. In this study, we investigated the discriminative value of six peptides that were previously proposed to be generated by breast cancer specific exoproteases: bradykinin, des-Arg(9)-bradykinin, Hyp(3)-bradykinin, and fragments of fibrinogen alpha-chain (Fib-alpha ([605-629])), complement component 4a (C4a ([1337-1350])), and interalpha trypsin inhibitor heavy chain 4 (ITIH4 ([666-687])). Their absolute serum concentrations were measured with a completely validated liquid chromatography-tandem mass spectrometric assay (LC-MS/MS) and compared between 62 newly diagnosed breast cancer patients and 62 controls matched for age and sample storage duration. Both ITIH4 ([666-687]) and des-Arg(9)-bradykinin showed statistically significantly higher median concentrations in breast cancer samples than in matched control samples. Additionally, we analyzed serum samples collected after surgical removal of the tumor, in which median ITIH4 ([666-687]) and des-Arg(9)-bradykinin concentrations were significantly decreased and not statistically significantly different from concentrations in the controls anymore. In a combined analysis, ITIH4 (666-687]) and des-Arg(9)-bradykinin independently contributed to the discrimination between cases and controls. In this study, we confirmed that the exoprotease breakdown peptides, ITIH4 (666-687]) and des-Arg(9)-bradykinin, differed between breast cancer cases and controls, supporting the potential of degradome markers for the diagnosis of breast cancer.

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry.

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The peptides include bradykinin, Hyp(3)-bradykinin, des-Arg(9)-bradykinin and fragments of fibrinogen alpha-chain (Fib-alpha([605-629])), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH(4[666-687])) and complement component 4a (C4a([1337-1350])). Ile(13)-ITIH(4[666-687]), d20-C4a([1337-1350]) and Sar-D-Phe(8)-des-Arg(9)-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10-500 ng/ml), Hyp(3)-bradykinin (4-200 ng/ml), des-Arg(9)-bradykinin (2-100 ng/ml), Fib-alpha([605-629]) (120-3000 ng/ml), ITIH(4[666-687]) (0.4-10 ng/ml) and C4a([1337-1350]) (1-25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.

Complement activation in patients with primary antiphospholipid syndrome.

OBJECTIVE: To investigate the significance of complement activation in patients with primary antiphospholipid syndrome (APS). METHODS: Thirty-six patients with primary APS, 42 control patients with non-systemic lupus erythematosus (SLE) connective tissue diseases, and 36 healthy volunteers were analysed retrospectively. Serum complement levels (C3, C4, CH(50)) and anaphylatoxins (C3a, C4a, C5a) were examined in all subjects, and serum complement regulatory factors (factor H and factor I) were measured in patients with primary APS. Plasma anticoagulant activity was determined in a mixing test using the activated partial thromboplastin time. RESULTS: Serum complement levels were significantly lower in patients with primary APS than in patients with non-SLE connective tissue diseases (mean (SD) C3: 81.07 (17.86) vs 109.80 (22.76) mg/dl, p<0.001; C4: 13.04 (8.49) vs 21.70 (6.96) mg/dl, p<0.001; CH(50): 31.32 (8.76) vs 41.40 (7.70) U/ml, p<0.001) or healthy volunteers. Only two healthy subjects with low serum C4 levels showed hypocomplementaemia, whereas most patients with primary APS showed raised serum C3a and C4a. No subjects showed raised C5a. Patients with primary APS with low serum C3 or C4 had significantly higher levels of C3a or C4a than healthy controls. No patients had low serum complement regulatory factors. Among patients with primary APS, hypocomplementaemia was significantly more common in those with high anticoagulant activity than in those with low or normal activity. CONCLUSION: Hypocomplementaemia is common in patients with primary APS, reflecting complement activation and consumption, and was correlated with anticoagulant activity, suggesting that antiphospholipid antibodies may activate monocytes and macrophages via anaphylatoxins produced in complement activation.

The use of ultrafiltration for inflammatory mediators removal during cardiopulmonary bypass in coronary artery bypass graf surgery.

OBJECTIVE: To investigate the effectiveness of ultrafiltration in removing inflammatory mediators released by cardiopulmonary bypass and to correlate ultrafiltration with alterations in organic function according to the Sequential Organ Failure Assessment Score. METHODS: Forty patients were included and randomized into two groups: "no ultrafiltration" (n=20; Group I) and "ultrafiltration" (n=20; Group II). Activated complement 3 and 4, interleukins 1beta, 6, 8 and tumor necrosis factor alfa were measured prior to anesthesia induction (Time 1), 5 minutes before cardiopulmonary bypass (Time 2), in the ultrafiltrated fluid (Time 3), 30 minutes (Time 4), and 6 (Time 5), 12 (Time 6), 24 (Time 7), 36 (Time 8) and 48 (Time 9) hours following cardiopulmonary bypass. Sequential Organ Failure Assessment Score was evaluated at Time 1, 6 and 9. Statistical significance was established at p < 0.05. RESULTS: In the ultrafiltrated fluid, only tumor necrosis factor alfa levels were detected. Levels of activated complement 3 at Times 5 and 7 and activated complement 4 at Times 5 and 6 were significantly higher in the unfiltered Group, and levels of interleukin 6 were higher in the filtered Group at Times 7 and 8. Interleukins 1beta, 8, tumor necrosis factor alfa, and the Sequential Organ Failure Assessment score were not significantly different between the groups. CONCLUSIONS: Ultrafiltration significantly filtered tumor necrosis factor alfa but did not influences serum levels of this cytokine. Ultrafiltration with the type of filter used in this study had no effect in organic dysfunction and should be used only for volemic control in patients undergo cardiopulmonary bypass.

Beneficial effects of C1 esterase inhibitor in ST-elevation myocardial infarction in patients who underwent surgical reperfusion: a randomised double-blind study.

BACKGROUND: The inflammatory cascade has been hypothesized to be an important mechanism of post-ischaemic myocardial reperfusion injury and several studies demonstrated that C1 esterase inhibitor (C1-INH) is effective in post-ischaemia myocardial protection. Therefore, we aimed to investigate prospectively in a randomised double-blind study the cardioprotective effects of C1-INH in ST segment elevation myocardial infarction (STEMI) in patients who underwent emergent reperfusion with coronary artery bypass grafting (CABG). METHODS: In this study, we enrolled 80 patients affected with STEMI who underwent emergent CABG. Patients were assigned in two groups (C1-INH group: receive 1000 UI of C1-INH; and placebo group: receive a saline solution). The effects of C1-INH on complement inhibition, myocardial cell injury extension and clinical outcome were studied. Haemodynamic data and myocardial function were monitored. C1-INH, C3a, C4a complement activation fragments and cardiac troponin I (cTnI) serum levels were measured before, during and after surgery. RESULTS: Patient characteristics were not different between the two groups. The overall in-hospital mortality rate was 6.2%. No statistical significant difference was observed between the two groups with regard to early mortality (p=0.36). Statistical significant difference between the two groups was showed for cardiopulmonary bypass support (p=0.04), administration of high dose of inotropes drugs (p=0.001), time of intubation (p=0.03), intensive care unit (ICU) stay (p=0.04) and in-hospital stay (p=0.03). A significant improvement in mean arterial pressure (p=0.03), cardiac index (p=0.02) and stroke volume (p=0.03) was showed in C1-INH group versus placebo group. The serum cTnI levels were significantly low in the C1-INH group versus placebo group after reperfusion, during the observation period. Plasma levels of C3a and C4a complement fragments were reduced significantly in C1-INH group. No drugs-related adverse effects were observed. CONCLUSIONS: The inhibition of the classic complement pathway by C1-INH appears to be an effective mean of preserving ischaemic myocardium from reperfusion injury as demonstrated by low serum cTnI levels in C1-INH group. Therefore, the use of C1-INH during CABG as a rescue therapy in STEMI patients is probably an effective treatment to inhibit complement activity and to improve cardiac function and haemodynamic performance without impacting early mortality. Large randomised study should be performed to support our results.

Common variable immunodeficiency and the complement system; low mannose-binding lectin levels are associated with bronchiectasis.

The importance of the innate immune system, including mannose-binding lectin and the complement system, in common variable immunodeficiency is unclear. The objective of this study was to evaluate mannose-binding lectin and the complement system in relation to clinical and immunological parameters in patients with common variable immunodeficiency. Circulating levels of mannose-binding lectin, complement components, complement activation products and functional capacity of complement pathways were correlated to clinical features within 71 patients and compared with 30 healthy controls. The main findings were; the patients had signs of increased complement activation significantly associated with signs of autoimmunity and immunological hyperactivity; there were no signs of deficiencies of the classical and alternative complement pathways in the patient group; the prevalence of lectin pathway deficiency was the same in patients and controls, but patients with increased frequency of lower respiratory tract infections or bronchiectasis had lower capacity of the lectin pathway than patients without these features (P = 0.002 and 0.004, respectively); the serum concentration of mannose-binding lectin was inversely correlated to the frequency of lower respiratory tract infections (P = 0.002) and bronchiectasis (P = 0.01). We conclude that patients with common variable immunodeficiency have no increased frequency of complement deficiencies but signs of increased complement activation. Our findings suggest that mannose-binding lectin and the lectin complement pathway may protect against lower respiratory tract infection and bronhiectasis in patients with common variable immunodeficiency.

A new poly-2-methoxyethylacrylate-coated cardiopulmonary bypass circuit possesses superior platelet preservation and inflammatory suppression efficacy.

BACKGROUND: Poly-2-methoxyethylacrylate (PMEA) is a new coating material, and several studies have revealed that PMEA-coated cardiopulmonary bypass (CPB) circuits have good biocompatibility. This study sought to compare this biocompatibility with those of heparin-coated and noncoated circuits. METHODS: Forty-five patients undergoing coronary artery bypass grafting were randomly assigned to PMEA-coated (group P, n = 15), heparin-coated (group H, n = 15), or noncoated (group N, n = 15) circuit groups. Clinical data and the following markers were analyzed: (1) platelet preservation by number of platelets; (2) complement (C) activation by C3a and C4a levels; (3) inflammatory response by interleukin-6 (IL-6) and interleukin-8 (IL-8) levels. RESULTS: Platelet numbers were significantly preserved in group P compared with groups N and H. Postoperative blood loss did not differ among the groups. During CPB, C3a values were significantly lower in group H (536 +/- 145 ng/mL) than in group P (1,458 +/- 433 ng/mL, p < 0.01) and group N (1,815 +/- 845 ng/mL, p < 0.01). The C4a values did not differ 60 minutes after CPB initiation among the groups. The IL-6 and IL-8 levels were significantly lower in group P and group H than in group N. CONCLUSIONS: The PMEA coating was superior to heparin coating and noncoating in preserving platelets, and was equivalent to heparin coating in terms of the perioperative clinical course and inhibition of inflammatory cytokines, but slightly inferior in reducing complement activation.

Complement activation in a model of chronic fatigue syndrome.

BACKGROUND: A need exists to identify biological markers in chronic fatigue syndrome (CFS). OBJECTIVE: To use an exercise and/or allergen challenge to induce the symptoms of CFS and to identify a biological marker that correlates with these symptoms. METHODS: Patients with CFS (n = 32) and age-matched, normal control patients (n = 29) exercised for 20 minutes on a stationary bike at 70% of their predicted max work load (Watts). Patients from each group with positive skin test results were also challenged with intranasally administered relevant allergens. Symptoms were recorded for 2 weeks before and 1 week after each challenge, using 3 different instruments. Blood samples were taken before, and 0, 1, 6, and 24 hours after challenges. Levels of complement split products, cell-associated cytokines, and eosinophilic cationic protein were measured. Mean preexercise and postexercise symptom scores were evaluated for each group. RESULTS: Exercise challenge induced significant increases of the complement split product C4a, but not C3a or C5a, at 6 hours after exercise only in the CFS group (P <.01), regardless of allergy status. Mean symptom scores were significantly increased after exercise through the use of a daily diary (P <.03) and a weekly diary (P <.01) for the CFS group only. Mean scores for the Multidimensional Fatigue Inventory categories "reduced activity" and "mental fatigue" were significantly increased in the CFS group only (P <.04 and P <.02, respectively). CONCLUSIONS: Exercise challenge may be a valuable tool in the development of diagnostic criteria and tests for CFS. Establishment of a role for complement activation products as markers or participants in production of illness require further study.

Relationship between complement components C4A and C4B diversities and two TNFA promoter polymorphisms in two healthy Caucasian populations.

The RP-C4-CYP21-TNX (RCCX) modules and the tumor necrosis factor (TNF) gene cluster are probably the most polymorphic genomic regions in the human central major histocompatibility complex (MHC). Using definitive methods for genotypic and phenotypic analyses of complement components C4A and C4B, determination of the RCCX length variants, and SSP-PCR/RFLP analyses of TNFA promoter polymorphisms at positions -308 and -238, we studied the complex relationships between the C4 and TNFA polymorphisms in two normal Caucasian populations. The patterns of the RCCX modular structures and the allelic frequency of -308A TNFA (TNF2) were similar between the Budapest (n = 125) and the Ohio (n = 80) Caucasians. However, the frequency of the -238A allele was significantly higher in the Ohio (11.3%) than in the Budapest (1.6%) study population (p < 0.0001). Marked features were found in the RCCX length variants in the TNF2 carriers and noncarriers. Strong associations were found between the C4AQ0 B1 haplotype from the monomodular short (mono-S) RCCX structure and the TNF2 allele, and between the C4A6 B1 haplotype from the bimodular long-short (LS) structure of the RCCX and the TNFA -238A allele. However, 36%-46% of the TNF2 carriers did not associate with a mono-S in both study cohorts, and 57.1% of the TNFA -238A carriers in Ohio did not associate with C4A6, which has a defective complement C5 convertase activity. The carriers of TNF2 allele had significantly lower C4A serum concentration (0.17 +/- 0.08 g/l) than noncarriers (0.23 +/- 0.09 g/l) (p < 0.001). The lowest C4A serum levels were found in TNF2 carriers with mono-S structures (0.14 +/- 0.06 g/l). In essence, our results demonstrated the heterogeneities of the TNFA promoter polymorphisms, and the linkage disequilibrium of TNFA -308A and -238A alleles with complement C4A deficiency and impaired C4A protein function, respectively.

[Effect of complement 4 genetic polymorphism on complement activation during cardiopulmonary bypass in open heart surgery among children].

OBJECTIVE: To evaluate the relation between complement 4 genetic polymorphism and complement activation during extracorporeal circulation in open heart surgery among children. METHODS: One hundred and fifty-six children to undergo open heart surgery with cardiopulmonary bypass in Wuhan and surrounding areas were investigated. Blood samples were obtained before the operation to analyze the C4 phenotypes by crossed immunoelectrophoresis. Before the initiation of CPB, at the end of CPB, and 10 minutes after protamine infusion, arterial blood samples were drawn to test the C4a and C3a with radioimmunoassay and lung compliance was recorded. RESULTS: The frequencies of C4 genetic phenotypes AABB, AOBB, OOBB, AABO, and AAOO were 51.28%, 17.95%, 4.49%, and 19.80% respectively among the investigated children. At the end of CPB, The C4a levels remained almost the same, and C3a levels increased significantly. Ten minutes after the protamine infusion, significant increase was seen in the levels of both C4a and C3a, especially in the OOBB phenotype group followed by AOBB phenotype group. The lung compliance began to decrease by the end of CPB, most significantly in the OOBB phenotype group, followed by the AOBB group, along with the increase of C4a level. CONCLUSION: The complement system is activated by the alternative pathway during CPB and by the classic way during the formation of protamine-heparin complexes. The complement system is activated and the lung function is damaged the most seriously during CPB among the persons with OOBB phenotype. The frequency of OOBB phenotype is higher among Chinese than among Caucasians.

Plasma C3a and C4a levels in liver transplant recipients: a longitudinal study.

Liver transplant patients were enrolled in a study designed to investigate correlations between plasma complement C3a or C4a levels and various postoperative complications. Longitudinal EDTA-plasma levels of C3a and C4a were measured by quantitative radioimmunoassay. Acute rejection gave a characteristic and marked increase in blood C3a, C4a and gamma-glutamyl transferase (gammaGT) levels, which rapidly resolved after high dose steroid treatment. Cytomegalovirus (CMV) infections in two of three patients gave an initial small increase only in C3a levels (i.e., alternative pathway activation) followed approximately 6 weeks later by a marked increase in C4a levels (i.e., classical or lectin pathway activation). In a third patient diagnosed for CMV infection, the complement activation profile was complicated by a coincident minor rejection episode. However, a late stage elevation in C4a was also noted. Two patients experiencing biopsy proven recurrent hepatitis C infections following transplantation exhibited increases in both gammaGT and C4a levels, without a significant increase in the level of C3a. Several hepatitis C and one hepatitis B patient had multiple late activation episodes involving marked elevation in both plasma C3a and C4a levels without detectable increases in the liver enzymes conventionally used to monitor organ function. We also showed that ex vivo activation of complement in EDTA plasma from all transplant patients was abnormally high. The classical or lectin pathway is believed to be responsible for this excessive ex vivo complement activation in the plasma of these patients. Therefore, subclinical rejection episodes and/or viral infections may be effectively detected or monitored by measuring C3a and C4a levels in plasma samples from liver transplant patients. Routine measurement of plasma complement products may provide an early non-invasive mode for detecting infections and also serve to monitor chronic or acute changes in the patient's immune system.

Complement activation by propofol and its effect during propofol anaesthesia.

We have examined whether propofol activates complement. In the first study, blood was mixed with saline, propofol or the lipid solvent for propofol, and the activated complement 3 (C3a) and 4 (C4a) concentrations in the supernatant were assayed. In the second study, blood and propofol were mixed with various levels of nafamostat mesilate (anti-complement agent) up to 0.3 mmol/l and the C3a was assayed. In the third study, the time course of plasma C3a concentration in patients during propofol anaesthesia was examined. The results showed that the lipid solvent activated complement and produced similar levels of C3a to propofol, probably via both the classical and alternative pathways. This activation was not inhibited by any of the nafamostat concentrations used. There was no significant change in plasma C3a concentration during propofol anaesthesia. These results suggest that C3a is generated by the lipid solvent, but its accumulation during propofol anaesthesia is minimal.