Leptin protects rat articular chondrocytes from cytotoxicity induced by TNF-α in the presence of cyclohexamide.

OBJECTIVE: Although leptin appears to be an important local and systemic factor influencing cartilage homeostasis, the role of leptin in chondrocyte death is largely unknown. Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine that plays a central role in the pathogenesis of articular diseases. This study examines whether leptin modulates TNF-α-induced articular chondrocyte death. METHODS: Primary rat articular chondrocytes were isolated from knee joint cartilage slices. To induce cell death, the chondrocytes were treated with TNF-α. To examine whether leptin modulates the extent of TNF-α-mediated chondrocyte death, the cells were pretreated with leptin for 3 h before TNF-α treatment followed by viability analysis. To examine the mechanism by which leptin modulates the extent of TNF-α-mediated chondrocyte death, we utilized mitochondrial membrane potential (MMP) measurements, flow cytometry, nuclear morphology observation, co-immunoprecipitation, western blot analysis and confocal microscopy. RESULTS: We demonstrated that leptin suppresses TNF-α induced chondrocyte death. We further found that apoptosis partially contributes to TNF-α induced chondrocyte death while necroptosis primarily contributes to TNF-α induced chondrocyte death. In addition, we observed that leptin exerts anti-TNF-α toxicity via c-jun N-terminal kinase (JNK) in rat articular chondrocytes. CONCLUSION: Based on our findings, we suggest that the leptin present in the articular joint fluid protects articular chondrocytes against cumulative mechanical load and detrimental stresses throughout a lifetime, delaying the onset of degenerative changes in chondrocytes. We can further hypothesize that leptin protects articular chondrocytes against destructive stimuli even in the joints of osteoarthritis (OA) patients.

Inhibitory effects of quercetin on aflatoxin B1-induced hepatic damage in mice.

Aflatoxin B(1) (AFB(1))-mediated hepatic damage is involved in production of AFB(1)-8,9-epoxide-bound DNA adducts and this is also affected by a pro-oxidant potential of the toxin. In this study we investigated the effects of quercetin on AFB(1)-treated HepG2 cells. We also examined the biochemical mechanisms associated with the effects of quercetin on AFB(1)-mediated liver damage in mice. Our results revealed that quercetin and isorhamnetin inhibit production of reactive oxygen species and cytotoxicity, and block the decrease of reduced glutathione (GSH) levels in AFB(1)-treated HepG2 cells. Isorhamnetin have inhibitory ability on lipid peroxidation stronger than quercetin in the cells. Oral supplementation with quercetin decreased serum lactate dehydrogenase levels, increased hepatic GSH levels and superoxide dismutase activity, and reduced lipid peroxidation in both the liver and kidney in AFB(1)-treated mice. However, quercetin did not show a significant reduction on serum levels of alkaline phosphate, alanine aminotransferase and aspartate aminotransferase that were increased in AFB(1)-treated mice. HPLC analysis revealed that quercetin in plasma is mainly present as glucoronides and/or sulfates of quercetin. Collectively, it is suggested that quercetin does not directly protect against AFB(1)-mediated liver damage in vivo, but exerts a partial role in promoting antioxidative defense systems and inhibiting lipid peroxidation.

Association between the HLA-DRB1 gene and clinical features of systemic sclerosis in Korea.

OBJECTIVE: To determine whether HLA-DR alleles are associated with the development and clinical features of systemic sclerosis (SSc) in Koreans. METHODS: Seventy-nine patients (74 women and five men; 45 diffuse types and 34 limited types; mean age at diagnosis 43.9 years) fulfilling the American College of Rheumatology (ACR) classification criteria for SSc were enrolled. The controls were 144 healthy, disease-free Koreans. HLA-DRB1 genotypes were assessed by the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. RESULTS: The HLA-DRB1*15 allele was increased in anti-topoisomerase I autoantibody (anti-topo I)-positive SSc patients [p = 0.003, p corrected (p(corr)) = 0.039, odds ratio (OR) = 3.43, 95% confidence interval (CI) 1.45-8.13] compared with controls. The DRB1*11 allele was also observed more frequently in anti-topo I-positive SSc than in controls (13.3% vs. 4.2%) but not statistically significant (p = 0.053, p(corr) = 0.689). In patients with SSc, the DRB1*04 allele was associated with subcutaneous calcinosis (p = 0.048, OR = 4.56, 95% CI 1.07-19.37). Patients with overlap syndrome showed a negative association with the DRB1*04 allele (p = 0.036, OR = 0.26, 95% CI 0.08-0.91). CONCLUSION: The HLA-DRB1*15 allele was associated with the development of anti-topo I-positive SSc in Koreans. In addition, the DRB1*04 allele was associated with certain clinical features in SSc patients.

Validation of the classification criteria commonly used in Korea and a modified set of preliminary criteria for Behçet's disease: a multi-center study.

OBJECTIVE: Recently we have proposed a modified set of criteria to settle the questions raised regarding the International Study Group (ISG) criteria for Behçet's disease (BD). The aim of the present study was to validate the two pre-existing criteria sets commonly used in Korea, the ISG criteria and the criteria of the Behçet's Disease Research Committee of Japan (Japanese criteria), as well as the proposed modified criteria. METHODS: The study population included 155 consecutive patients with BD and 170 controls with non-Behçet's rheumatic diseases. Detailed data for all of the subjects were recorded prospectively by the participating physicians on a standard form that listed the clinical features of BD. The sensitivity, specificity, and accuracy of each set of the criteria were measured. RESULTS: Of the three criteria sets employed, the modified criteria were the most accurate, with an accuracy of 96.3%. The ISG criteria often failed to classify the following patients with BD: patients with only oral and genital ulcerations, certain patients with intestinal ulcerations, patients who did not manifest oral ulcerations, and patients with acute disease but fewer than three recurrent oral ulceration relapses in a 1-year period. The Japanese criteria also failed to categorize the following patients with BD: patients with oral and genital ulcerations, and patients with oral ulcerations, skin lesions, and a positive pathergy reaction. In addition, the Japanese criteria misclassified some of the control subjects with non-Behçet's uveitis as having BD. CONCLUSIONS: The results of this study suggest that there are some points that need to be reconsidered in the clinical application of the two pre-existing sets of criteria. Although the modified criteria were the most accurate, further validation studies will be required in other ethnic populations.

Endothelial nitric oxide synthase gene polymorphisms in Behçet's disease and rheumatic diseases with vasculitis.

OBJECTIVE: To assess potential associations between Korean Behçet's disease (BD) or other rheumatic diseases with vasculitis and two polymorphisms of the endothelial nitric oxide synthase (eNOS) gene, which include the Glu298Asp polymorphism in exon 7 and a variable number of tandem repeats (VNTR) polymorphism in intron 4. METHODS: 65 patients with BD, 27 with rheumatic diseases with vasculitis, and 80 controls were studied. Analyses of the Glu298Asp polymorphism in exon 7 and VNTR polymorphism in intron 4 of the eNOS gene were made by the polymerase chain reaction (PCR)-restriction fragment length polymorphism technique and PCR genotyping, respectively. Additionally, HLA-B51 typing was performed in the BD group and controls by a two step PCR sequence-specific primers method. RESULTS: Significant differences in Glu298Asp genotype frequencies were found between the BD or vasculitis groups and the controls (BD group v controls: p(corr)=0.006; vasculitis group v controls: p<0.001). The Asp298 frequency was much higher in the BD and vasculitis groups than in the controls. Even after stratification of the BD group based on the results of HLA-B51 testing, a significant association of the Glu298Asp polymorphism was still found (p=0.002, Mantel-Haenszel weighted odds ratio 4.3, 95% confidence interval 1.7 to 10.9). Distribution of the genotype frequencies in two eNOS gene polymorphisms was similar in connective tissue diseases-associated vasculitis and primary vasculitic syndromes. In contrast, distribution of alleles and genotypes of VNTR polymorphism did not differ between BD or vasculitis groups and the controls. CONCLUSION: The Glu298Asp polymorphism in exon 7 of the eNOS gene seems to be a susceptibility gene for Korean BD and other rheumatic diseases.

Association between HLA-DR B1 and clinical features of adult onset Still's disease in Korea.

OBJECTIVE: To determine whether HLA-DR alleles are associated with the development and clinical features of Adult Onset Still's Disease (AOSD) in Korea. METHODS: Forty-seven patients (41 women, 6 men, mean age at diagnosis 31.6 yr) meeting Yamaguchi's criteria for AOSD and 144 healthy controls were enrolled in this study. The patients with AOSD were subdivided into groups according to their chronicity: monocyclic systemic, polycyclic systemic, and chronic destructive type, and were furthermore classified as non-articular, oligoarticular or polyarticular types (having arthritis involving 5 or more joints) according to the extent of articular involvement. HLA-DRB1 genotypes were assessed by PCR-SSOP. RESULTS: Patients with AOSD had more frequent DRB1*12 (p = 0.028, relative risk (RR) = 2.27, 95% confidence interval (CI): 1.08-4.80) and DRB1*15 (p = 0.013, RR = 2.16, 95% CI: 1.17-4.00). They had less frequent DRB1*04 (p = 0.006, RR = 0.35, 95% CI: 0.16-0.75) compared to controls. DRB1*14 (p = 0.011, RR = 3.80, 95% CI: 1.27-11.31) were associated with the monocyclic systemic type. CONCLUSION: Korean AOSD patients had more frequent DRB1*12 and DRB1*15, and less frequent HLA-DRB1*04. The patients with the monocyclic systemic type had more frequent DRB1*14 alleles. This study suggests that Korean AOSD patients have distinct immunogenetic profiles, and that it would be valuable to assess the relationships between HLA-DRB1 genes and polymorphisms of proinflammatory cytokines in the pathogenesis of AOSD.

The use of acupunture to control side effects and pain during treatment for childhood cancer

At the Hospital do Câncer in São Paulo, Brazil, appoximately 300 patients per year are diagnosed with childhood câncer. Generally 60 children/adolescents are seen in the outpatient clinics each day (25 receive chemotherapy). The multidisciplinary team includes a consulting acupuncturist (a specialty recognized by the Federal Medical Council of Brazil) to address the side effects of the treatment of childhood cancer. Six children/adolescents, 2 to 20 years of age, were chosen during 2000 to receive this treatment. Diagnoses included soft tissue sarcoma, retinoblastoma, and Hodgkin’s disease, nd one third of the patients were in palliative care. The indications for acupuncture included nause/vomiting, pain, constipation, stress, and aversion to chemotherapy. Children/adolescents, identified by the nurse as appropriate for the treatment, met with the acupuncturist to discuss the indications and carefully explain the procedure. Each needle application took 30 minutes, while the patient was observed by the nursing staff. The nurses removed the needles, gave the discharge instructions, and monitored the child/adolescent during follow-up.

Approximately 70% of the children/adolescents achieved complete relief of their symptoms. An example of the patients’ satisfaction is as follows. &dquo;The first time I was a little worried about the needles, but after that I felt so good that I wanted to repeat the application. Nowadays I set my chemo for the days I can do a session of acupuncture before.&dquo; Acupuncture is more cost effectivethan pharmacologic interventions for our unit. It is particularly welcome in palliative care since we use minimal pharmacology during this phase tobe less invasive. Acupuncture provides better control of side effects and enhances the quality of treatment, and so it improves our nursing practice.From a patient/family perspective it has reduced the psychological damage caused by the side effects,increased oral intake, and given patientsadditional self-confidence.

FcgammaRIIa/IIIa polymorphism and its association with clinical manifestations in Korean lupus patients.

The aim of this study was to determine the distribution of the FcgammaRlla and FcgammaRIIIa polymorphisms and their association with clinical manifestations in Korean lupus patients. Three hundred SLE (systemic lupus erythematosus) patients (48 male, 252 female) meeting 1982 ACR criteria and 197 Korean disease-free controls were enrolled. Genotyping for FcgammaRlla 131 R/H and FcgammaRIIIa 176 F/V was performed by PCR of genomic DNA using allele-specific primers and the FcgammaRIIIa genotype was confirmed by direct sequencing of PCR product in some cases. There was significant skewing in the distribution of the three FcgammaRIIa genotypes between the SLE and the controls (P=0.002 for R/R131 vs R/H131 and H/H131, OR 2.5 (95% Cl 1.4-4.5), but not in FcgammaRIIIa genotypes. FcgammaRIIa-R allele was a significant predictor of lupus nephritis, as compared with SLE patients without nephritis (P=0.034 for R131 vs H131, OR 1.4 (95% Cl 1.03-1.9)), but proliferative nephritis (WHO class III and IV) was less common in patients with FcgammaRlla-R/R131 and in FcgammaRIIa-R allele. In 300 SLE patients, high binding allele combination H131/V176 was less common in SLE with nephritis than in SLE without nephritis. Hemolytic anemia was less common in R131/F176 allele combination among four FcgammaRIIa/FcgammaRIIIa allelic combinations. Male SLE patients showed a higher frequency of renal involvement, serositis, thrombocytopenia, malar rash and discoid rash than female SLE, and male SLE had a higher frequency of FcgammaRIIa-R/R131 or R131-allele than male controls, but FcgammaRIIa or FcgammaRIIIa genotypes had no association with renal involvement in male SLE patients. FcgammaRIIa-H/H131 showed a higher frequency of hemolytic anemia and less pulmonary complications in male SLE. Female SLE patients showed higher frequency of any hematologic abnormality, lymphopenia, anticardiolipin antibody (+) and anti-Ro antibody (+) than male SLE, and had earlier onset of first symptoms. There was no skewing in FcgammaRIIa or FcgammaRIIIa genotypes between female SLE and female controls, but FcgammaRIIa-R131 allele showed skewing between female SLE with nephritis and female SLE without nephritis. The age at onset of thrombocytopenia was earlier in FcgammaRIIa R/R131 among three FcgammaRIIa genotypes, and serositis in FcgammaRIIIa-F/F176 among three FcgammaRIIIa genotypes. FcgammaRIIa-R131 homozygote was a major predisposing factor to the development of SLE and FcgammaRIIa-RI31 homozygote and R131 allele were a predisposing factor, and H131/V176 was a protective allele combination in lupus nephritis. In contrast to other ethnic patients, in our study cohort, clinical manifestation was different between male and female, and FcgammaRIIa and FcgammaRIIIa showed somewhat different clinical associations between the genders.

Effect of mycelial culture broth of Ganoderma lucidum on the growth characteristics of human cell lines.

Two types of purified samples, water-soluble (sample A; M. W, 1.2 x 10(6) dalton) and water-insoluble (sample C; M. W., 1.0 x 10(6) dalton) samples, were obtained through consecutive separation processes from the culture broth of Ganoderma lucidia mycelium. It was found that both samples from the culture broth were very effective in inhibiting the growth of several human cancer cell lines, having a 93-85% growth inhibition on Hep3B, AGS and A549 with the least cytotoxicity on the normal human lung cell line, WRL68 of less than 25% the highest supplementation concentration of 1.0 mg/l. In general, the sample C showed greater inhibition of cancer cell growth than the sample A. The same trend was also observed in antimutagenicity using the Chinese hamster ovary cell line (CHO test) or Salmonella typhimurium (Ames test). The CHO test showed that sample C had higher antimutagenicity on mutagens 4NQO or MMNG than sample A (approximately 40% vs approximately 25%). The percentage of antimutagenicity from the Ames test was lower than that from the CHO test, possibly due to the difference in the sensitivity of mutagens. The water-insoluble sample greatly enhanced the growth of the human T cell line (H9) up to 1 x 10(5) with sample supplementation at 1.0 mg/l concentration from 4.3 x 10(4) without sample supplementation as well as improved the secretion level of both IL-6 and TNF-alpha up to 100 pg/ml from approximately 40 pg/ml without sample supplementation. The kinetics of response to the immune cell growth was illustrated by the response time obtained when the sample concentration was increased. The water-insoluble sample can be used for effectively treating cancer in that it accelerated apoptosis of human carcinoma cells up to 70% compared to less than 50% for the control. The sample also increased the differentiation ratio of HL-60 cells up to 58% after four days of cultivation, compared to 18% in the case of no sample supplementation. These results can be used in implying that the insoluble part of G. lucidium mycelium culture broth must be related to controlling signal transduction, resulting in the regulation of cancer cell growth.

Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Anti-tumor and immune activation activities.

Immune modulating activity of ethanol extracts from Glycyrrhiza uralensis Fisch was investigated by conserving growth characteristics of several human cell lines. All of the samples did not show severe cytotoxicity on normal human liver cell line, WRL-68, showing less than 25% inhibition of cell growth. The crude extract and its fractionized samples (F1 and F3) inhibited the growth of human hepatoma, Hep3B, down to ca. 70% of normal cell growth in adding 1.0 g l(-1) of fraction F3. The result of anticancer experiments was well matched to the results of antimutagenicity using Chinese Hamster Lung cell lines(CHL V79). In adding 1.0 g l(-1) of fraction F1, the growth of human B cell was enhanced, up to 60% of control growth. The secretion of two kinds of cytokines, Interleukin-6 and Tumor Necrosis Factor-alpha from human B cells was also enhanced in adding the crude extract, but not the standards such as Glycyrrhizin (GL) or 18,beta-glycyrrhetinic acid (GM). It was found that both of the apoptosis and differentiation were more accelerated in supplementing the crude extract and fraction F1 than in adding the standards. A spot was found only in the crude extract and fractions, not standards by Thin Layer Chromatography(TLC) analysis. It tells that there must be another unknown component in crude and/or fraction F1 as a possible candidate of immune modulators. This component seems to be a derivative of a monomer, GM since its R(f) was close to the monomer. It was also interesting that glycyrrhizin, a major component in G. uralensis Fisch was biologically activated by first being hydrolyzed by an enzyme.

Primary Sjögren's syndrome manifested as multiple sclerosis and cutaneous erythematous lesions: a case report.

Sjögren's syndrome is a chronic autoimmune disorder characterized by lymphocytic infiltration of the lacrimal and salivary glands, leading to dryness of eyes (kerato-conjunctivitis sicca) and mouth (xerostomia). The skin lesions in Sjögren's syndrome are usually manifested as xeroderma, but sometimes appear as annular erythema or vasculitis. Central nervous system symptoms may be presented as one of extraglandular manifestations, though rare in incidence, and need differential diagnosis from multiple sclerosis. We report a case of a 45-year-old woman diagnosed as multiple sclerosis at first but later as neurologic manifestation of primary Sjögren's syndrome, showing signs of multiple sclerosis and cutaneous erythematous lesions.