C4B gene influences intestinal microbiota through complement activation in patients with paediatric-onset inflammatory bowel disease.

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota.

Resolution of HLA-B*44:02:01G, -DRB1*14:01:01G and -DQB1*03:01:01G reveals a high allelic variability among 12 European populations.

Within the framework of the EU-funded HLA-NET action, an analysis of three G-group alleles, HLA-B*44:02:01G, DRB1*14:01:01G and DQB1*03:01:01G, was undertaken in 12 European populations. Ambiguities were resolved by polymerase chain reaction-sequence-specific amplification (PCR-SSP) or PCR-sequence-based typing (PCR-SBT) in a total of 5095 individuals. The results of the DRB1*14:01/14:54 ambiguity showed high relative ratios (24-53%) of DRB1*14:01 in Bulgarians, Croatians, Greeks, Italians and Slovenians, contrasting with low ratios (6-13%) in Austrians, Finnish, French, Hungarians, Norwegians and Swiss. Resolution of the B*44:02/44:27 ambiguity showed that B*44:27 had a high relative ratio in Slovenians (25.5%) and Bulgarians (37%) and low in French and Swiss (0.02-1%), and was not observed in Greeks and Italians. The highest relative ratio of DQB1*03:19 was found in Portuguese (11%), by contrast with low ratios (0-3%) in the other five populations. Analysis of the A, B, DRB1 phenotypes and family-derived haplotypes in 1719 and 403 individuals positive for either HLA-B*44:02G or DRB1*14:01G ambiguities, respectively, showed some preferential associations, such as A*26∼DRB1*14:01, B*35∼DRB1*14:01, B*38∼DRB1*14:01 and B*44:27∼DRB1*16. Because these ambiguities are located outside the peptide-binding site, they may not be recognized by alloreactive T-cells. However, because of strong linkage disequilibrium (LD), the DRB1*14:01 vs DRB1*14:54 and the B*44:02 vs B*44:27 mismatches are associated to DRB3-, and C-mismatches, respectively. These results are informative for algorithms searching unrelated hematopoietic stem cell donors. For B*44:27-positive patients, searches are expected to be more successful when requesting donors from Southeastern-European ancestry. Furthermore, the introduction of human leukocyte antigen (HLA)-typing strategies that allow resolving exon 4 (for class I) and exon 3 (for class II) polymorphisms can be expected to contribute significantly to population genetics studies.

Lymphotoxin alpha LTA+496C allele is a risk factor for periodontitis in patients with coronary artery disease.

Periodontitis and coronary artery disease (CAD) are inflammatory diseases and associated with each other. The major histocompatibility complex (MHC) region carries genes involved in immune response and inflammation. We investigated whether the MHC genes correlate with the presence of periodontitis or with the occurrence of periodontal pathogens in patients with CAD. Blood and saliva samples from CAD patients (n = 106) were collected at the time of hospitalization. Nine MHC genetic markers [human leukocyte antigen (HLA)-A, HLA-B, HLA-DRB1, lymphotoxin alpha (LTA) +253(a/g), +496(C/T), +633(c/g), +724(C/A), C4A and C4B)] were typed. Based on panoramic tomography, patients were categorized into nonperiodontitis and periodontitis groups. Two major periodontal pathogens, Aggregatibacter (Actinobacillus) actinomycetemcomitans and Porphyromonas gingivalis, were cultivated and polymerase chain reaction-amplified from salivary samples. Serum immunoglobulin (Ig)A and IgG antibody levels to these pathogens were measured. In the univariate analysis, LTA+496C allele (OR = 5.29; 95% CI = 2.07-13.51, P = 0.00027), and the occurrence of P. gingivalis in saliva (OR = 4.74; 95% CI = 1.64-13.70; P = 0.002) were more frequent in periodontitis when compared with nonperiodontitis. Similarly, serum IgA antibody level against the pathogen was increased in periodontitis (P = 0.048). In the multiple logistic regression analysis, when a wide range of covariates was included, the LTA+496C allele (OR = 10.87; 95% CI = 3.23-36.60; P = 0.00012) and the elevated serum IgA antibody level against P. gingivalis (OR = 1.56; 95% CI = 1.05-2.30; P = 0.026) remained as significant risk factors for periodontitis. In conclusion, the major finding of this study is that the LTA+496C allele is associated with periodontitis in patients with CAD.

Human MHC region harbors both susceptibility and protective haplotypes for coronary artery disease.

Aiming to study the role of human major histocompatibility complex (MHC) region on coronary artery disease (CAD), we enrolled two separate patient materials and controls. First, heart transplantation recipients (n = 276) were divided into three subgroups according to the severity of atherosclerosis. The human leukocyte antigen (HLA)-A-B-DR haplotype and gene frequencies were compared between groups. Second, patients with acute coronary syndrome (ACS) (n = 100) and healthy controls (n = 74) were assessed by nine genetic MHC markers (HLA-A, HLA-B, HLA-DRB1, LTA+253(a/g), LTA+496(C/T), LTA+633(c/g), LTA+724(C/A), C4A and C4B), and the frequencies were compared. In the heart transplantation recipients, HLA-DR1 was strongly associated with CAD [severe vs no evidence, odds ratio (OR) 2.37; 95% confidence interval (CI) 1.33-4.25; P = 0.003]. Similarly, in the patients with ACS, HLA-DRB1*01 was associated with CAD (patients vs controls, OR 2.36; 95% CI 1.25-4.44; P = 0.007). HLA-DRB1*01 was associated with low-density-lipoprotein cholesterol (OR 5.32; 95% CI 1.64-17.26; P = 0.005) and smoking habit (OR 3.13; 95% CI 1.09-9.03; P = 0.035) as risk factors. The strongest protective gene was HLA-B*07 alone (OR 0.46; 95% CI 0.24-0.88; P = 0.02) or together with the haplotype LTA+253a-LTA+633g-C4A3-C4B1 (OR 0.36; 95% CI 0.22-0.57; P = 0.00001). In conclusion, human MHC region harbors genes that protect from and predispose to CAD.

Complement and c4 null alleles in severe chronic adult periodontitis.

Severe forms of chronic periodontitis affect up to 10% of adults. Tumour necrosis factor and lymphotoxin-alpha genes in the major histocompatibility complex are associated with severe periodontitis. Complement factor C4 is a nearby, polymorphic, functionally relevant gene region. Although associated with chronic mucosal infections, C4 deficiencies have not been assessed in adult periodontitis patients. We tested whether complement levels are systemically altered and C4 deficiencies associated with severe chronic periodontitis. In a case-control study, we analysed levels of plasma C3, and C4, serum classical pathway haemolytic activity, C4 allotypes and C4 gene numbers in 37 patients with severe chronic periodontitis and in 150 voluntary controls. Plasma levels of C3 were higher, and classical pathway haemolytic activity was lower in patients than in controls. Partial C4 gene deficiencies were more frequent in patients than in controls (odds ratio 2.4, 95% confidence interval 1.1-5.5, P = 0.032). Changes in complement levels may reflect chronic, recurring inflammation. C4 gene deficiencies are associated with predisposition to chronic periodontitis.

Immunoglobulins and complement factor C4 in adult rhinosinusitis.

We assessed whether complement and its factor C4 or abnormal immunoglobulin levels are associated with chronic or recurrent rhinosinusitis. We used multiple patient and control groups to obtain clinically meaningful data. Adult chronic or recurrent rhinosinusitis and acute purulent rhinosinusitis patients were compared with unselected adults and controls without previous rhinosinusitis. Associated clinical factors were reviewed. Levels of immunoglobulins, plasma C3, C4 and classical pathway haemolytic activity were analysed. C4 immunophenotyping was used to detect C4A and C4B deficiencies as null alleles. Complement was up-regulated in rhinosinusitis. C4A nulls and low IgA, IgG, IgG1, IgG2, IgG3 and IgG4 levels were all more common in chronic or recurrent rhinosinusitis patients than in unselected and healthy controls. We searched for relevant differences between the patient groups. According to stepwise logistic regression analysis, nasal polyposis [odds ratio (OR) 10.64, 95% confidence interval (CI) 2.5-45.7, P = 0.001], bronchial asthma (OR 8.87, 95% CI 2.3-34.9, P = 0.002), C4A null alleles (OR 5.84, 95% CI 1.4-24.9, P = 0.017) and low levels of IgG4 together with either IgG1 or IgG2 (OR 15.25, 95% CI 1.4-166.8, P = 0.026) were more common in chronic or recurrent rhinosinusitis than in acute rhinosinusitis patients. Isolated low IgG subclasses had limited value in patient assessment. C4A null alleles are associated with chronic or recurrent rhinosinusitis, potentially through their effect on immune defence and inflammation control. Multiple clinical and immunological parameters may need to be evaluated when searching for prognostic variables.

Inherited complement C3 deficiency: reduced C3 mRNA and protein levels in a Laotian kindred.

To determine the molecular basis of complement C3 deficiency in a Laotian kindred, the homozygous C3-deficient male propositus was studied. By ELISA, this individual's serum was determined to contain approximately 4 microg/ml C3 (0.3% of normal). In accord with this result, anti-C3 immunoprecipitation of [35S]-methionine-labeled fibroblasts from this C3D individual revealed pro-C3 of normal size (180,000 Mr), but in significantly reduced amounts (approximately 1% of normal fibroblasts), that was processed and secreted with normal-size alpha- and beta-chains. In addition, C3-specific mRNA of normal size (5.2 kb) but in reduced quantity (approximately 1% of normal) was detected in this individual's fibroblasts by Northern analysis. The nucleotide sequence of the transcriptional initiation site, the promoter, and the IL-1beta/IL-6 cis-regulatory elements of the C3-deficient gene are normal in this C3-deficient individual, indicating that the low C3 mRNA and protein levels are not caused by reduced C3 transcription that is the result of a cis-mutation. Moreover, cDNA sequencing studies revealed no defect in the C3-deficient mRNA, including the areas mutated in four previously characterized C3-deficient patients. These data indicate that (1) C3 protein deficiency in this Laotian patient results from reduced levels of C3-specific mRNA, (2) the small amount of expressed C3 protein is processed and secreted normally from the deficient cells, and (3) the molecular genetic defect(s), although not yet delineated, is different from those described in other C3-deficient individuals, thereby providing additional evidence for numerous mutations that cause inherited C3 deficiency in humans.

Tumour necrosis factor B gene polymorphism in relation to complotype in couples with spontaneous abortions and in control families.

NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor B (TNFB) gene gives two allelic fragments of 5.5 and 10.5 kb, corresponding to the TNFB*1 and TNFB*2 alleles, respectively. The frequencies of these alleles were analysed in 121 healthy Finns and 56 Finnish couples suffering from recurrent spontaneous abortions (RSA). In the healthy Finns the frequency of TNFB*1 was 37% and that of TNFB*2 63%, of which the frequency of TNFB*1 was significantly increased compared with the Danish population. No deviation was seen between the RSA couples and the Finnish controls. TNF genes are located in major histocompatibility complex class III region close to complement (BF, C4A and C4B) genes. Some complotypes associated most often with the TNFB*1 (S01, S30, S02) and some (S31, F320) with the TNFB*2 allele in the healthy Finns. The combination of the TNFB and the C4 'null' allele differed between the RSA couples and the Finnish controls.