Sequencing-based typing of HLA-B*51 alleles and the significant association of HLA-B*5101 and -B*5108 with Behçet's disease in Greek patients.

Behçet's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101. As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD. After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method. The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108. In contrast, all of the 9 B51-positive normal controls carried B*5101. This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.

Localization of the pathogenic gene of Behçet's disease by microsatellite analysis of three different populations.

PURPOSE: Behçet's disease (BD) is known to be associated with HLA-B51 in many ethnic groups. However, the pathogenic gene responsible for BD is as yet unknown. To localize the critical region of the pathogenic gene, microsatellite markers distributed around the HLA-B gene were investigated. The BD patients studied were of three ethnic origins: Japanese, Greek, or Italian. METHODS: The total group consisted of 172 BD patients, of whom were 95 Japanese, 55 Greek, and 22 Italian. Eight polymorphic microsatellite markers distributed within 1100 kb of the HLA-B gene were analyzed using PCR and subsequent automated fragment detection by fluorescent-based technology. RESULTS: Among the eight markers, allele 348 of the MIB microsatellite was remarkably common in all three BD populations (Japanese, PC: = 0.000014; Greek, PC: = 0. 00047; Italian, PC: = 0.11). However, HLA-B51 was found to be the marker most strongly associated with BD in each population (Japanese, PC: = 0.000000000017; Greek, PC: = 0.00000032; Italian, PC: = 0. 0074). In genotypic differentiation between the patients and controls, only HLA-B51 was found to be significantly associated with BD in all three populations. Stratification analysis suggested that significant associations of BD with MICA and other microsatellites resulted from a linkage disequilibrium with HLA-B51. CONCLUSIONS: These results suggest that the pathogenic gene of BD is HLA-B51 itself and not other genes located in the vicinity of HLA-B.

Adamantiades-Behçet's disease: interleukin-8 is increased in serum of patients with active oral and neurological manifestations and is secreted by small vessel endothelial cells.

The serum levels of several cytokines were determined in 94 patients with Adamantiades-Behçet's disease (ABD), aged 36.1+/-11.0 years, during the active stage (n = 75) and the inactive stage (n = 19) of the disease. A group of 75 healthy individuals matched for age and sex served as controls. Cytokine levels were determined using commercially available ELISA kits. Of the 75 patients with active disease and 19 with inactive disease, 38 (51%) and 4 (21%), respectively, and 23 healthy controls (31%) were found to have detectable levels of interleukin 8 (IL-8) in their serum (P < 0.05). Also, increased IL-8 serum levels were found in patients with active disease (median 12 pg/ml, P = 0.010) compared to patients with inactive disease (< or = 10 pg/ml) and to healthy controls (< or = 10 pg/ml). In particular, patients with oral aphthous ulcers (n = 51, 34 pg/ml) and neurological features (n = 4, 71 pg/ml) exhibited increased IL-8 levels. In contrast, there was no correlation between disease activity and the serum levels of IL-1alpha, IL-1beta, tumor necrosis factor alpha (TNF-alpha), soluble intercellular adhesion molecule-1 or basic fibroblast growth factor (bFGF). In a second set of experiments, the involvement of dermal microvascular endothelial cells in IL-8 secretion was investigated. Immortalized human dermal microvascular endothelial cells (HMEC-1 cells) were maintained for 4 h in vitro with serum from 18 ABD patients or with IL-1beta, a known stimulator of IL-8 synthesis, TNF-alpha or their combination at five- to tenfold higher concentrations than those found in the serum of ABD patients. Increased IL-8 secretion was found after incubation with ABD patients' serum (median 20 pg/ml), but IL-1beta, TNF-alpha and IL-1beta + TNF-alpha failed to induce IL-8 secretion by HMEC-1 cells (< or = 1-1.2 pg/ml) in biologically relevant concentrations. Our study showed increased IL-8 serum levels in ABD patients with active oral and neurological manifestations. Human microvascular endothelial cells may, at least partially, be responsible for the enhanced IL-8 secretion in the active stage of the disease.

Hepatitis B and C viruses in the etiology of hepatocellular carcinoma; a study in Greece using third-generation assays.

OBJECTIVES: The purpose of this study was to describe the role of hepatitis B virus (HBV) and hepatitis C virus (HCV) in the etiology of hepatocellular carcinoma (HCC). METHODS: During a 4-year period from January 1995 to December 1998, blood samples and questionnaire data were obtained from 333 incident cases of HCC from Athens, Greece, as well as from patients in two control groups, also from Athens. Controls were 272 metastatic liver cancer (MLC) patients and 360 patients hospitalized for injuries or eye, ear, nose or throat conditions. Coded sera were tested for hepatitis B surface antigen (HBsAg) and antibodies to hepatitis C virus (anti-HCV) by third-generation enzyme immunoassays. RESULTS: The odds ratios (with 95% confidence intervals) in logistic regression modeling comparing the HCC cases to the combined control series were 48.8 (30.5-78.3) for the presence of HBsAg and 23.2 (11.4-47.3) for the presence of anti-HCV. The odds ratio for concurrent infection with HBV and HCV was 46.2 (9.9-216.6) compared to infection with neither virus. CONCLUSIONS: Although HBV and HCV are both important causes of HCC in this study population the data do not suggest, neither do they conclusively refute, a super-additive interaction between the two infections in the development of this malignancy. In this population, 58% of HCC cases can be attributed to HBV, 12% to HCV, and 3% to dual infection with these viruses.

Tobacco smoking, alcohol consumption and their interaction in the causation of hepatocellular carcinoma.

During a 4-year period from January 1995 to December 1998, blood samples and questionnaire data were obtained from 333 incident cases of hepatocellular carcinoma (HCC), as well as from 360 controls who were hospitalized for eye, ear, nose, throat or orthopedic conditions in Athens, Greece. Coded sera were tested for hepatitis B surface antigen (HBsAg) and antibodies to hepatitis C virus (anti-HCV) by third-generation enzyme immunoassays, and information on smoking habits and beverage consumption was obtained. We found a significant dose-response, positive association between smoking and HCC risk [>/= 2 packs per day, odds ratio (OR)=2.5]. This association was stronger in individuals without chronic infection with either HBV or HCV (>/= 2 packs per day, OR=2.8). Consumption of alcoholic beverages above a threshold of 40 glasses per week increased the risk of HCC (OR=1.9). We also found evidence of a strong, statistically significant and apparently super-multiplicative effect of heavy smoking and heavy drinking in the development of HCC (OR for both exposures=9.6). This interaction was particularly evident among individuals without either HBsAg or anti-HCV (OR for both exposures=10.9). Coffee intake was not positively associated with HCC risk, but the reverse could not be excluded for the subgroup of chronically infected individuals. In conclusion, tobacco smoking and heavy alcohol consumption are associated with increased risk of HCC, especially when these 2 exposures occur together.

Genetic polymorphism of IL-12 p40 gene in immune-mediated disease.

Understanding of the genetic basis of autoimmune diseases is currently incomplete. Cytokine gene polymorphisms warrant consideration as factors explaining variation in the human immune and inflammatory responses and as candidate susceptibility genes for related pathological states. Interleukin 12 (IL-12) is a key regulator of the polarisation of immune responses to T helper 1 or 2 categories and plays a role in autoimmune and infectious diseases. Using a bioinformatic strategy, we aligned cDNA and expressed sequence tag sequences to identify putative polymorphic regions of the IL-12 p40 gene. Position 1188 in the 3' untranslated region (UTR) was polymorphic with the frequency of the common allele around 80% in healthy UK Caucasoids. PCR genotyping of multiple Caucasoid groups and an African group showed significant population variation. In a case-control design, the polymorphism was not associated with rheumatoid arthritis, Felty's syndrome or large granular lymphocyte syndrome with arthritis or multiple sclerosis. A nonsignificant increase in the B allele frequency was observed in the rare large granular lymphocyte syndrome without arthritis (odds ratio 2.02 95% CI 0.95-4.3). This new genetic marker could be useful in anthropological studies and should be investigated in other autoimmune, allergic, inflammatory and infectious diseases.

Dietary factors in relation to rheumatoid arthritis: a role for olive oil and cooked vegetables?

BACKGROUND: Although several studies showed that risk of rheumatoid arthritis (RA) is inversely associated with consumption of n-3 fatty acids, the one study showing that olive oil may have a protective role has not yet been confirmed. OBJECTIVE: We examined the relation between dietary factors and risk of RA in persons from southern Greece. DESIGN: We studied 145 RA patients and 188 control subjects who provided information on demographic and socioeconomic variables, prior medical and family history, and present disease status. Subjects responded to an interviewer-administered, validated, food-frequency questionnaire that assessed the consumption of >100 food items. We calculated chi-square statistics for linear trend and odds ratios (ORs) for the development of RA in relation to the consumption of olive oil, fish, vegetables, and a series of food groups classified in quartiles. RESULTS: Risk of developing RA was inversely and significantly associated only with cooked vegetables (OR: 0.39) and olive oil (OR: 0.39) by univariate analysis. A significant trend was observed with increasing olive oil (chi-square: 4.28; P = 0.03) and cooked vegetable (chi-square: 10. 48; P = 0.001) consumption. Multiple logistic regression analysis models confirmed the independent and inverse association between olive oil or cooked vegetable consumption and risk of RA (OR: 0.38 and 0.24, respectively). CONCLUSIONS: Consumption of both cooked vegetables and olive oil was inversely and independently associated with risk of RA in this population. Further research is needed to elucidate the underlying mechanisms of this finding, which may include the antioxidant properties or the high n-9 fatty acid content of the olive oil.

Dietary fat and carbohydrates are independently associated with circulating insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 concentrations in healthy adults.

PURPOSE: To evaluate and quantify the association between consumption of specific food groups/macronutrients and concentrations of serum insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 3 (IGFBP-3). SUBJECTS AND METHODS: Data from a comprehensive food-frequency questionnaire administered to 115 healthy subjects were used to study cross-sectionally the relationship between nutritional factors and circulating IGF-1 and IGFBP-3 concentrations. Adjustment for the effect of total energy intake and a series of epidemiologic parameters (age, sex, height, body mass index, smoking, alcohol consumption, and coffee drinking) was implemented through multivariate linear regression. RESULTS: We observed that serum levels of IGF-1 are positively associated with consumption of red meats, fats, and oils. In addition, serum levels of IGF-1 are independently and positively associated with energy intake from lipids and negatively associated with energy intake from carbohydrates. Finally, serum levels of IGFBP-3 are independently and negatively associated with energy intake from saturated fat. CONCLUSION: Serum IGF-1 and/or IGFBP-3 concentrations are associated with red meat, carbohydrate intake, and fat intake and, thus, may mediate the effect of these dietary factors on the pathogenesis of several disease states. Additional studies are needed to further quantify these associations and elucidate the underlying mechanisms.

Association of MICA gene and HLA-B*5101 with Behçet's disease in Greece.

PURPOSE: Behçet's disease (BD) is known to be associated with HLA-B51 in many different ethnic groups. Recently MICA, a member of a novel family of the human major histocompatibility complex (MHC) class I genes termed MIC (MHC class I chain-related genes), was identified near the HLA-B gene, and a triplet repeat microsatellite polymorphism was found in the transmembrane (TM) region. Because a strong association with BD of one particular MICA-TM allele, A6, was shown in a Japanese population, the present study was conducted to investigate microsatellite polymorphism in Greek patients with BD to know whether this association is generally observed in BD occurring in other populations. METHODS: Thirty-eight Greek patients with BD and 40 ethnically matched control subjects were examined for MICA microsatellite polymorphism using polymerase chain reaction (PCR) and subsequent automated fragment detection by fluorescent-based technology. RESULTS: Similar to the Japanese patients with BD, the phenotype frequency of the MICA-TM A6 allele was significantly increased in the Greek patients with BD (50.0% in control subjects versus 86.8% in BD cases), with an odds ratio (OR) of 6.60 (P = 0.0012). The MICA-A6 allele was found in a high frequency both in males and females (weighted OR = 6.68; P = 0.0017). No association was found between the A6 allele and several disease features. A strong association exists between the MICA-TM A6 allele and the B*5101 allele in both the control subjects and patients with BD (weighted OR = 44.39; P = 0.0000023). CONCLUSIONS: This study revealed in Greek patients a strong association of BD with a particular MICA-TM allele, MICA-A6, providing insight into the molecular mechanism underlying the development of BD.

Age, sex, and smoking are predictors of circulating insulin-like growth factor 1 and insulin-like growth factor-binding protein 3.

PURPOSE: Insulin-like growth factor (IGF-1) and its major binding protein (IGF-BP3) have recently been implicated in the pathogenesis of several malignancies. However, anthropometric and lifestyle predictors of these hormones have not been elucidated. Here we report the results of a cross-sectional study. SUBJECTS AND METHODS: This cross-sectional study examines the relationship of a series of epidemiologic parameters (age, sex, height, body mass index, smoking, alcohol consumption, and coffee drinking) with IGF-1 and IGF-BP3 in a sample of 130 healthy adults. RESULTS: We observed that serum levels of IGF-1 are higher, whereas levels of IGF-BP3 are lower, in men than in women. In addition, serum levels of IGF-1 are independently and negatively associated with age and positively associated with pack-year history of smoking. Finally, serum levels of IGF-BP3 are independently and negatively associated with the number of cigarettes smoked per day or pack-year history of smoking. CONCLUSION: Age, sex, and smoking are independent predictors of IGF-1 and/or IGF-BP3. The influence of these epidemiologic variables on the pathogenesis of disease states associated with IGF-1 and IGF-BP3 warrants further exploration.

HLA-B*5101 in Greek patients with Behçet's disease.

Behçet's disease (BD) is a recurrent systemic vasculitis of unknown etiology. Genetic factors and infectious agents seem to be related to the etiology and pathogenesis of the disease. BD is strongly associated with HLA-B51 antigen in many ethnic groups. As there are differences in HLA profile in different ethnic groups, we designed this case-control study to examine the association of HLA-B51 alleles and BD as well as to investigate the influence of sex, age at development of the International Study Group (ISG) for Behçet's Disease criteria and certain features of disease severity on the strength of this association. The study includes 62 Greek BD patients who fulfill the ISG criteria for Behçet's disease and 87 controls. Serological HLA Class-I typing was performed by standard microlymphocytotoxicity technique. HLA-DNA typing for the B5 group was performed in all B51 subjects and controls by PCR-SSO. Allele B*5101 was found in 80% of BD patients and in 26% of controls (odds ratio (OR) 10.48, p < 10[-6]). Males who carry this allele have a higher risk than females for BD (OR 16.97 and 5.74 respectively). B*5101 predisposes to BD at a younger age in both sexes and to the development of erythema nodosum (OR = 11, p = 0.004). This was confirmed by multiple logistic regression analysis. A weak but not significant association was found between B*5101 and uveitis (OR = 2). No association was found between B*5101 and vasculitis or skin lesions in either sex. It was concluded that in the Greek population allele B*5101 is a predisposing marker for BD, as in most ethnic groups, and that this allele predisposes to the development of the disease at a younger age in both sexes and to the development of erythema nodosum.

HLA-DRB1 alleles in Greek rheumatoid arthritis patients and their association with clinical characteristics.

The association of certain HLA-DRB1 alleles in Green rheumatoid arthritis (RA) patients with several features of the disease, the gender of the patient and the age at onset was investigated. This case control study includes 86 Greek RA patients and 130 healthy controls unrelated to the patients. HLA typing was performed by polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotide (SSO) probes. HLA-DR4 was significantly increased in RA patients. The alleles *0101, *0401, *0405 and *1001 were associated with a higher risk of RA. The *0408 allele was absent from our patients. Sixty-five per cent of RA patients carried the 'sharp epitope' (SE) compared with 31.5% of controls. The risk for RA in individuals carrying a single allele positive for SE was 2.85 times higher, and for those carrying two alleles positive for SE 8.57 times higher, than in SE-negative individuals. The risk was higher in those carrying the *0401 allele, followed by *0405 and *0101, while the genotype *0401/*0404 was absent. Alleles positive for SE comprise a predisposing factor for RA at an early age, particularly in men, and are associated with positive rheumatoid factor, nodules and erosions.

Family history as a risk factor for rheumatoid arthritis: a case-control study.

OBJECTIVE: To investigate the risk of rheumatoid arthritis (RA) in the first degree relatives and to investigate whether the sex of the parent influences the pattern of inheritance. METHODS: An interview based case-control study, with subjects serially matched for age and sex. We analyzed 126 cases (hospital cases) and 94 controls (derived from the same hospitals), who gave information for family history of RA. Data concerning RA history among siblings and parents were computerized and analyzed univariately and multivariately. RESULTS: The odds ratio (OR) for developing RA is 4.4 (p < 0.001) if a first degree relative reported having the disease and 5.4 (p < 0.01) if a female first degree relative reported having the disease. For females the OR is 7.0 (p < 0.01) if the first degree relative is female. When the analysis was restricted to parents only, it was found that mothers with RA predispose their daughters and sons to develop RA more (OR = 8.6, p < 0.01, for daughters and 4.8, p < 0.05, for both sexes) than fathers (OR = 1.1 and 1.9, respectively). CONCLUSION: This case-control study confirms the familial clustering of RA and suggests that mothers confer susceptibility to RA on their offspring more often than fathers.

Hepatitis C virus 1b is the dominant genotype in HCV-related carcinogenesis: a case-control study.

In an ongoing case-control study in Athens on the etiology of hepatocellular carcinoma (HCC), an analysis was made in order to assess whether HCV genotype 1b is associated with hepatocellular carcinoma (HCC). The HCV genotype was determined in 17 HCC patients, 87 patients with chronic hepatitis C (CHC) without cirrhosis (NC-CHC) and 23 patients with CHC and cirrhosis (C-CHC). HCV genotype 1b was detected in 14/17, 16/23 and 23/87 of HCC, C-CHC and NC-CHC respectively. The age- and gender-adjusted odds ratios contrasting HCC with NC-CHC and C-CHC with NC-CHC were 8.3 and 3.8 respectively. These data strongly support the hypothesis that HCV 1b is a stronger liver carcinogen than other HCV genotypes, probably through increased HCV replication and enhanced liver cytopathicity.

A case-control study of hepatitis B and C virus infections in the etiology of hepatocellular carcinoma.

During a 16-month period in 1991-1992, blood samples and questionnaire data were obtained from 65 incident cases of hepatocellular carcinoma (HCC) as well as from 2 control groups of hospitalized patients matched on gender and age, which included 65 metastatic liver cancer (MLC) patients and 65 patients hospitalized for eye, ear, nose or throat conditions. Coded sera were tested for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen, antibody to HBsAg and antibody to hepatitis C virus (anti-HCV) by enzyme immunoassay. The odds ratios (with 95% confidence intervals) in logistic regression modeling comparing the HCC cases to the combined control series were 18.8 (8.2-43.2) for the presence of HBsAg and 7.7 (1.7-35.1) for anti-HCV. In the present hospital-based case-control study anti-HCV testing was conducted on recently collected sera, using a second-generation enzyme immunoassay with confirmation by immunoblot assay. Comparisons with previous work in a similar population demonstrated that, when second-generation anti-HCV assays are applied to sera stored for 7-15 years, confirmatory assays or a higher diagnostic cut-off point may be necessary to ensure that the testing is specific.

The effect of Mycoplasma arthritidis infection on the kinetics of colloidal carbon clearance in mice.

The activity of phagocytes from A/J mice was estimated by the carbon clearance test following injection of Mycoplasma arthritidis. Phagocytic activity was significantly depressed 12 h post-infection (P = 0.001) and returned to normal values at 24 h. For animals examined 2 and 7 days post-infection, the overall phagocytic activity increased significantly (P < 10(-4). Phagocytic activity gradually decreased and returned to that of the control group by the end of the fourth week. The relative weights of liver and spleen were significantly increased from the 2nd day post infection (P = 0.0028 and P = 0.0014 respectively) and remained increased until the end of the experiment. The early depressive effect on phagocytic activity may be related to superantigen activity with the production of mediators such as macrophage deactivating factor. The later expansion of the macrophage population might bring about the stimulation of autoreactive clones of T and B cells and be responsible for the chronic arthritis that developed in the mycoplasma treated mice.

Hepatitis C virus infection in Greece and its role in chronic liver disease and hepatocellular carcinoma.

The aim of this study was to evaluate the prevalence of hepatitis C virus infection (HCV) in Greece, to estimate its frequency in chronic liver disease and to explore the role of HCV infection in the aetiology of hepatocellular carcinoma. A series of 1034 patients with chronic liver disease of various aetioloigies and 299 patients with hepatocellular carcinoma allocated to two case-control studies was tested for anti-HCV. Twelve recent reports on HCV infection in Greece were reviewed and analyzed. The results of the present study indicate the existence of a large pool of HCV infection in Greece and an impressive spread of the virus in high-risk groups. Chronic HCV infection was found to account for 83.6% of patients with chronic non-A, non-B hepatitis parenterally transmitted, 56.5% of cases of sporadic community-acquired disease and for almost 1/4 of all patients with chronic liver disease. The relative risks for development of hepatocellular carcinoma of patients with chronic HCV infection was 6.3 in the first and 13.7 in the second case-control study, increasing to 20.0 and 18.7, respectively, when hepatitis B surface antigen (HBsAg) was positive. Serum HBV-DNA was positive and/or anti-HBc IgM levels were high in 12 of 15 (80%) patients with hepatocellular carcinoma positive only for HBsAg, and in 7 of 15 (47%) positive both for HBsAg and antibodies to HCV. The present data support the view that hepatitis B and C virus have an interacting role in the origin of hepatocellular carcinoma.