Anti-glycolipid antibodies in patients with neuropathy: a diagnostic assessment.

Anti-glycolipid antibodies are associated with immune-mediated neuropathies and screening is often performed as part of the diagnostic assessment for patients presenting with peripheral neuropathy. We report our experience in testing for immunoglobulin (Ig) G and IgM anti-glycolipid (GM1, GM2, GM3, GM4, GD1a, GD1b, GD2, GD3, GT1a, GT1b, GQ1b, sulfatides) antibodies in 290 consecutive patients presenting with neuropathy. Anti-glycolipid antibodies were detected significantly more often (43%) in patients who were diagnosed with definite immune-mediated neuropathy than in patients without a final diagnosis of immune-mediated neuropathy (control group) (23%). With positive and negative predictive values of 22% and 90%, respectively, anti-glycolipid antibodies are not a very reliable diagnostic tool in early patient contact. Certain antibodies (IgM to GM2, GT1a and IgG to GM3, GD3 and GT1b) were equally or more prevalent in the control group; clinicians should be aware of this distribution when receiving positive screening results for these antibodies. Concomitant IgG and IgM reactivities were found for GM1, GM2, GD1b and sulfatides, and were detected more frequently in patients with definite immune-mediated neuropathies.

Gamma heavy chain disease in a patient with rheumatoid arthritis--a laboratory evaluation.

INTRODUCTION: Heavy chain diseases (HCD) are neoplastic proliferations of B cells which secrete truncated immunoglobulin heavy chains without associated light chains. Being rare and probably underdiagnosed diseases the aim of this report is to show an additional case of gamma heavy chain disease in a 48 year old female patient with rheumatoid arthritis focusing on the laboratory presentation. MATERIALS AND METHODS: Laboratory work-up included agarose gel electrophoresis (AGE), capillary zone electrophoresis (CZE), immunofixation and nephelometrically determined immunoglobulin and immunoglobulin subclasses of the patient's serum. Urine samples were also subjected to immunofixation and to a SDS-PAGE with consecutive immunoblot. RESULTS: Nephelometrically measured elevated IgG concentrations were noted in combination with a decreased gamma globulin region and an increased beta globulin region on AGE. A definite monoclonal spike was not identified on AGE but at least suspected on CZE; finally serum and urine immunofixation demonstrated a monoclonal gamma heavy chain devoid of any corresponding light chains confirming the diagnosis of HCD. Analysis of the gamma heavy chain (HC) with means of SDS-PAGE revealed proteins of 40 kD and 80 kD most likely presenting a truncated HC in its monomeric and dimeric form and possibly leading to the failure of IgG-subclass typing with the applied IgG subclass antisera. CONCLUSION: This case report illustrates a new case of gamma HCD demonstrating variable laboratory manifestations and therefore the need for heightened awareness concerning this disease when confronted with abnormal and discrepant protein profiles in routinely applied laboratory tests.

Ma2 antibodies: an evaluation of commercially available detection methods.

BACKGROUND: Ma2 antibodies belong to the onconeuronal antibodies which define a "definite" paraneoplastic neurological syndrome (PNS). Because of the clinical relevance, use of two separate methods (indirect immunofluorescence technique--IFT--and immunoblot) is advocated; however, with an increasing number of commercially available assay systems, usually only one assay is performed. METHODS: We compared IFT and three commercially available immunoblots (ravo Diagnostika, Euroimmun, Milenia Biotec) on sera from 35 patients with clinically suspected PNS. 17 were Ma2 antibody associated as defined by consensus result (showing positive reactivity in 2 assays), 18 were Ma2 antibody negative controls. RESULTS: Sensitivity/specificity for single assays were for IFT 94%/94%, for ravo Diagnostika PNS blot 88%/100%, for Euroimmun Neuronal Antigens Profile blot 100%/89%, and for Milenia Biotec MTR blot 94%/100%. CONCLUSIONS: Our data confirm, although all tests performed well, a combination of 2 independent assays is still advisable for Ma2 antibody detection in order to achieve higher sensitivity and specificity rates.

TAK1 mediates the collagen-II-dependent induction of the COX-2 gene and PGE2 release in primary human chondrocytes.

We investigated the role of transforming growth factor-beta activated kinase 1 (TAK1) in collagen II signaling in primary human chondrocytes (PHCs). We asked whether TAK1 acts as a modulator of collagen II signaling with respect to collagen-II-dependent induction of cyclooxigenase-2 (COX-2) in PHCs and release of PGE2 from PHCs. Therefore, PHCs were incubated with collagen II, and cells were then analyzed by RT-PCR for the expression of COX-2. ELISA was used to quantify PGE2 release. To examine the influence of TAK1 on these events, TAK1 gene silencing was performed by RNAi in PHCs prior to collagen II treatment. Results indicated that COX-2 gene expression and PGE2 release are specific outcomes of collagen II signaling and that both depend on TAK1 mediation. These findings are promising in that therapeutic inhibition of TAK1 might be used to reduce pain and relieve inflammatory symptoms that are common in osteoarthritis.

The relationship and prognostic impact of low-T3 syndrome and NT-pro-BNP in cardiovascular patients.

OBJECTIVES: Low-T3 syndrome is highly prevalent and independently prognostic in cardiovascular patients. The relationship and prognostic impact with the cardiac marker NT-pro-BNP have not been thoroughly investigated. METHODS: Thyroid hormone levels and NT-pro-BNP were assessed in 615 consecutive patients hospitalized for cardiovascular disease. Patients with primary overt or latent thyroid disorder, hormone replacement, thyreostatic and amiodarone therapy were excluded. The association with and predictive impact on mortality were examined. RESULTS: 36 (7.1%) patients had low-T3 syndrome. After adjustment for known confounders, NT-pro-BNP was significantly associated with fT3 and low-T3 syndrome. fT3 (HR 0.58, 95%CI 0.34-0.98) and low-T3 syndrome (HR 3.0, 95%CI 1.4-6.3) were predictive for mortality after adjustment for NT-pro-BNP levels and other cardiovascular prognostic variables. In patients with fT3 levels within the normal range, fT3 and NT-pro-BNP stratified by median values showed complementary prognostic information with the highest risk for mortality in patients with low normal fT3 and high NT-pro-BNP (HR 10.5, 95%CI 3.2-34.6). CONCLUSIONS: fT3 and low-T3 syndrome are significantly related to NT-pro-BNP in patients with cardiovascular disease, but are predictors of mortality independently of NT-pro-BNP and other known cardiovascular risk parameters.

Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes.

Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1beta, TNFalpha, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1beta. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.

Discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes.

We deciphered constituent parts of a signal transduction cascade that is initiated by collagen II and results in the release of various pro-inflammatory cytokines, including interleukin-6 (IL-6), in primary human chondrocytes. This cascade represents a feed-forward mechanism whereby cartilage matrix degradation is exacerbated by the mutually inducing effect of released collagen II fragments and pro-inflammatory cytokines. We previously proposed discoidin domain receptor 2 as a central mediator in this event. Since this cascade plays a prominent role in the pathogenesis of osteoarthritis, our study further investigates the hypothesis that discoidin domain receptor 2 is a candidate receptor for collagen II, and that transcription factor NFkappaB, lipid kinase PI3K, and the MAP kinases are constituent parts of this very signal transduction cascade. To accomplish this, we selectively knocked down the molecules of interest in primary human chondrocytes, induced the specified cascade by incubating primary human chondrocytes with collagen II, and observed the outcome, specifically the changes in interleukin-6 release. Knockdown was performed by siRNA-mediated gene silencing in the case of discoidin domain receptor 2 (DDR2) or by using specific inhibitors for the remainder of the molecules. Results indicated that discoidin domain receptor 2 mediates the collagen II-dependent release of interleukin-6 in primary human chondrocytes and that MAP kinases p38, JNK and ERK, as well as transcription factor NFkappaB, are integral components of intracellular collagen II signalling. Given the detrimental role of these molecules in osteoarthritis, our findings provide new targets for more specific therapeutics, which may have fewer side effects than those currently applied.

A critical role for collagen II in cartilage matrix degradation: collagen II induces pro-inflammatory cytokines and MMPs in primary human chondrocytes.

We report a process that results in the acceleration of matrix degradation in human articular cartilage, a phenomenon commonly observed in osteoarthritis (OA). The study was conducted by (1) examining the potential of collagen II in modulating the gene expression profile of primary human chondrocytes (PHCs), and (2) investigating the involvement of pro-inflammatory signaling cascades. We first tested the collagen II-dependent induction of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) in PHCs. PHCs were incubated with or without monomeric (i.e., nonfibrillar) collagen II. Cells were then analyzed by RT-PCR for the expression of MMP1, MMP3, MMP13, MMP14, and IL-1beta. ELISA was used to quantify IL-6 and IL-8 release. To examine the influence of collagen II signaling, specifically the role of MAPK p38, a p38-inhibitor was added prior to collagen treatment. Changes in IkappaB concentration were monitored by immunoblot analysis to detect NFkappaB signaling. Results indicated that incubation of PHCs with collagen II did produce a dose-dependent induction of MMP1, MMP3, MMP13, MMP14, as well as cytokines IL-1beta, IL-6, and IL-8. At the same time, inhibition of p38 and IkappaB degradation revealed that collagen II-dependent gene induction also involves MAPK p38 and NFkappaB signaling. Thus, we provide evidence for a collagen II-dependent feed-forward mechanism whereby collagen II induces first MMPs and pro-inflammatory cytokines and then release of collagen II fragments from mature collagen II fibers. This, in turn, induces more pro-inflammatory cytokines and MMPs, and the process is repeated, which results in the acceleration and perpetuation of cartilage matrix degradation.

Decreased number of circulating progenitor cells in obesity: beneficial effects of weight reduction.

AIMS: Cardiovascular risk factors are associated with decreased levels of circulating progenitor cells (CPC). The aim of this study was to determine whether the number of CPC is an independent correlate of body mass index (BMI) and whether weight loss leads to an increase in CPC. METHODS AND RESULTS: CD34 positive and KDR/CD34, CD133/CD34, and CD117/CD34 double positive cells were measured by fluorescence activated cell sorting (FACS) analysis in peripheral blood of 149 volunteers (52.5 +/- 12.0 years, BMI 21.5-52.7 kg/m(2), mean 31.6 +/- 5.1 kg/m(2)) participating in a weight reduction program offered by German pharmacies. In addition, carotid intima media thickness (IMT) and brachial artery flow-mediated dilatation were determined. After a diet and sports program for 6 months, 86 representing subjects were re-evaluated (mean weight loss 5.8 +/- 5.2 kg). There was an inverse correlation between BMI as well as waist circumference and CPC, especially CD34 positive, KDR/CD34 positive, CD133/CD34 positive, and CD117/CD34 positive cells. This decrease in CPC in obesity held true not only for the absolute cell numbers, but also for the relative fractions of KDR, CD133, and CD117 positive cells within the CD34 positive cells, indicating a specific down regulation of these progenitor cell types. Multiple regression analysis revealed that BMI was a more prominent predictor of CPC regulation than blood pressure, LDL cholesterol, triglycerides, fasting glucose, and smoking. IMT increased in dependence on BMI (P < 0.001) and was inversely correlated with the number of CD34 positive cell (P < 0.05). After diet, there was a significant increase of CD34 and CD117/CD34 positive cells, which correlated with the decrease in BMI. Also, weight loss was accompanied by a decrease in IMT (P = 0.015), which also correlated with the increase in CPC (P < 0.001). The increase in the number of CPC was independent from whether weight loss was achieved by increased physical exercise or by reduced calorie intake only. CONCLUSION: Obesity is associated with decreased numbers of CPC and increased IMT. Diet and weight loss lead to an increase in CPC count, which might contribute to regression of IMT.

Impaired mitochondrial Ca2+ homeostasis in respiratory chain-deficient cells but efficient compensation of energetic disadvantage by enhanced anaerobic glycolysis due to low ATP steady state levels.

Energy-producing pathways, adenine nucleotide levels, oxidative stress response and Ca(2+) homeostasis were investigated in cybrid cells incorporating two pathogenic mitochondrial DNA point mutations, 3243A>G and 3302A>G in tRNA(Leu(UUR)), as well as Rho(0) cells and compared to their parental 143B osteosarcoma cell line. All cells suffering from a severe respiratory chain deficiency were able to proliferate as fast as controls. The major defect in oxidative phosphorylation was efficiently compensated by a rise in anaerobic glycolysis, so that the total ATP production rate was preserved. This enhancement of glycolysis was enabled by a considerable decrease of cellular total adenine nucleotide pools and a concomitant shift in the AMP+ADP/ATP ratios, while the energy charge potential was still in the normal range. Further important consequences were an increased production of superoxide which, however, was neither escorted by major changes in the antioxidative defence systems nor was it leading to substantial oxidative damage. Most interestingly, the lowered mitochondrial membrane potential led to a disturbed intramitochondrial calcium homeostasis, which most likely is a major pathomechanism in mitochondrial diseases.

RNAi in primary human chondrocytes: efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression.

RNAi-mediated gene silencing is a recent, powerful tool to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as IkappaB degradation and NFkappaB activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation.

FOXD4a and FOXD4b, two new winged helix transcription factors, are expressed in human leukemia cell lines.

Winged helix factors are important regulators of embryonal development and tissue differentiation. They are also involved in translocations found in acute leukemias and solid tumors. We have detected transcripts from five known and four novel winged helix genes in leukemia cell lines and CD34(+) blood progenitor cells by reverse trancription-polymerase chain reaction with degenerate primers on the highly conserved DNA binding domain. The genomic clones coding for two new winged helix proteins, FOXD4a and FOXD4b were isolated by high-stringency hybridization of a human phage library. FOXD4a and FOXD4b are encoded by a 1319 and 1250 bp single exon coding for a winged helix DNA binding domain, an amino-terminal acidic region and a carboxy-terminal proline- and alanine-rich region which correspond to putative transcriptional regulatory motifs. TATA box, CCAAT box, and transcription factor binding motifs have been identified in the 5' region of the genes. In addition, foxD4a and foxD4b cDNA has been isolated from NB-4 mRNA. The fox genes are transcribed in a tissue-restricted pattern in adult and fetal human tissues. FoxD4a and foxD4b mRNA was expressed in the leukemia cell lines KG-1, Kasumi, NB-4, HL-60, U937, THP-1, HEL, U266, Jurkat, and Raji. It has already been shown that winged helix factors are also involved in carcinogenesis. Based upon these studies, our results suggest that FOXD4a and FOXD4b may play a role in leukemogenesis.

Comparison of three dsDNA-ELISAs with regard to their efficiency in the diagnosis of systemic lupus erythematosus.

We compared the performance of three commercially available anti-double-stranded DNA antibody ELISA kits with respect to their precision, accuracy, linearity, and the detection limit. We tested six sets of patients (lupus erythematosus, scleroderma, rheumatoid arthritis, psoriatic arthritis, hepatitis C, malignancies) to assess specificity, sensitivity, and diagnostic efficiency of the three assays. The diagnostic efficiency of the Crithidia luciliae immunofluorescence test was analyzed as a reference. The sensitivity of the evaluated enzyme immunoassays ranged from 55.6 to 66.7%, the specificity from 98.4 to 100%, and the diagnostic efficiency from 90.2-92.7%. All ELISAs were superior to the immunofluorescence test, both with respect to sensitivity, specificity, and diagnostic efficiency. The commercial kits tested were comparable with respect to their performance characteristics.