Serum fetuin-A levels inversely correlate with the severity of arterial calcification in patients with chronic lower extremity atherosclerosis without renal disease.

AIM: Fetuin-A is a hepatic glycoprotein that inhibits extraosseous calcification. Lower serum fetuin-A concentration was associated with severe arterial calcification in patients with end stage renal disease. We evaluated the association of serum fetuin-A levels and the severity of atherosclerosis in patients with peripheral vascular disease having normal renal function. METHODS: In this cross-sectional study among 93 chronic atherosclerotic patients with lower extremity vascular disease, systemic atherosclerosis and calcification was assessed by ultrasound (carotid intima-media thickness/IMT/, calcification at the abdominal aorta, carotid and femoral bifurcations, aortic and mitral valves) and angiography (Bollinger score). Standard serum markers of inflammation, diabetes, renal function, ankle-brachial indexes and traditional risk factors for atherosclerosis were noted and Fontaine classification was applied for the severity of symptoms. RESULTS: The patients mean (SD) age was 59.95 (7.61) years, 78% were men, 35% had diabetes. Serum fetuin-A level showed significant negative correlation with ultrasound calcification score (P=0.018, r=-0.257) and Bollinger angiographic score (P=0.035, r=-0.347). Fetuin-A did not correlate with IMT or Fontaine classification. Fetuin-A also showed significant correlation with albumin, transferrin and hemoglobin A1c (r=0.287, 0.305 and 0.219, respectively at P<0.05). Logistic regression analysis confirmed the association between fetuin-A and calcification score (OR: 3.03, CI: 1.05-8.7), P=0.039) independent of traditional risk factors. CONCLUSION: Our data show that serum fetuin-A levels inversely correlate with the severity of atherosclerosis in nonuremic patients with symptomatic chronic lower limb ischemia. These data support a putative protective role for fetuin-A in the development of arterial calcification.

Association between early onset and organ manifestations of systemic lupus erythematosus (SLE) and a down-regulating promoter polymorphism in the MBL2 gene.

The serum concentration of mannose-binding lectin (MBL) is genetically determined by a series of allelic polymorphisms in the MBL2 gene. Since several polymorphisms of the MBL2 gene have been suggested to be risk locus for systemic lupus erythematosus (SLE), we investigated MBL2 polymorphisms in 315 SLE patients from Hungary and 182 geographically matched healthy controls. Within the group of patients, we found that homozygotes for an MBL2 down-regulating promoter polymorphism at position -221 (YA to XA) (rs7096206) were significantly (p=0.017) younger at diagnosis than the other patients. The frequency of juvenile-onset SLE (

High prevalence of IgG and IgA antibodies to 19-kDa Helicobacter pylori-associated lipoprotein in chronic urticaria.

BACKGROUND: Chronic urticaria has been described in patients with Helicobacter pylori infection. We studied the titer of IgG and IgA type antibodies against H. pylori in patients with and without urticaria of unknown etiology. We also investigated the prevalence of antibodies against H. pylori-associated lipoprotein 20 (lpp20) in patients with and without chronic urticaria. METHODS: The concentration of anti-H. pylori antibodies (IgG and IgA) was determined by the RIDA test. The level of anti-lpp20 antibodies was determined by Western blot using various H. pylori antigens (from 19 to 120 kDa). RESULTS: Patients with chronic urticaria and H. pylori infection (subgroup 1, n = 33) had high IgG and IgA titers whereas all patients with chronic urticaria and without H. pylori infection (subgroup 2, n = 23) were seronegative (P = 0.0128 for IgG and P = 0.003 for IgA). Titers in subgroup 1 did not differ significantly from a control group (n = 33) with severe H. pylori-associated gastritis without urticaria. The prevalence of the anti-lpp20 antibodies was significantly higher in subgroup 1 compared to the control group (93.9 vs 21.2%, P < 0.0001 for IgG, and 46.1 vs 6.3%, P < 0.0029 for IgA). CONCLUSIONS: We suggest that IgG and IgA antibodies to H. pylori-associated lpp20 may play role in the pathogenesis of chronic urticaria.

High levels of antibodies against Clq are associated with disease activity and nephritis but not with other organ manifestations in SLE patients.

OBJECTIVE: Serum concentration of antibodies to C1q (C1qAb) has been reported to be elevated in a high percentage of patients with systemic lupus erythematosus (SLE). The associations of high C1qAb levels with different clinical manifestations and the activity of the disease, however, are not definitely understood. METHODS: We measured the levels of IgG type C1qAb in the sera of 137 patients with SLE using an ELISA method. RESULTS: Serum concentrations of C1qAb were found to be higher (p < 0.0001) in SLE patients than in healthy controls. High titer (> 66 AU/ml) C1qAb was found in 40/137 (29.2%) SLE patients, and 4/192 (2.1%) healthy controls (p < 0.0001). A strong negative correlation (R = -0.4, p < 0.0001) between the age of the patients and the C1qAb titers could be detected. C1qAb levels in clinically active SLE patients significantly (p < 0.0001) exceeded those measured in the sera of patients in the inactive stage of the disease. A significant positive correlation was detected between C1qAb levels and the laboratory activity markers (anti-DNA, low C3 level) of the disease. We found a significant negative correlation between levels of C1qAb and a negative acute phase protein, alpha2-HS-glycoprotein. Renal involvement was present in 11/40 (27.5%) and 11/97 (11%) of the patients with high and low titers of C1qAb, respectively (p = 0.038). The prevalence of other organ manifestations was, however, the same in the patients with or without high titer C1qAb. CONCLUSION: These findings indicate that C1qAb measurement is a useful method for detecting the activity of SLE and predicting renal manifestations, but not other organ involvement in the disease.

Cytokine regulation of the acute-phase protein levels in multiple myeloma.

BACKGROUND: Interleukin (IL) 6 has an important role in the regulation of acute-phase proteins (APPs) during an acute-phase response. We studied IL-6 and other cytokines to determine if they regulate serum APP levels in the same way under the condition of the aberrant, long-lasting 'acute-phase response' that occurs in patients with chronic inflammation and cancer. METHODS: Serum levels of nine positive APPs [CRP, SAA, C1-INH, Bf, C5, C8, C9, alpha 1-acidic glycoprotein (AGP) and haptoglobin] and two negative APPs [transferrin and alpha 2-HS glycoprotein (AHSG)] were measured using immunochemical methods in 59 multiple myeloma patients and in 72 healthy control subjects. Serum IL-6 and tumour necrosis factor (TNF) alpha levels were determined by bioassays. RESULTS: IL-6 was negatively correlated with five out of nine (C1-INH, C8, C9, AGP and haptoglobin) positive APPs but positively correlated with C-reactive protein (CRP). When patients with high and low IL-6 serum concentration were compared, CRP levels were higher, AGP and haptoglobin levels were lower in the high- than in the low-L-6 group, whereas no significant difference between the two groups was found in levels of the other positive and negative APPs. TNF-alpha levels were negatively correlated with transferrin and AHSG levels. No difference in the levels of positive APPs was observed between patients with low and high TNF-alpha serum concentration. By contrast, levels of both transferrin and AHSG were significantly lower in the high- than in the low-TNF-alpha group. CONCLUSIONS: These findings indicate that, except for regulation of the negative APPs by TNF-alpha, the mechanism of APP regulation is different under the conditions of the short-term and the chronic, long-lasting 'acute-phase reaction'.

Human recombinant alpha 2-HS glycoprotein is produced in insect cells as a full length inhibitor of the insulin receptor tyrosine kinase.

Human Alpha 2-HS glycoprotein (AHSG), a glycoprotein synthesized by hepatocytes, was expressed in insect cells using the recombinant baculovirus system. The protein was purified from the cell supernatant, and appeared as a single band at about 52 kDa. Western blot using a specific antibody to the B-chain of AHSG indicated that the connecting peptide was present in the protein. When incubated with solubilized insulin receptors, recombinant AHSG inhibited the tyrosine kinase activity of the receptors in a dose-dependent fashion at concentrations in the range of those of the circulating protein. AHSG did not interfere with the binding of insulin to its receptor. These results indicate that human AHSG represents a natural inhibitor of the insulin receptor tyrosine kinase, is active as a single-chain protein and possesses a biological role similar to that of its homologue in rats, pp63, described by Auberger et al. (1).

The acute phase reaction syndrome: the acute phase reactants (a review).

The acute phase reaction of the organism, which determines the response that follows the injury of tissues and organs and helps the survival of the individual is reviewed. The main participants, the induction of the reaction and the regulatory mechanisms are shortly discussed, with special respect to the prominent role of interleukin-6. The relationship between the production and synthesis of the acute phase reactants under physiological and pathophysiological conditions is analyzed. The particular functions of orosomucoid and alpha 2HS-glycoprotein are summarized. Finally, the common biological actions of IL-6 and the acute phase reactants are mentioned.

Isolation of human alpha 2HS-glycoprotein synthesized by Sf9 cells.

The human alpha 2HS-glycoprotein is a negative acute phase protein synthesized by hepatocytes. Because of its fragility and the difficulty of its purification, we used recombinant DNA techniques to produce the protein in order to investigate its biological effects. The cDNA coding for the whole alpha 2HS-glycoprotein from human liver library had been cloned into the baculovirus expression vector system using the pVL 1392 transfer vector and Sf9 cells. The recombinant protein was synthesized in the Sf9 cells and was isolated on a hydroxiapatite column from the culture medium. Western blot analysis indicated that the cells synthesized large quantities of the recombinant human protein. The molecular mass of the recombinant AHSG was the same as that of the protein in human plasma but was slightly lower than that of AHSG in the cell culture supernatants of HepG2 and higher than that of AHSG from Hep3B cell cultures, respectively.

[Role of the thymus and normal microbial flora on plasma fibronectin concentration in mice treated with immunomodulatory drugs]. / A thymus és a mikrobaflóra plazma fibronektin szintet befolyásoló szerepének vizsgálata immunmoduláns készítményekkel kezelt egerekben.

The plasma fibronectin (pFN) concentration (cc)-determined by electro-immunodiffusion method-of untreated genetically or artificially athymic mice, or treated with TP-4 (thymus hormone sequence analog synthetic preparation) showed no significant difference from their euthymic or untreated controls. In contrast, the pFN cc in mice with different microbiological state showed significant alterations; the highest level occurred in conventional mice. The lower level in germfree mice was increased by bacterial monocontamination. The alternation from SPF into conventional state in athymic mice or treatment of athymic and euthymic mice with Bordetella pertussis vaccine also resulted in the increase of the pFN cc. Based on these and earlier results, it was assumed that the pFN cc is independent from the presence or absence of the thymus, but it depends on the actual microbiological state of the macroorganism.

Electrophoretic and isoelectric focusing analysis of human recombinant alpha 2-HS glycoprotein produced in insect cells: analysis of the post-translational events.

Alpha 2-HS glycoprotein (AHSG) is a human serum glycoprotein synthesized by liver cells. It is a natural inhibitor of the insulin receptor tyrosine kinase activity. We produced this protein in insect cells by using a recombinant baculovirus expressing the whole coding sequence of the protein. By analyzing AHSG on isoelectric focusing and on sodium dodecyl sulfate (SDS) gels, followed by immunoblot, AHSG produced in insect cells was found to be phosphorylated and to possess the connecting peptide between the A and the B chains. The same features were found in the protein produced by Hep3B, a human liver cell line that synthesizes AHSG. By contrast, no phosphorylation could be detected in AHSG present in normal human plasma, and the connecting peptide was clipped. As the protein produced in insect cells is active on insulin receptors, in contrast to the plasma protein, our results suggest that the biological activity of the protein may be associated with its single chain form together with its phosphorylation.

Neurointermediate pituitary lobe cells synthesize and release interleukin-6 in vitro: effects of lipopolysaccharide and interleukin-1 beta.

The cytokine interleukin-6 (IL-6) is produced by a variety of cells, including macrophages, T-cells, and B-cells. Recent studies have confirmed a neuroendocrine role for IL-6 in the regulation of anterior pituitary (AP) hormone release. Because the neurointermediate pituitary lobe (NIL) may modulate AP hormone release, we investigated the production of IL-6 by NIL cells in vitro. NIL tissue removed from pituitary glands of male Long-Evans rats was enzymatically and mechanically dispersed, and the cells were subsequently cultured in 96-well tissue culture plates for 4-6 days in 10% serum-containing RPMI-1640. Test incubations were performed in serum-free RPMI-1640, and IL-6 concentrations were determined using the 7TD1 cell bioassay. Preliminary studies revealed a cell-dependent release of IL-6: increasing the number of NIL cells per well from 6.25 to 50 x 10(3) revealed detectable basal release of IL-6 between 25-50 x 10(3) cells/well. The endotoxin lipopolysaccharide (LPS; 100 ng/ml) and IL-1 beta (100 ng/ml) stimulated IL-6 release at 25 and 50 x 10(3) cells/well. Subsequent studies used a cell density of 50 x 10(3) cells/well and demonstrated time-dependent 3- to 6-fold inductions of IL-6 release by 100 ng/ml IL-1 beta and LPS. Concentration-response studies revealed maximal stimulation of IL-6 release by 1 ng/ml and a minimally effective concentration of 1 pg/ml for both IL-1 beta and LPS. Treatment of NIL cells with 1-10 mM (Bu)2cAMP increased IL-6 release by 7- to 14-fold. Endotoxin and IL-1 beta also enhanced the accumulation of IL-6 messenger RNA in these cells. Vasopressin and oxytocin (1 microM) inhibited LPS and IL-1 beta stimulation of IL-6 release from NIL cells, but did not inhibit IL-6 release from AP cells. Immunofluorescent dual labeling of NIL cells for flow cytometry revealed that greater than 95% of the cells did not stain for CD11b/c (common epitope found on monocytes, granulocytes, and macrophages) or CD45 (leukocyte common antigen). These results demonstrate for the first time the synthesis and release of IL-6 from cultured NIL cells. Agents that enhance IL-6 release [LPS, IL-1 beta, and (Bu)2cAMP] from other cell types also increase IL-6 release from NIL cells. Vasopressin and oxytocin inhibition of IL-6 release suggests a role for these neuropeptides in feedback inhibition in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of the thymus and the normal microflora on the plasma fibronectin concentration in mice.

The plasma fibronectin (pFN) concentration (cc) of untreated genetically or artificially athymic mice, or treated with TP-4 (thymus hormone sequence analog synthetic preparation) showed no significant difference from their euthymic or untreated controls. In contrast, the pFN cc in mice with different microbiological state showed significant alterations; the highest level occurred in conventional mice and the lower level in germfree mice was increased by bacterial monocontamination. The alternation from SPF into conventional state in nude mice also resulted in the increase of the pFN cc. Based on these and earlier results, it was assumed that the pFN cc is independent from the presence or absence of the thymus, but it depends on the actual microbiological state of the macroorganism.

[Diagnostic value of the determination of serum alpha2-HS-glycoprotein]. / A szérum alpha 2-HS-glycoprotein meghatározások diagnosztikus értékéröl.

Opsonic glycoprotein, alpha 2-HS-glycoprotein concentration was studied in the serum of 753 patients with various hematological, malignant, immunological, metabolic, endocrine and liver diseases and 68 healthy controls. Decreased serum alpha 2-HS-glycoprotein levels were detected in patients with acute leukemias, chronic granulocyte and myelomonocyte leukemias, lymphomas, myelofibrosis, multiple myeloma, metastatizing solid tumors, systemic lupus erythematosus, rheumatoid arthritis, acute alcoholic hepatitis, fatty liver, chronic active hepatitis, liver cirrhosis, acute and chronic pancreatitis, and Crohn's disease. Elevated levels were measured in patients with B and NANB/C hepatitis. Further decreased levels were observed in some groups with secondary infections. Serum alpha 2-HS-glycoprotein levels are affected by many factors, influencing the synthesis and elimination of the protein. The detection of serum alpha 2-HS-glycoprotein concentration has no specific diagnostic value as a marker for tumors or other diseases, however, its determination can be useful for the assessment of a non-specific regulator of the host defence.

[Effect of estrogen on the blast transformation of lymphocytes and interleukin-2 production in lupus erythematosus]. / Oestrogen hatása a lymphocyta blastos transformatióra és az interleukin-2 termelésre systemás lupus erythematosusban.

The mitogenic response of peripheral lymphocytes was investigated in 12 patients with systemic lupus erythematosus and in healthy female volunteers who were on 11 and without 9 contraceptive pills. The effect of estrogen (ethinyl-estradiol 10(-5)-10(-6)-10(-7)M) was studied on Phytohaemagglutinin and Pokeweed mitogen induced blastogenic transformation and interleukin-2 production of peripheral lymphocytes in vitro. We observed a significantly depressed Phytohaemagglutinin induced lymphoblastic transformation both in patients and women taking oral contraceptive in presence of 10(-5)M estrogen as compared to normal controls. However there was no significant alteration neither in the response of lymphocyte nor in the production of interleukin-2 using of Pokeweed mitogen. The stimulataneous inhibition of the interleukin-2 production proved to be moderate. Marked significant correlation (r greater than = 0.8) vas detected between lymphoblastic transformation and interleukin-2 production in healthy females. Correlation coefficient measured in females taking oral contraceptive (r less than = 0.64) and patients with systemic lupus erythematosus (r less than = 0.34) suggest that in these groups the inhibition of lymphoblastic transformation is due to the inhibition effect of estrogen on the interleukin-2 production.

PIP: 12 patients with systemic lupus erythematosus (SLE) (8 in the active stage) with an average age of 26 years (17-54) and 20 healthy control subjects (9 were aged 18-49 years and 11 were oral contraceptives [OC] users aged 17-44) were studied to assess the inhibiting effect of estrogen in vitro on phytohemagglutinin (PHA) and Pokeweed (PWM) mitogen induced blast transformation of lymphocytes (lymphoblastic transformation=LBT) gained from periphral blood and simultaneous interleukin-2 (IL-2) production. 8 women were taking Anteovin, 2 Ovidon, and 1 Rigevidon. The average duration of OC use was 5.2 years. 1 SLE patient did not need immunosuppressive treatment, 3 patients received corticosteroid maintenance therapy, and 4 patients were also taking 50 mg of Imuran. In 4 active SLE patients the tests were done before raising the dose of immunosuppressive drugs, and in the case of 2 other patients the administration of 75 mg and 25 mg/die Prednisolone was necessary in addition to 50 mg and 100 mg/die Imuran. LBT decreased significantly in patients and OC users. The LBT values induced by PWM were similar but not significant. The IL-2 production induced by PHA decreased in all 3 groups but not significantly. I1-2 production was 6 E/ml in patients, 5 E/ml in OC users, and 11.5 E/ml in nonusers, but the differences did not prove significant because of wide individual fluctuations. The amount of IL-2 produced by lymphocytes at PWM stimulation was almost the same in all 3 groups with or without estrogen. There was a positive, significant relationship between the extent of LBT and the amount of IL-2 produced in the healthy group of nonusers, it was less solid in the OC users, and in the SLE group trhe low correlation coefficient of .34 suggested the reduction of IL-2 through the inhibition of LBT.

Serum alpha 2-HS glycoprotein concentration in patients with hematological malignancies. A follow-up study.

We observed significantly reduced serum alpha 2-HS glycoprotein concentrations in patients with acute lymphocytic, acute nonlymphocytic, chronic granulocytic and chronic myelomonocytic leukemias, Hodgkin's and non-Hodgkin's lymphomas, myelofibrosis, and multiple myeloma, but not in patients with chronic lymphocytic leukemia and polycythemia vera, as compared with healthy controls. We followed the serum level of the protein for 18 months. Patients with infectious complications, those receiving cytostatic treatment, and those in the preterminal period had further reduced serum alpha 2-HS glycoprotein levels. The reduction of serum alpha 2-HS glycoprotein concentration was primarily due to decreased production caused by infiltration of the liver, a hepatotoxic effect of cytostatic treatment, and, to a lesser degree, to increased consumption. We found statistically significant negative correlations between serum alpha 2-HS glycoprotein concentration and erythrocyte sedimentation rate, serum aspartate aminotransferase and alkaline phosphatase activities, and IgG and IgM concentrations. The determination of the alpha 2-HS glycoprotein concentration is useful for the assessment and follow-up of the clinical status and therapy of patients with hematological malignancies and also has prognostic significance.

[The role of the central nervous system in lupus erythematosus]. / A központi idegrendszer részvétele systemás lupus erythematosusban.

Central nervous system involvement of systemic lupus erythematosus was observed in 34 (36%) of the 94 patients studied between 1970-1990. A review of the diagnostic methods and therapy for central nervous system lupus is presented. The diagnosis of primary cerebral lupus was based on the history, physical examination and on the results of the cerebrospinal fluid analysis, CT-scan and EEG. Intractable headache (22/34), behavioural abnormalities (18/34), cranial neuropathy (16/34) occurred most frequently among neuropsychiatric symptoms. Immunoglobulin analysis of the cerebrospinal fluid proved to be the most sensitive method for detecting clinical activity (in 20/23). Central nervous system involvement was suggested by conventional serological test to a lesser degree. Alterations on CT scan and EEG were found in 17/27 and 14/26 of cases, respectively. IgM, IgA, and IgG indexes (indicators of intratechal immunglobulin synthesis) decreased when the central nervous system events subsided after successful treatment but the CT abnormalities (e. g. atrophy) were not altered.

The effect of the alpha 2-HS-glycoprotein on the mitogen-induced lymphoblastic transformation and IL-2 production.

Authors investigated the in vitro effect of alpha 2-HS-glycoprotein on the lymphoblastic transformation and interleukin-2 production. The observations were made on the peripheral blood lymphocytes of healthy persons, on those of women taking oral contraceptives and on those of patients with systemic lupus erythematosus. The alpha 2-HS-glycoprotein inhibited the mitogen responses both in physiologic and in the halved concentrations. The inhibition of the interleukin-2 production seemed less remarkable.

PIP: Whether blast transformation of human lymphocytes from healthy volunteers, women taking oral contraceptives, and women with systemic lupus erythematosus (SLE) would be affected by alpha2-HS-glycoprotein (A2HSGP) was explored by incubating lymphocytes stimulated by phytohemagglutinin or pokeweed mitogen with or without the glycoprotein. A2HSGP did indeed inhibit blast transformation in these cell populations. Release of the peptide interleukin-2 into the culture medium was also inhibited to a lesser extent, slightly more so in cells from healthy persons, and slightly less so in women with SLE. The mechanism and biological significance of this observation is unexplained.

Correlations between serum alpha 2-HS-glycoprotein concentration and conventional laboratory parameters in systemic lupus erythematosus.

Serum alpha 2HS-glycoprotein (A2HSG) concentrations of 63 patients with systemic lupus erythematosus (SLE) were determined, and found to be significantly low compared to those of 59 healthy blood donors. The diminution of serum A2HSG concentration was proportional to the degree of activity of SLE, and was not influenced by secondary infections. There was a statistically significant positive correlation between serum A2HSG and the C3 complement component levels. A negative correlation between serum A2HSG and IgG, IgA concentration and anti-DNA activity was observed. Serum A2HSG was significantly low in cases of positive for the following laboratory parameters: anti-nuclear antibodies, circulating immune complexes and LE cell phenomenon. We found no correlation between serum IgM concentration, cryoglobulins, latex agglutination and serum A2HSG levels. The unusually good negative correlation between A2HSG pathogenetical role of this glycoprotein in SLE. The determination of A2HSG concentration may be of clinical importance in SLE.

Effect of mannozym treatment on plasma fibronectin concentration in germfree and conventional mice.

As a result of a single intraperitoneal treatment with 1 mg/ml of Mannozym (M) the plasma fibronectin (FN) level was significantly increased both in germfree (Gf) and conventional (Cv) mice, and though it started from a lower value in Gf mice, it rose to a similar level to that of Cv animals. The maximum of FN level was observed on the first day after treatment both in Gf and Cv mice. It had returned to normal by the 7th day in Cv mice, but it was higher in Gf mice as compared to untreated controls even on the 14th day. Thus the increase of FN level was of higher degree and longer duration following M treatment in Gf mice than in Cv animals. In treated Gf mice the plasma FN concentration was in the same range as in untreated Cv mice even at the termination of experiment. Both in Gf and Cv mice, there was a relative spleen weight increase, the degree of which was similar, but the duration was longer in Gf mice than in Cv ones.