Autoantibodies to box A of high mobility group box 1 in systemic lupus erythematosus.

Autoantibodies to nuclear structures are a hallmark of systemic lupus erythematosus (SLE), including autoantibodies to nuclear protein high mobility group box 1 (HMGB1). HMGB1 consists of three separate domains: box A, box B and an acidic tail. Recombinant box A acts as a competitive antagonist for HMGB1 and might be an interesting treatment option in SLE. However, antibodies to box A might interfere. Therefore, levels of anti-box A were examined in SLE patients in association with disease activity and clinical parameters. Serum anti-box A was measured in 86 SLE patients and 44 age- and sex-matched healthy controls (HC). Serum samples of 28 patients with primary Sjögren's syndrome and 32 patients with rheumatoid arthritis were included as disease controls. Anti-HMGB1 and anti-box B levels were also measured by enzyme-linked immunosorbent assay during quiescent disease [SLE Disease Activity Index (SLEDAI) ≤ 4, n = 47] and active disease (SLEDAI ≥ 5, n = 39). Anti-box A levels in active SLE patients were higher compared to quiescent patients, and were increased significantly compared to HC and disease controls. Anti-box A levels correlated positively with SLEDAI and anti-dsDNA levels and negatively with complement C3 levels. Increased levels of anti-box A antibodies were present in the majority of patients with nephritic (73%) and non-nephritic exacerbations (71%). Antibodies to the box A domain of HMGB1 might be an interesting new biomarker, as these had a high specificity for SLE and were associated with disease activity. Longitudinal studies should be performed to evaluate whether these antibodies perform better in predicting an exacerbation, especially non-nephritic exacerbations.

Lungurra Ngoora--a pilot model of care for aged and disabled in a remote Aboriginal community--can it work?

INTRODUCTION: The literature on the health of and services for older Aboriginal and Torres Strait Islander populations is relatively sparse. This study explored the development and implementation of a locally designed community service model of care for older people, and people with disability and/or mental health problems in remote Aboriginal Australia. METHODS: Based on extensive community consultation with older people, families, carers, community members and stakeholders, a model of care was developed to address unmet needs for the target population and their carers in the remote community of Looma, in the Kimberley region of Australia. The model was implemented and evaluated over 12 months. The main outcome measures included the number of services (including home services, meals, transport, respite, personal care and advocacy) provided. Outcomes of community participation, capacity building, resources, partnerships, workforce, service delivery and cultural protection were assessed qualitatively by an external evaluator. RESULTS: The number of people receiving community care services in Looma increased from eight to 22, and services increased in all domains from 140 total services delivered for 1 month at baseline to 2356 by the final month of the program. CONCLUSIONS: The Lungurra Ngoora community care service model pilot project demonstrated a successful collaborative service model that addressed the care needs of older persons, those with disability and mental illness, and their carers in this remote community. The developmental approach, and model structure, could serve as a template for future delivery of services in remote Aboriginal communities.

Effects of pioglitazone versus simvastatin on biomarkers of inflammation in patients on high cardiovascular risk.

High levels of fetuin-A has been linked to cardiovascular disease, possibly via modulating low-grade systemic inflammation. We performed a subanalysis from the PIOSTAT study to investigate a possible link between fetuin-A and the inflammatory biomarker hs-CRP. 66 nondiabetic individuals at cardiovascular risk were randomized to either pioglitazone, simvastatin, or the combination of both, and followed for 12 weeks. At study endpoint, correlations between serum fetuin-A, hs-CRP, blood lipids, PAI-1, MMP-9, HOMA-IR, and liver transaminases were investigated by Spearman rank correlation. Changes in fetuin-A concentration did not correlate to changes in hs-CRP (r=0.19, p=0.16). A positive correlation was found for change of HOMA-IR value (r=0.33, p=0.01) and for the AST/ALT ratio (p<0.05). Our data suggest that the previously observed correlation between elevated circulating fetuin-A and hs-CRP in epidemiological studies may not reflect a causal relationship in nondiabetic patients on high cardiovascular risk.

Effect of acarbose on postmeal mononuclear blood cell response in patients with early type 2 diabetes: the AI(I)DA study.

So far little is known about how the antidiabetic drugs acting at the level of gastrointestinal mucosa may affect immune and cellular response to food intake. The following study investigated the association between acarbose treatment and postprandial metabolism, immune- and inflammatory activity in patients with early type 2 diabetes: The Acarbose action on low grade Inflammation and Immune response in type 2 Diabetes on Atherosclerosis risk (AIIDA) study. Middle-aged patients (n=87) with early type 2 diabetes (2 h-plasma-glucose >or=11.1 mmol/l and/or HbA1c >or=6.5%) and sub-clinical inflammation (leucocytes >or=6.2 GPt/l and/or hsCRP >or=1.0 mg/l) underwent a mixed meal load (527 kcal). Metabolic parameters and markers of subclinical inflammation were measured at fasting (0'), 2 h-postprandial (2-hpp) and 4-hpp before and after 20 weeks of treatment with acarbose or placebo. Leukocytes and lymphocytes excursion after 20 weeks of treatment was significantly reduced with acarbose 4 h after testmeal [GPt/l] (7.5 vs. 7; p<0.05; and 2.29 vs. 2.14; p<0.05, respectively). Acarbose had only marginal effects on pp glucose, FFA, triglycerides, and insulin excursion. Biomarkers of inflammation (hsCRP, MBL, and PAI1) were not affected by acarbose. Multivariate analysis reveals only baseline leukocytes and of acarbose as independent determinant of 4-h leucocytes excursion. Postprandial metabolic and inflammatory parameters were strongly interrelated. These results suggest pleiotropic effects of acarbose, which may contribute to its vasoprotective potentials.

Effects of the alpha glucosidase inhibitor acarbose on endothelial function after a mixed meal in newly diagnosed type 2 diabetes.

Endothelial dysfunction (ED) has been suggested as a possible causal link between postprandial hyperglycemia and cardiovascular events in patients with type 2 diabetes. Recent trials demonstrated a reduction of cardiovascular events by treatment with alpha-glucosidase inhibitor acarbose - a drug which mainly reduces postprandial glucose excursions. We were interested to know whether patients with newly diagnosed type 2 diabetes showed postprandial ED and if so whether acarbose was able to improve this condition. Forearm blood flow (FBF) measurements for assessment of ED were performed in the fasting and postprandial state in 20 newly diagnosed type 2 diabetic patients and 10 healthy control subjects. After baseline examination, patients were randomly assigned to a 20-week treatment of acarbose 100 mg t.i.d or matching placebo, thereafter FBF measurements were repeated. FBF of patients in the fasting state was significantly impaired compared to healthy control subjects (max. FBF 5.3+/-0.7 vs. 8.0+/-0.9 ml/100 ml, p<0.02) and did not change in the postprandial state (max. FBF 5.6+/-0.7 ml/100 ml). In contrast, healthy controls showed a significant improvement of FBF in the postprandial state (11.5+/-1.2 ml/100 ml), which is compatible with postprandial ED in the group of patients. Twenty weeks of acarbose treatment did not affect either fasting or postprandial FBF in patients. Early type 2 diabetes is a state of both fasting and postprandial ED, which is not sensitive to acarbose treatment. Protective cardiovascular effects of acarbose might involve other mechanisms.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Sulfonato-imino copper(ii) complexes: fast and general Chan-Evans-Lam coupling of amines and anilines.

Sulfonato-imine copper complexes with either chloride or triflate counteranions were prepared in a one-step reaction followed by anion-exchange. They are highly active in Chan-Evans-Lam couplings under mild conditions with a variety of amines or anilines, in particular with sterically hindered substrates. No optimization of reaction conditions other than time and/or temperature is required.

IRF-1 induced cell growth inhibition and interferon induction requires the activity of the protein kinase PKR.

Expression of the tumor suppressor IRF-1 results in the inhibition of cell growth and transcriptional activation of the IFN-beta gene. IFN production is not responsible for the IRF-1 mediated cell growth inhibition. It is shown here that activation of the IRF-1 causes induction of PKR expression. PKR is a serine/threonine kinase with tumor suppressor activity. IRF-1 mediated cell growth inhibition and IFN induction correlates with PKR expression. A catalytically inactive dominant negative PKR mutant abolishes both activities of IRF-1. These data demonstrate that the tumor suppressor activity of IRF-1 is mediated, at least in part, by PKR.

Immunohistological changes in dilated cardiomyopathy induced by immunoadsorption therapy and subsequent immunoglobulin substitution.

BACKGROUND: Immunoadsorption (IA) and subsequent immunoglobulin (Ig) G substitution represent an additional therapeutic approach in dilated cardiomyopathy (DCM). It remains to be elucidated whether this treatment modulates myocardial inflammation, which is possibly a causal factor of ventricular dysfunction. METHODS AND RESULTS: From 25 DCM patients (EF <30%), 12 patients were randomized for IA therapy and subsequent IgG substitution at 1-month intervals until month 3. Before (<7 days) and after IA therapy, right ventricular biopsies were obtained from all patients. Biopsies were also obtained at intervals of 3 months from 13 patients without IA/IgG treatment (controls). IA/IgG treatment induced improvement in left ventricular ejection fraction from 21.3+/-1.7% (+/-SEM) to 27.0+/-1.3% (P<0.01 versus baseline/controls) and reduction of the beta-receptor autoantibody serum levels (P<0.01 versus baseline/controls). The number of CD3 cells decreased from 5.7+/-0.8 to 2.9+/-0.5 cells/mm(2) (P<0.01 versus baseline/controls). This decline was paralleled by a decrease in CD4 (P<0.01 versus baseline/controls) and CD8 (P<0.05 versus baseline/controls) lymphocytes. The number of leukocyte common antigen-positive cells (leukocytes) was reduced from 20.0+/-3.2 to 9.9+/-2.8 cells/mm(2) (P<0.01 versus baseline/P<0.05 versus controls). HLA class II expression decreased from 2.1+/-0.7% to 1.1+/-0.4% (P<0.05 versus controls/baseline). The number of immunopositive cells and the expression of HLA class II in controls remained stable. In both groups, the degree of fibrosis remained unchanged. CONCLUSIONS: IA and subsequent IgG substitution mitigate myocardial inflammation in DCM.

Increased intimal-medial thickness in newly detected type 2 diabetes: risk factors.

OBJECTIVE: To examine carotid intimal-medial thickness (IMT) and its determinants in newly detected type 2 diabetic subjects, classified according to the new criteria of the American Diabetes Association, in comparison with age- and sex-matched control subjects with normal glucose tolerance. RESEARCH DESIGN AND METHODS: This study was case-controlled, with matched pairs for 71 newly diagnosed type 2 diabetic individuals. Subjects aged 40-70 years were recruited from a risk population for diabetes seen in the Risk Factors in IGT for Atherosclerosis and Diabetes (RIAD) Study. Standard risk factors, 75-g oral glucose tolerance test with real insulin, proinsulin, and C-peptide, and ultrasound measurement of the IMT of the common carotid artery were performed. RESULTS: The diabetic subjects, both men and women, displayed carotid intimal-medial thickening, even in the subgroup with fasting plasma glucose between 7.0 and 7.8 mmol/l. HbA1c was significantly increased in the diabetic patients (6.33 vs. 5.48%). Insulin, proinsulin, and C-peptide were also significantly higher. Among the coronary risk factors, triglycerides and plasminogen activator inhibitor were significantly increased. After age and sex adjustment. IMT in the diabetic group was correlated to triglycerides and the total-to-HDL cholesterol ratio. In the total group, IMT was significantly correlated to blood pressure, 2-h glucose in oral glucose tolerance testing, triglycerides, albuminuria, and the total-to-HDL cholesterol ratio, and inversely correlated to HDL cholesterol. No independent determinant of IMT was found in the diabetic group by multivariate analysis. CONCLUSIONS: Newly detected type 2 diabetic patients exhibit a higher degree of early atherosclerosis than normal glucose-tolerant subjects matched for age and sex. Our data suggest that hyperglycemia, together with a clustering of risk factors, and in particular dyslipidemia, may cause intimal-medial thickening in the early phases of diabetes.

A new hybrid promoter directs transcription at identical start points in mammalian cells and in vitro.

A hybrid promoter which generates large amounts of mRNAs with transcription start points (tsp) differing in one nt, both in mammalian cells and in vitro, was constructed by integrating the promoter of bacteriophage T7 in between the TATA box and the tsp of the retroviral myeloproliferative sarcoma virus (MPSV) long terminal repeat (LTR). This promoter was designed for sequence and secondary structure studies of 5' untranslated regions (UTR) with respect to mRNA stability and translatability.

A multifunctional vector family for gene expression in mammalian cells.

A family of different promoters and vector backbones for multiple use in mammalian cells in vivo and in vitro experiments has been created. The elements allow high-level constitutive, as well as adjustable expression with mono- and dicistronic transcription units and several in vitro manipulations of the inserted ORFs. The vector components are designed to be combined in a simple cloning step.

Constitutive internalization and association with adaptor protein-2 of the interleukin-6 signal transducer gp130.

The transmembrane protein gp130 is the common signalling receptor subunit for the interleukin-6 (IL-6)-type cytokines. It has recently been shown that the cytoplasmic domain of gp130 contains a dileucine internalization motif and that endocytosis of gp130 occurs signal-independent. Here, we have studied whether gp130 itself undergoes constitutive internalization or whether its endocytosis is stimulated by formation of the IL-6/IL-6R/gp130 complex. Using two different assays, we found that gp130 is internalized independent from IL-6/IL-6R stimulation. In addition, we show that gp130 is constitutively associated with the cell surface adaptor complex AP-2. Our findings strongly suggest endocytosis of gp130 to be constitutive.

Hepatocyte growth factor/scatter factor (HGF/SF) signals via the STAT3/APRF transcription factor in human hepatoma cells and hepatocytes.

Acute phase protein expression is regulated by a variety of cytokines such as IL-1, IL-6, IL-11, tumour necrosis factor alpha, interferon-gamma, oncostatin-M, leukemia inhibitory factor, ciliary neurotrophic factor and cardiotrophin-1. Presently, IL-6 is regarded as the most potent mediator of acute phase protein (APP) synthesis. It was shown that IL-6 and IL-6-type cytokines activate the so-called JAK/STAT pathway and finally regulate APP expression in liver cells. Since HGF/SF is also capable of regulating APP expression, we asked whether it might also signal via the JAK/STAT pathway. Here we show that incubation of human hepatocytes as well as hepatoma cells (HepG2) with HGF/SF results in activation of the transcription factor STAT3. This STAT3 activation after HGF/SF did not occur before 5-7 h and was maintained up to 28 h. These observations are in contrast to the rapid and transient activation of STAT1 and STAT3 mediated by IL-6.

Differential inhibition of IL-6-type cytokine-induced STAT activation by PMA.

Prior activation of mitogen-activated protein kinases by phorbol 13-myristate 12-acetate (PMA) results in an inhibition of interleukin (IL)-6-induced activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway which is most likely mediated by the induction of suppressor of cytokine signaling-3 and requires the specific SHP2 binding site Y759 of the IL-6 signal transducer gp130. In this study, we demonstrate that PMA inhibits STAT activation by IL-6 and the related cytokine leukemia inhibitory factor (LIF) but not by oncostatin M (OSM). Since the LIF receptor also contains an SHP2 recruitment site whereas the OSM receptor lacks such a module, we propose that two SHP2 binding modules within a homo- or heterodimeric receptor are necessary to mediate the PMA inhibitory effect.

LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages.

Recent findings indicate that cytokine signaling can be modulated by other mediators of simultaneously activated signal transduction pathways. In this study we show that LPS and TNFalpha are potent inhibitors of IL-6-mediated STAT3 activation in human monocyte derived macrophages, rat liver macrophages and RAW 264.7 mouse macrophages but not in human hepatoma cells (HepG2) or in rat hepatocytes. Accordingly, LPS and TNFalpha were found to induce the expression of SOCS3 mRNA in each of the investigated type of macrophages but not in HepG2 cells. Using a specific inhibitor, evidence is presented that the p38 MAP kinase might be involved, especially for the inhibitory effect of TNFalpha.

In vivo formation of IRF-1 homodimers.

Interferon regulatory factor 1 (IRF-1) is a transcriptional activator which exerts different biological activities. IRF-1 activates interferon induced genes as well as genes which are not directly linked to the interferon system, such as the ICE protease gene. IRF-1 activity is post-transcriptionally regulated in addition to transcriptional regulation by interferons, cytokines, hormones and many other factors. This includes heterodimerisation with activators and repressors of transcription. These protein interactions modulate the transactivating capacity of IRF-1. By using a two-hybrid system, we demonstrate that IRF-1 forms homodimers in vivo. The homodimerization domain was determined to be located in the N-terminal part of IRF-1 which belongs to the DNA-binding domain. Since this sequence is highly conserved between members of the IRF-family, our observation raises the question of homodimerization of other IRFs through this domain.

Diminished susceptibility to in vitro oxidation of low-density lipoproteins in hypercholesterolemia: key role of alpha-tocopherol content.

Oxidizability of low-density lipoproteins (LDL) in hypercholesterolemia is of clinical relevance, but previous studies revealed diverging results. Therefore, we studied ex vivo oxidation of LDL in plasma samples of 57 hypercholesterolemic and 20 normocholesterolemic volunteers. LDL were isolated by ultracentrifugation and analyzed for lipids and alpha-tocopherol. The formation of conjugated dienes, lipoperoxides and malondialdehyde was measured at 0.1 micromol/l LDL, 3.2 micromol/l CuSO4. We found prolongation of the lagtime (53.6/65.8/71.4 min) with tertiles (< or = 3.17/3.89/14.2 mmol/l) of LDL-cholesterol (LDL-C). Regression analysis revealed that the lagtime increased with the apparent concentrations of cholesterol (P = 0.003) and alpha-tocopherol (P = 0.001) in the oxidation assay. A multiple regression model with the apparent concentrations of alpha-tocopherol, triglycerides and cholesterol explained 40% of the variation in lagtime. The close relationship between plasma concentrations of LDL-C and LDL-alpha-tocopherol (P = 0.002) indicated that LDL contained more of this antioxidant in hypercholesterolemia. This might provide an explanation for the positive relationship between lagtime and LDL-C. The latter was independent of whether LDL-C or LDL-protein was chosen for standardization of the oxidation assay. The formation of conjugated dienes (P = 0.000), lipoperoxides (P = 0.038) and malondialdehyde (P = 0.001) increased with the cholesterol level in the assay. This may be due to the increased load of LDL with cholesterol esters as a substrate for oxidation in hypercholesterolemia. Our data do not support the opinion that hypercholesterolemia is characterized by increased susceptibility of LDL to oxidation.

Postprandial plasma glucose is an independent risk factor for increased carotid intima-media thickness in non-diabetic individuals.

Postprandial (pp) hyperglycemia--frequently associated with an increase in cardiovascular risk factors--may be damaging for the endothelium. So far, little information exists how glucose, insulin and lipids may affect atherosclerosis in the pp state. Therefore, we evaluated the relationship of pp hyperglycemia, insulin secretion and coronary risk factors to intima-media thickness (IMT) in a non-diabetic risk population. In 403 subjects (147 males, 256 females), aged 40-70 years, in the majority relatives of index cases with type 2 diabetes--a 75 g oral glucose tolerance test was performed together with measurement of insulin fractions, various risk factors and IMT of the common carotid artery. We found a continuous rise of 2h pp insulin fractions along the quintiles of 2h pp plasma glucose. A significant increase of body mass index, waist to hip ratio, triglycerides and decrease of HDL-cholesterol was observed in the top quintile of 2h pp plasma glucose (8.24 > or = pp plasma glucose < 11.1 mmol/l). Albuminuria was significantly enhanced in the 5th quintile. In parallel, IMT was significantly increased in the 5th quintile versus the bottom quintile of 2 h and maximal glucose (range 11.7-15.3 mmol/l) postprandially. After age and sex adjustment pp glucose and C-peptide, total cholesterol, triglycerides and HDL-cholesterol but not fasting plasma glucose were significantly correlated to IMT. In multivariate analysis age, male sex, pp plasma glucose, total and HDL-cholesterol were found to be independent risk factors for increased IMT. In conclusion, our data in a non-diabetic European risk population show that the two top quintiles of pp plasma glucose are associated with a clustering of standard risk factors. Corresponding to this clustering of risk factors IMT was significantly increased in the top quintile of 2 h and maximal pp plasma glucose. These data show that pp hyperglycemia may exert a noxious impact on the arterial wall together with a cluster of anomalies typical for the metabolic syndrome.

A new peptide-affinity tag for the detection and affinity purification of recombinant proteins with a monoclonal antibody.

A monoclonal anti-peptide antibody (2E11) was raised against the synthetic peptide 38 (C-L-D-K-S-G-L-P-S-D-R-F-F-A) representing a part of the variable region of the Vbeta 6.2 T-cell receptor. This mAb (IgG(1), kappa light chain) bound very specifically to peptide 38 as shown by ELISA but did not recognize the corresponding native Vbeta 6.2 T-cell receptor on T-cells. For epitope analysis, overlapping peptides of 4-10 amino acids in length corresponding to the sequence of peptide 38 were synthesized and assayed by SPOT synthesis on cellulose sheets. The shortest peptide recognized was L-P-S-D-R. The specificity of mAb 2E11 was examined with 100 different peptides comprising other parts of the different variable Vbeta domains of the human T-cell receptor that do not include the epitope region L-P-S-D-R. None of these peptides were recognized. The chemical synthesis of a peptide with the sequence L-P-S-D-R on Sepharose beads allowed to efficiently purify the mAb 2E11 in a single step by affinity chromatography. An equilibrium binding constant of 4.9x10(6) l/mol was determined for mAb 2E11 by using rhodamine-green-labelled peptide 38 in fluorescence correlation spectroscopy. In order to demonstrate that peptide 38 can be used as an affinity-tag, it was fused to the carboxyl-terminus of interferon regulatory factor-1 (IRF-1). It could be shown that in vitro translated peptide 38 tagged IRF-1 was immunoprecipitated by the mAb 2E11 and that the fusion protein could be purified by immunoaffinity chromatography. Additionally peptide 38 was fused to the amino-terminus of the Taq polymerase. This recombinant protein was expressed in E. coli and specifically detected in a Dot blot and Western blot using mAb 2E11.