Tumor necrosis factor alpha induces expression of genes for matrix degradation in human chondrocyte-like HCS-2/8 cells through activation of NF-kappaB: abrogation of the tumor necrosis factor alpha effect by proteasome inhibitors.

Tumor necrosis factor alpha (TNF-alpha) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-alpha activates a transcription factor nuclear factor kappaB (NF-kappaB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-alpha markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP-3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-kappaB. IkappaB-alpha and IkappaB-beta, cytoplasmic molecules preventing the nuclear translocation of NF-kappaB, were degraded rapidly by TNF-alpha followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IkappaB-alpha and IkappaB-beta and prevented the TNF-alpha-dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-alpha-dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-kappaB by TNF-alpha plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

[Evaluation of myocarditis using T1-201 myocardial scintigraphy with bull's eye functional map].

In order to evaluate myocardial damage in a patient with myocarditis, rest thallium-201 myocardial single photon emission computed tomography (SPECT) was performed in 15 patients with myocarditis. For qualitative and semiquantitative analysis, Bull's eye functional maps were made up with SPECT images. In the functional map, the abnormal area, where T1 uptake is less than mean-2SD of the T1 uptake of normal subjects, is generally distributed in the myocarditis group. But focal and sequential abnormal areas were recognized more often in the clinically severe cases. Abnormal area tended to be observed commonly at the antero-septal wall, but it was uncommon at the lateral wall. Extent score, i.e. degree of extension of abnormal area, and severity score, i.e. degree of abnormality, were in good negative correlation with left ventricular ejection fraction (r = 0.6, r = 0.7). Furthermore, existence of abnormal area was in good correlation with the left ventricular regional wall motion. Abnormal area existed 100% in the akinetic region, 71% in the region of severe hypokinesis, and 27% in the region of hypokinesis. Abnormal area occupied 30% of the normokinetic region in the myocarditis group, which was a higher rate than in the normal control group (p less than 0.05). It was suggested that latent myocardial damage existed in the normokinetic myocardium with myocarditis. Thus, rest T1-201 SPECT with Bull's eye map is useful for clinical diagnosis in patients with myocarditis.

Increased serum high mobility group box-1 level in Churg-Strauss syndrome.

Churg-Strauss syndrome (CSS) is a rare form of systemic vasculitis occurring in patients with asthma and hypereosinophilia; however, its mechanisms involved in the severe tissue inflammation with vasculitis are poorly understood. High mobility group box 1 (HMGB1) protein, originally identified as a DNA binding protein, also has potent pro-inflammatory and proangiogenic properties. In this study, we hypothesized that HMGB1 might be associated with CSS, and examined serum HMGB1 levels and compared those of asthma patients and healthy volunteers. We also investigated HMGB1 expression in the lesion, and eosinophil HMGB1 amount in CSS patients. We found that the serum HMGB1 levels in CSS patients were significantly higher than those of asthma patients and healthy volunteers. Eosinophils in the CSS lesion expressed HMGB1 and HMGB1 level in eosinophils from CSS patients was significantly higher than that of asthma patients, while there was no significant difference in HMGB1 levels in peripheral mononuclear cells. The serum HMGB1 level in CSS patients decreased after the steroid therapy, and showed significant positive correlations with several molecules, including soluble interleukin-2 receptor, soluble thrombomodulin, and eosinophil cationic protein in sera. We propose that HMGB1 might contribute to the pathogenesis of CSS.

Calcification of human articular knee cartilage is primarily an effect of aging rather than osteoarthritis.

OBJECTIVE: Pathologic calcification of articular cartilage in human knees is often associated with advanced age and conditions of osteoarthritis (OA). Coincidently, most studies that have characterized calcification in joint cartilage have examined populations that are aged and presenting with clinical symptoms. Generally, these studies rely upon relatively insensitive plain radiographs or synovial fluid crystal analyses to quantify calcium levels. The purpose of this study was to examine the relationship between cartilage calcification and aging in an unselected donor population of diverse age using highly sensitive calcification imaging. METHODS: A group of 106 knee blocks were obtained from 56 individual donors (25 females and 31 males, aged 12-74, avg. 50.3 years). Condylar surfaces were graded on a 4-point OA grading scale for cartilage degeneration. The condyles were cut into approximately 7-10mm thick slabs. Using a Faxitron radiography system, high-resolution images were taken of the slabs to specifically image calcification in cartilage. The quantified calcification areas were then analyzed and correlations with both OA grade and age were assessed. RESULTS: Every knee presented some measurable calcification. The relative calcium deposition had a significant positive correlation with age. This same positive correlation was seen between condyles showing grade 1 and 2 changes. OA grades higher than 2 did not present any further significant increase in calcium levels. CONCLUSION: These observations indicate that age rather than OA is the predominant factor driving progressive pathologic calcification in articular cartilage.

Clinical investigation: increased serum stromal derived factor 1 alpha levels in pulmonary tuberculosis.

Pulmonary tuberculosis, a granulomatous disease, has few serological markers for its activity. Recently, an increased plasma level of stromal derived factor 1 alpha (SDF-1alpha), which can induce strong chemotaxis of cells through its receptor CXCR4, was detected in patients with tuberculosis. In this study we investigated serum SDF-1alpha levels and CXCR4 expression on peripheral blood mononuclear cells (PBMCs). Fifty-five active tuberculosis patients, 30 resolved tuberculosis patients, 27 acute bronchitis patients and 8 healthy volunteers were examined. Histological expression of SDF-1alpha in the tuberculosis lesion and CXCR4 expression of PBMCs were also analysed. Serum SDF-1alpha levels in active tuberculosis patients were significantly higher than other groups. The sensitivity and specificity for the diagnosis of active tuberculosis was 88.5% and 85.3% (cutoff value = 650 pg/ml), respectively. CXCR4 expression levels on PBMCs showed a significant negative correlation with serum SDF-1alpha levels. Inflammatory cells including multinuclear giant cells in the lesion expressed SDF-1alpha. Measurement of serum SDF-1alpha could be a useful screening marker for the identification of active pulmonary tuberuculosis. We propose that interaction of SDF-1alpha and CXCR4 might be involved in the pathogenesis of pulmonary tuberculosis.

Uptake and antioxidant effects of ergothioneine in human erythrocytes.

Ergothioneine is a fungal metabolite that may have antioxidant functions in mammalian cells. Although it accumulates to low millimolar concentrations in liver and other tissues, it is not thought to be taken up by mature erythrocytes. During a study of the function of ergothioneine as an antioxidant in human erythrocytes, we found that these cells do take up ergothioneine from the surrounding medium. Ergothioneine concentrations in freshly prepared erythrocytes were 2-9-fold higher than in plasma from the same donor. Slow but progressive accumulation of ergothioneine to about 125% of basal levels was observed in erythrocytes over a 4 h incubation. After a 2 h incubation, intracellular ergothioneine concentrations rose on addition of increasing amounts of ergothioneine to the incubation medium, although saturation was not evident in cells from all donors. Both initial levels and rates of ergothioneine uptake varied in erythrocytes from different donors. Intracellular ergothioneine was stable to depletion of GSH by N-ethylmaleimide and to a more severe oxidant stress induced by hydrogen peroxide in the presence of catalase. These results show that human erythrocytes do take up ergothioneine; however, the GSH results do not support an antioxidant role for ergothioneine in erythrocytes.

GABA(A) receptor alpha1, alpha4, and beta3 subunit mRNA and protein expression in the frontal cortex of human alcoholics.

Animal studies have shown that chronic ethanol consumption produces physical dependence upon ethanol and alters gamma-aminobutyric acid-A (GABA(A)) receptor subunit gene expression in brain. Although extensive investigation has been conducted in animal models, relatively little work has been performed directly on human alcoholic brain tissue to determine if there are alterations in GABA(A) receptor gene expression. In this study, GABA(A) receptor alpha1, alpha4, and beta3 subunit mRNA and peptide expression in postmortem frontal cortex from human alcoholics (n = 15) and age- and sex-matched controls (n = 13) were measured by quantitative, competitive reverse transcription polymerase chain reaction and Western blot analysis. GABA(A) receptor beta3 subunit mRNA expression was 35% greater (p < 0.05) in alcoholics, compared with nonalcoholic controls. We found no significant difference in alpha1 and alpha4 subunit mRNA levels between groups. However, there was a trend toward greater (21%) alpha1 subunit mRNA expression. There was no difference in alpha1, alpha4, or beta2/3 subunit peptide levels in frontal cortex between controls and alcoholics. Neither the age of the subjects nor the postmortem interval correlated with mRNA or peptide levels. Blood ethanol content also did not correlate with mRNA or peptide expression in alcoholic samples. These data suggest that GABA(A) receptor adaptations, resulting from prolonged alcohol consumption in human alcoholics, may differ from animal models of alcohol dependence. These differences may be related to the longevity of alcohol exposure in human alcoholics, as well as variability in the dependence/withdrawal state of the human subjects. Therefore, further studies in human postmortem brain tissue are warranted.

Effects of chronic ethanol consumption and withdrawal on the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one in male and female rats.

Prolonged alcohol (ethanol) consumption leads to the development of alcohol tolerance and cross-tolerance to some benzodiazepines and barbiturates. In contrast, rats undergoing alcohol withdrawal are sensitized to the anticonvulsant effects of the endogenous GABA(A) receptor modulator, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP). Alterations in endogenous, cerebral cortical levels of 3alpha,5alpha-THP during alcohol withdrawal could contribute to the observed sensitization to 3alpha,5alpha-THP. Therefore, this study investigated plasma and brain levels of 3alpha,5alpha-THP, progesterone, and corticosterone during alcohol dependence and withdrawal in the rat. Plasma corticosterone, progesterone (a precursor of 3alpha,5alpha-THP) and 3alpha,5alpha-THP levels were unchanged in alcohol-dependent animals. Cerebral cortical levels of 3alpha,5alpha-THP decreased in dependent male animals, but not in dependent female rats. During alcohol withdrawal, plasma corticosterone and progesterone levels increased in male, but not female rats. However, neither plasma nor cerebral cortical 3alpha,5alpha-THP levels were altered from control levels in male or female rats during alcohol withdrawal. Plasma and brain levels of 3alpha,5alpha-THP were markedly higher in female compared with male rats. Cerebral cortical levels of 3alpha,5alpha-THP during the diestrus phase of the estrus cycle were approximately 4 to 6 ng/g, a concentration that may approach physiological relevance. These findings suggest that sensitization to 3alpha,5alpha-THP during alcohol withdrawal is not mediated by elevations in brain levels of endogenous 3alpha,5alpha-THP in male or female rats. However, elevations in circulating corticosterone and progesterone levels during ethanol withdrawal in male rats may underlie gender differences in allopregnanolone sensitivity during ethanol withdrawal.