Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders.

Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.

Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF-alpha?

Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF-alpha). Since human adipose tissue produces TNF-alpha, to further characterize the relationship of PAI-1 to TNF-alpha, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF-alpha through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10(-3)M, the same inhibitory effect caused by Genistein at 100 microg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF-alpha production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF-alpha antibody (at a concentration able to inhibit 100 UI/ml human TNF-alpha activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8+/-19.0% decrease, P = 0.04, n = 7). In conclusion, the results of this study demonstrate that PAI-I expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF-alpha. Furthermore our data support the possibility of a main role of endogenous TNF-alpha on human adipose tissue PAI-1 expression. This cytokine, produced by human adipose tissue and causing insulin resistance, may be a link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels.

Pulmonary mucosa-associated lymphoid tissue lymphoma and myasthenia gravis. A case report.

We describe a low-grade, MALT-lymphoma with multiple, unusually large opacities involving both the lungs in a woman suffering from myasthenia gravis. Unlike other autoimmune diseases, myasthenia gravis has never been associated with MALT-lymphoma thus far. After cyclophosphamide treatment, a complete detersion of the pulmonary opacities was obtained.

Chronic parvovirus B19 infection induces the production of anti-virus antibodies with autoantigen binding properties.

Human parvovirus B19 infection in adults shows some clinical features similar to those found in autoimmune connective tissue diseases. To better clarify the relationship between viral infection and autoimmunity, we have evaluated the ability of anti-parvovirus antibodies to specifically recognize autoantigens in ten patients with chronic symmetric arthritis resembling rheumatoid arthritis or with recurrent episodes of arthritis and cutaneous manifestations and persistence of specific IgM antibodies against B19 parvovirus. We synthetized a 24-amino acid immunodominant peptide corresponding to a part of the virus protein 1 and virus protein 2 overlapping region. The peptide has been used to test patients' sera at different time points with an enzyme-linked immunosorbent assay (ELISA) and to purify anti-virus antibodies by affinity chromatography on a peptide-Sepharose column. Eluted immunoglobulins recognized the B19 peptide in both direct and competitive ELISA. Affinity-purified anti-parvovirus antibodies were then tested on a panel of autoantigens including human keratin, collagen type II, thyreoglobulin, single-strand (ss)DNA, cardiolipin and ribonucleoprotein antigen Sm. Eluted antibodies specifically recognized keratin, collagen type II, ssDNA and cardiolipin. Autoantibody activity was not detected in the immunoglobulin fraction after complete removal of anti-peptide antibodies and in antibodies eluted from normal donors. Epstein-Barr virus-transformed cell clones obtained from two subjects produced antibodies which simultaneously recognize the viral peptide and several autoantigens. To further confirm the role of the virus in inducing an autoantibody response, eight BALB/c mice were immunized with the viral peptide coupled to a carrier protein. Autoantibody activity against keratin, collagen II, cardiolipin and ssDNA was detected in six of the eight mice which developed a strong anti-virus response. Together, these data indicate that B19 parvovirus may be linked to the induction of an autoimmune response.

[Pulmonary mucosa-associated lymphoid tissue lymphoma in a patient with myasthenia gravis]. / Linfoma polmonare del tessuto linfoide associato alla mucosa in una paziente affetta da miastenia grave.

Mucosa-associated lymphoid tissue lymphomas are a subgroup of non-Hodgkin's lymphoma. The lung is the most frequent non-gastrointestinal organ they affect. Pulmonary mucosa-associated lymphoid tissue lymphoma usually appears as a solitary mass often accidentally discovered on chest radiography. Diffuse, bilateral involvement is rare. The association of mucosa-associated lymphoid tissue lymphoma with autoimmune diseases has been reported, and a pathogenetic role has been suggested for the autoimmune process in its development. Optimum management has not yet been standardized. The case described here is a mucosa-associated lymphoid tissue lymphoma with multiple, unusually large opacities involving both lungs. The patient, a 55-year-old woman, also suffered from myasthenia gravis, an autoimmune disease characterized by an autoaggressive process against the acetylcholine receptors. Whereas other autoimmune diseases such as rheumatoid arthritis, polymyositis, and fibrosing alveolitis have been correlated with mucosa-associated lymphoid tissue lymphoma, an association between this lymphoma and myasthenia gravis has not yet been reported. Complete resolution of the pulmonary opacities was obtained with cyclophosphamide treatment. It continues at 15 months after the suspension of therapy.

Serum laminin and type III procollagen in chronic hepatitis C. Diagnostic value in the assessment of disease activity and fibrosis.

Laminin P1 (pepsin-resistant fragment of laminin) and aminoterminal peptide of type III procollagen are measurable in serum and are now considered useful serum markers of fibrogenesis and inflammation in chronic liver diseases. However, very few studies thus far have focused on assessing the diagnostic value of these markers in detecting fibrosis and necro-inflammatory activity in chronically diseased liver. The aim of the present study was therefore to investigate the correlations of laminin and type III procollagen with liver histology and to compare their diagnostic value in detecting the degree of liver fibrosis and necro-inflammatory activity in a homogeneous group of 99 patients suffering from chronic hepatitis C, and lacking other factors which can directly affect the serum levels of the two markers. Both these serum markers were measured by radioimmunoassay, employing commercially available kits. The three main aspects of liver pathology, i.e. portal-periportal activity, lobular activity and fibrosis, were histologically evaluated and semiquantitatively expressed by numerical scores. The results of this study show that laminin and type III procollagen were both positively correlated with the histological scores for portal-periportal activity and with those for fibrosis, whereas no significant correlation was observed between each of the two serum markers and the histological scores for lobular activity. The sensitivity and specificity of laminin and type III procollagen in detecting histological aspects of fibrosis and disease activity in liver, computed at various cut-off levels, showed overlapping trends for the two markers; however, the diagnostic value was in general rather low, whatever the cut-off considered. We therefore conclude that the 'static' measurement of both serum laminin and type III procollagen is of limited value for individual diagnosis of liver damage.

Analysis of the T cell receptor repertoire in rheumatoid arthritis.

OBJECTIVE: To evaluate whether there is a restricted T cell receptor repertoire in rheumatoid synovium and to analyse the CDR3 region of the V beta families found to be more expressed in the synovial membrane than in the peripheral blood, in order to ascertain the presence of clonotypic expansion of T lymphocytes. METHODS: The level of expression of individual V beta and V alpha families of the TCR was evaluated in paired synovial membrane and peripheral blood T cells from 8 female patients affected by rheumatoid arthritis, using the RT-PCR method. Nucleotide sequences of the CDR3 region of some V beta families were analysed in order to identify the presence of conserved sequences. Sequencing was carried out with the dideoxy chain termination method using modified T7 DNA polymerase. RESULTS: All of the V alpha and V beta families were amplified in both compartments of the 8 patients. Four patients did not show any preferential expression of the TCR alpha or beta chains in synovium compared with peripheral blood. The other 4 patients showed increased expression of one or more V alpha and/or V beta families in the synovium. We did not find any correlation between the duration of disease, rheumatoid factor status, HLA-DR type and the V gene families which were elevated in the synovium. Analysis of the CDR3 region showed the presence of conserved amino acid sequences in the synovium, but not in the peripheral blood. CONCLUSION: The V families found to be increased in 4 of the 8 patients studied were different, except for V beta 1 which was more highly expressed in 2 patients. The presence of conserved amino acid sequences in the CDR3 region is consistent with an antigen-driven T cell expansion at the site of autoimmune inflammation. These findings do not support our original hypothesis of the possible usefulness of therapy based on the inactivation or elimination of presumed pathogenic T cells using TCR-derived peptides or monoclonal antibodies against particular TCRs.

Effects of five-day versus one-day infusion of iloprost on the peripheral microcirculation in patients with systemic sclerosis.

OBJECTIVE: To evaluate the effects of iloprost infusion on the microcirculation in patients suffering from severe Raynaud's phenomenon secondary to systemic sclerosis. METHODS: Eight patients received a 7-hour infusion of iloprost for five consecutive days and then for one day 3 months later. The effects on vascular distensibility were evaluated by piezoelectric plethysmography before and after the treatment and at 2, 4 and 6 weeks. RESULTS: The beneficial effects on the peripheral microcirculation were statistically significant after five days of infusion (distensibility index: 0.18 +/- 0.01 vs 0.23 +/- 0.01, p < 0.002) and lasted for less than four weeks, whereas no difference (0.22 +/- 0.04 vs 0.24 +/- 0.02, p: ns) was seen after one day of treatment. One patient suffered from typical angina pectoris with electrocardiographic changes of the ST wave detected during the infusion. CONCLUSION: Our results show that a five-day infusion of iloprost has an effect which lasts from two to four weeks; after four weeks the distensibility index returned to the baseline value. The one-day infusion had no effect on the vascular bed, studied by the piezoelectric pletysmographic method. Treatment with five consecutive days of infusion every four weeks is an impracticable scheme to adopt, however. We have therefore instituted a treatment schedule of a single daily infusion every four weeks with the aim of maintaining the effects induced by the initial five-day infusion. The preliminary results obtained with this schedule are reported.

Ultrasonographic intra-abdominal depth and its relation to haemostatic factors in healthy males.

OBJECTIVE: To examine the relationship of intra-abdominal fat to plasma haemostatic factors. SUBJECTS: 60 healthy, predominantly nonobese, male volunteers aged 38 years. MEASUREMENTS: Anthropometry, sonographic intra-abdominal depth (IAD), as an indicator of intra-abdominal fat, plasma lipids, plasma insulin (at fasting and after glucose load), various plasma haemostatic factors. RESULTS: Sonographic IAD correlated positively with plasma fibrinogen (r = 0.33; P < 0.01), PAI-1 activity (r = 0.52; P < 0.0001) and antigen (r = 0.32; P < 0.05), and negatively with t-PA activity, at baseline and after stasis (r = -0.28 and r = 0.31; P < 0.05). Factor VII levels did not correlate significantly with sonographic IAD. Haemostatic variables were also closely associated with BMI, plasma triglyceride and insulin concentrations. Most correlations of haemostatic factors with IAD disappeared after adjustment for either BMI or insulin or triglycerides, except PAI-1 levels which maintained a significant association even after simultaneous adjustment for all potential confounders. CONCLUSIONS: These results, obtained by sonography, confirm our previous findings of significant associations of haemostatic variables with visceral fat accumulation by using computed tomography, and highlight the role of the intra-abdominal fat as an independent predictor of PAI-1 activity.

Serum laminin P1 in chronic viral hepatitis: correlations with liver histological activity and diagnostic value.

Laminin is a major basement membrane-associated, non-collagenous glycoprotein of the extracellular matrix and is deposited in the space of Disse during sinusoidal capillarisation. Laminin P1, a pepsin-resistant fragment originating from the central portion of the cross-shaped laminin molecule, is detectable in serum and has been related to liver fibrosis and portal hypertension. In this study we investigated the behaviour of serum laminin P1, measured by radioimmunoassay, in a homogeneous group of 95 patients suffering from chronic viral hepatitis, types C or B, in order to determine the relationships between serum laminin P1 and each of the main histological aspects of the disease process (i.e. portal-periportal activity, lobular activity and fibrosis), which were assigned numerical scores. Moreover, we computed, at several cut-off levels, the sensitivity, specificity and positive and negative predictive values of laminin P1 in detecting both necroinflammatory activity and fibrosis in the liver. The results show that serum laminin P1 levels parallel the severity of liver disease, the highest laminin concentrations being observed in cirrhotic patients. They suggest also that serum laminin P1 should be considered a marker of the liver disease process as a whole, rather than a marker exclusively linked to fibrosis. Nevertheless, the usefulness of serum laminin P1 measurement, as investigated in this study, seems too limited to be recommended for routine clinical practice.

Liver steatosis and its relation to plasma haemostatic factors in apparently healthy men--role of the metabolic syndrome.

The relationship between liver steatosis, evaluated by ultrasonography, and various plasma haemostatic factors was examined in 64 apparently healthy males, aged 38 years. Plasma levels of factor VII clotting activity (F-VIIc), plasminogen activator inhibitor-1 (PAI-1) activity and antigen, tissue-type plasminogen activator (t-PA) activity significantly differed in men with liver steatosis (n = 31) as compared with those without steatosis (n = 33). No significant differences were found in t-PA antigen and F-VII antigen. The men with liver steatosis also had significantly higher body mass index (BMI), plasma triglyceride and 2 h post-load insulin concentrations. While the differences in plasma haemostatic factors were substantially unchanged after adjustment for BMI, they totally disappeared when further allowance was made for plasma triglyceride and 2 h insulin concentrations. In conclusion, these results indicate that liver steatosis correlates specifically with increased PAI-1, F-VIIc and decreased t-PA levels, and suggest that such a relation is largely mediated by concomitant alterations in plasma triglyceride and insulin concentrations.

The white blood cell count: its relationship to plasma insulin and other cardiovascular risk factors in healthy male individuals.

OBJECTIVES: To evaluate the relationships of total and differential white blood cell (WBC) count to the components of the so-called insulin resistance syndrome. SUBJECTS AND DESIGN: The study population consisted of a random sample of 90 38-year-old healthy men with normal glucose tolerance. INTERVENTIONS: A 75 g oral glucose tolerance test was performed in all participants. MAIN OUTCOME MEASURES: Total and differential WBC count, lipids, blood pressure, plasma glucose, C-peptide and insulin (at fasting and 2 h after glucose load). RESULTS: Total WBC count correlated consistently with plasma 2-h glucose (r = 0.38; P < 0.001), fasting and 2-h postload insulin (r = 0.26 and r = 0.33; P < 0.01-0.001, respectively) and C-peptide (r = 0.28 and r = 0.32; P < 0.01-0.001) concentrations. Smokers had significantly higher total leukocytes (P < 0.01), neutrophils and lymphocytes than nonsmokers. Furthermore, total WBC count correlated positively with body mass index, blood pressure, plasma triglycerides, fibrinogen, and negatively with HDL cholesterol concentration. As differential WBC count, most variables correlated essentially to neutrophils and/or lymphocytes, whereas plasma insulin and C-peptide concentrations correlated essentially to lymphocytes and monocytes, but not to neutrophils. In a multiple linear regression analysis, only 2-h plasma glucose (P < 0.01) and fibrinogen (P < 0.05) were positive predictors of total WBC count after adjusting for all potentially confounding variables. CONCLUSIONS: The results indicate that increased, albeit normal, WBC count associates with the cluster of metabolic and haemodynamic disorders typical of the insulin resistance syndrome, and suggest that increased WBC count may be yet another component of this syndrome.

Plasma factor VII and its relation to adipose tissue fatty acids and other atherogenic risk factors in healthy men.

In this study the authors examined the relationships of plasma factor VII (F-VII) to adipose tissue fatty acid composition, as an objective index of the habitual dietary fat intake, as well as to a number of other atherogenic risk factors in 60 healthy male volunteers (aged 38 years). Significant positive correlations were found between plasma F-VII [measured as antigen (F-VIIAg) and coagulant activity, using bovine thromboplastin (F-VIIbt)] and body mass index (BMI), waist-thigh girth ratio (WTR), cigarette smoking and plasma triglyceride concentration. After adjustment for BMI, only plasma triglycerides remained positively correlated with F-VII (r = 0 center dot 27, P = 0 center dot 03, and r = 0 center dot 29, P < 0 center dot 01, for F-VIIbt and F-VIIAg respectively). A significant positive relation was found between F-VII and the total proportion of fatty acid as monounsaturated fatty acid (r = 0 center dot 26, P < 0 center dot 05, for F-VIIAg), whereas inverse relations were found between F-VII, the total proportion of fatty acid as polyunsaturated fatty acid (r = -0 center dot 26 and r = -0 center dot 25, P < 0 center dot 05, for F-VIIbt and F-VIIAg respectively), polyunsaturated-saturated fat ratio (r = -0 center dot 25, P < 0 center dot 05, for F-VIIbt) and, more significantly, between F-VII and adipose-tissue alpha-linolenic acid (r = -0 center dot 29, P < 0 center dot 01, for F-VIIbt and r = -0 center dot 49, P < 0 center dot 001, for F-VIIAg). All these correlations remained significant after matching for BMI. In a multiple linear regression analysis, only adipose tissue alpha-linolenic acid was a negative and independent predictor of F-VIIAg (P = 0 center dot 004) and, at borderline significance, of F-VIIbt (P = 0 center dot 061) when allowance was made for BMI, WTR, smoking and plasma triglycerides. In conclusion, this study shows significant relations between F-VII and adipose tissue fatty acid composition in healthy male individuals; it supports the possibility that adipose tissue poly-unsaturated fatty acids, derived from dietary intake, play a role in the relation between F-VII and coronary heart disease (CHD), thus suggesting that high dietary polyunsaturated fatty acid intake (especially alpha-linolenic acid) may reduce the risk for CHD by an improvement of a number of risk factors, including a lowering of plasma F-VII (both activity and antigen).

Moderate alcohol consumption and its relation to visceral fat and plasma androgens in healthy women.

OBJECTIVE: To study the inter-relationships between daily alcohol intake, fat distribution and plasma androgens in order to verify whether daily alcohol intake correlates with abdominal body fat and, if so, to what extent such a relation is mediated by plasma androgens. SUBJECTS: A random sample of 87 clinically healthy women (aged 38 y) with a light-moderate alcohol consumption and without clinical evidence suggestive of any endocrine disorder. MEASUREMENTS: Anthropometric and computed tomography (CT scans made at the level of L4-L5 in a subgroup of 18 women) measurements of body fatness and adipose tissue distribution, main behavioural factors, including daily alcohol intake and plasma androgens (i.e. total and free testosterone levels). RESULTS: After adjustment for BMI, cigarette smoking and physical activity, significant differences were found in waist circumference and waist-hip ratio as well as in plasma androgens with increasing daily alcohol intake. Waist-thigh ratio tended to parallel waist-hip ratio, but did not achieve statistical significance. In simple linear regression analysis, abdominal visceral fat area, derived from CT, correlated positively with both plasma free testosterone and alcohol intake. While the above reported difference in body fat distribution totally disappeared after controlling also for free testosterone level, the differences in plasma androgens with increasing alcohol intake remained essentially unchanged when allowance was made also for waist-hip ratio. In multiple linear regression analysis, daily alcohol intake appeared to be positively and independently correlated to both plasma total and free testosterone levels. Neither BMI nor waist-hip ratio nor fasting insulin made any significant contribution to the prediction of plasma androgens after daily alcohol intake had been taken into account. CONCLUSIONS: The results of this study show that moderate alcohol consumption correlates with abdominal distribution of body fat, likely due to enlarged visceral fat area, and increased plasma androgenicity (i.e. higher total and free testosterone levels) in adult healthy women. These data also suggest that the relation between alcohol intake and fat distribution may be, at least in part, mediated by plasma androgens.

Visceral fat accumulation and its relation to plasma hemostatic factors in healthy men.

The associations between abdominal visceral fat and the plasma hemostatic system were examined in 38-year-old healthy men (n=52) with a wide range of fatness and fat distribution. Plasma hemostatic factors and metabolic parameters, including glucose tolerance, were measured, and body fatness and adipose tissue distribution were assessed by using computed tomography. The men with more visceral fat (ie, higher than the median value [n=26]) had a less favorable metabolic profile than the men with less visceral fat (n=26). They also had significantly (P<.05) higher plasma fibrinogen, factor VIII clotting activity, tissue-type plasminogen activator antigen, and plasminogen activator inhibitor-1 (PAI-1) activity (19.2+/-2.4 versus 8.5+/-1.6 AU/mL, P<.001) and lower basal tissue-type plasminogen activator activity. After adjustment for plasma insulin, the men with larger abdominal visceral fat area still had significantly higher plasma PAI-1 activity, but no difference was found in any of the other hemostatic factors. In multiple linear regression analysis, abdominal visceral fat area was a positive predictor of plasma PAI-1 activity, but it failed to show any significant association with other hemostatic factors after controlling for plasma insulin. These results suggest the presence of relationships between abdominal visceral fat and several plasma hemostatic factors that are largely mediated by concomitant alterations in plasma insulin concentration. In addition, our results suggest that abdominal accumulation of visceral fat is an independent predictor of plasma PAI-1 activity.