Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens.

Selenium is an essential element with antioxidant roles in immune regulation, but there is little understanding of how Se acts in apoptosis in the immune organs of birds. The aim of study was to evaluate the influence of Se deficiency on oxygen free radicals, NO and apoptosis in immune organ of chickens. 160 1-day-old chickens were randomly assigned to two groups of 80 each and were fed on a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se), respectively. OFR production in blood was determined on days 30, 45, 60 and 75, respectively. The iNOS-NO system activity in immune organ (thymus, spleen, bursa of fabricius) was identified by NO content and NOS activity assay on days 30, 45, 60 and 75, respectively. Apoptosis was measured by DNA ladder analysis, ultrastructural observations, TdT-mediated dUTP nick end labeling TUNEL assay and flow cytometric analysis of apoptotic DNA. The transcription of factor-associated suicide, caspase-3 mRNA was tested by fluorescence quantitative PCR. The results showed that OFR production, NO and inducible NO synthases (iNOS) activity in the low-Se group were significantly increased (p < 0.05) than in the control group. In addition, apoptosis was observed in chicken immune organ in the low-Se group. The degree and the number of apoptotic cells rose in a time-dependent manner. The expression of Fas and caspase-3 mRNA increased (p < 0.05) than in the control group. It indicated that the oxidative stress and NO played a causative role in the apoptosis of immune tissues induced by selenium deficiency.

Treatment of refractory anti-melanoma differentiation-associated gene 5 anbibody-positive dermatomyositis complicated by rapidly progressing interstitial pulmonary disease: Two case reports.

BACKGROUND: Anti-melanoma differentiation-associated gene 5 antibody-positive (anti-MDA5 Ab+) dermatomyositis complicated with rapidly progressive interstitial lung disease (anti-MDA5 Ab+ DM-RP-ILD) has an unclear underlying mechanism with no recommended unified treatment plan. Herein, one of the cases that we report (Case 2) was successfully treated with tocilizumab despite having lung infection. CASE SUMMARY: Case 1 was a 30-year-old woman who was admitted due to recurrent rash for 5 mo, fever and cough for 1 mo, and chest tightness for 3 d. She was diagnosed with non-myopathic dermatomyositis (anti-MDA5 Ab+) and interstitial pneumonia, and was treated with the combination of hormone therapy and cyclophosphamide followed by oral tacrolimus. Case 2 was a 31-year-old man admitted due to systemic rash accompanied by muscle weakness of limbs for more than 1 mo, and chest tightness and dry cough for 4 d. He was diagnosed with dermatomyositis (anti-MDA5 Ab+) and acute interstitial pneumonia with Pneumocystis jirovecii and Aspergillus fumigatus infections and was treated with hormone therapy (without cyclophosphamide) and the combination of tocilizumab and tacrolimus. The condition of both patients eventually improved and they were discharged and showed clinically stable condition at the latest follow-up. CONCLUSION: Tocilizumab could be a salvage treatment for patients with anti-MDA5 Ab+ DM-RP-ILD who are refractory to intensive immunosuppression.

[Serum metalloproteinase-3 levels in assessing efficacy of Etanercept in patients with ankylosing spondylitis].

OBJECTIVE: To investigate the value of metalloproteinase-3 (MMP-3) levels in assessing efficacy of etanercept treatment in patients with ankylosing spondylitis (AS). METHODS: The serum and synovial fluid levels of MMP-3 were measured by enzyme linked immunosorbent assay (ELISA) in 48 patients with AS in week 0, 6 and 12; and also measured in 30 serum samples and 10 synovial fluid samples from healthy controls. RESULTS: The serum levels of MMP-3 in AS patients were significantly higher than those in controls. In AS patients, the MMP-3 levels in synovial fluid were significantly higher than those in serum levels. The serum MMP-3 levels in AS patients with peripheral arthritis were higher than those with exclusively axial involvement; while C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR) did not differ between these 2 groups of AS. At week 6 and week 12 of etanercept treatment, the serum MMP-3 levels were significantly decreased (p<0.01) with the declining trend of ESR, CRP, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) (all p<0.01). Before the etanercept treatment (week 0), serum levels of MMP-3 were correlated with ESR, CRP, BASDAI and BASFI (p<0.05). ESR was also correlated with CRP and BASFI, but not with BASDAI (r=0.361, P=0.071). At weeks 12, serum MMP-3 levels were still correlated with ESR, CRP and BASDAI (P<0.05), but not with BASFI (P=0.339); ESR was correlated with CRP, but not with BASDAI and BASFI. There was a significant correlation between BASDAI and BASFI (r=0.818,P=0.001). CONCLUSION: Serum MMP-3 levels are closely related to disease activity and may serve as an useful indicator for efficacy of etanercept treatment in AS patients.

[Alendronate prevents steroid-induced osteoporosis in patients with rheumatic diseases].

OBJECTIVE: To investigate the effects of alendronate (Alen) on the prevention of systemic glucocorticoid-induced osteoporosis in patients with rheumatic diseases. METHODS: 140 patients suffering from rheumatic diseases, including systemic lupus erythematosus, polymyositis, dermatomyositis, and Sjögren's syndrome, with normal bone mineral density (BMD) and treated with oral glucocorticoids were randomly divided into 2 groups: Alen + calcium group (n = 74) receiving Alen 10 mg once a day and castrate D 600 0.6 g once a day for 24 weeks and control group (n = 66) receiving castrate D 600 0.6 g once a day for 24 weeks. The BMD and biomarkers of bone turnover were measured at baseline and 24 weeks after initiating glucocorticoid therapy. RESULTS: After 24 weeks, the BMD values at lumbar spine, femoral neck, major trochanter, and Ward' s triangle increased by 6.1%, 6.3%, 3.3%, and 2.2% respectively compared with those at baseline (all P<0.05), however, those of the control group decreased by 8.7%, 9.1%, 7.7%, and 6.4% respectively (P<0.01, P<0.05), and the BMD levels at lumbar spine and femoral neck 24 weeks later of the Alen + calcium group were both higher than those of the control group (P<0.01, P<0.05). 24 weeks later the level of urine cross linked N-telopeptides of type I collagen (NTX) of the Alen + calcium group decreased (P<0.05), and the blood osteocalcin (BGP) of the Alen + calcium group increased, however, not significantly (P>0.05). There were no significant differences in serum AKP and BGP and urine NTX 24 weeks later between these 2 groups. CONCLUSION: Improving BMD, alendronate plays an important role in the prevention of glucocorticoid-induced osteoporosis. However, calcium treatment alone fails to prevent the loss of bone.

[Clinical significance of CD4+CD25+ T cells in peripheral blood of patients in systemic lupus erythematosus].

OBJECTIVE: To investigate the expressions of C(4)D(+)CD(25)(+) T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE) and to identify the relationship between the levels of CD(4)(+)CD(25)(+) T cells and the disease activity and progression of SLE. METHODS: Fifty-three SLE patients were enrolled in the study. Flow-cytometric assay was employed for detection of CD(4)(+)CD(25)(+) T cells and CD(4)(+)CD(25)(bright) T cells were defined according to fluorescence intensity of CD(25) expression exceeding 100. Meanwhile, correlation analysis was performed between their expression and the scores of SLE disease active index (SLEDAI), C(3), dsDNA and antinuclear antibody titles. RESULTS: The levels of peripheral blood CD(4)(+)CD(25)(+) T cells in SLE were (7.84 +/- 1.85)%, which were significantly lower than those in a group of healthy control [(9.18 +/- 2.01)%, P < 0.05]. The levels of CD(4)(+)CD(25)(+) T cells in a group of active SLE [(6.72 +/- 1.16)%] were higher than those in a group of stable SLE [(8.57 +/- 1.91)%, P < 0.01]. There was no difference between the active and stable groups of SLE in peripheral blood CD(4)(+)CD(25)(bright) T cells [(0.85 +/- 0.24)% vs (0.91 +/- 0.25)%, P = 0.686], but they were significantly lower than those in the group of healthy controls [(1.43 +/- 1.08)%, P < 0.01]. With the reduction of the SLEDAI scores in SLE patients after relevant treatment, the levels of peripheral blood CD(4)(+)CD(25)(bright) T cells did not change. No correlation was found between the levels of CD(4)(+)CD(25)(bright) T cells and SLEDAI scores, antinuclear antibody titles, dsDNA and C(3), respectively (rho = -0.188, P = 0.178; rho = -0.216, P = 0.121; rho = 0.082, P = 0.560; rho = 0.010, P = 0.944). CONCLUSION: The reduction of CD(4)(+)CD(25)(+) T cells in peripheral blood may participate in the pathogenesis of SLE.

Therapeutic effect of okra extract on gestational diabetes mellitus rats induced by streptozotocin.

OBJECTIVE: To explore the effect of okra extract on gestational diabetes mellitus (GDM) rats and its probable molecular mechanism. METHODS: A total of 30 female SD rats were caged with male rats for pregnancy, 27 pregnant rats were obtained and weighed. The pregnant rats were equally randomized into the control group, GDM group and intervention group. Once the pregnancy was verified, GDM group and intervention group were given 45 mg/kg streptozotocin by peritoneal injection for inducing GDM, control group was given equal volume of citrate buffer. Once the model was established successfully, intervention group was administered orally the solution containing 200 mg/kg/d okra extract, the other groups were given the diet and water only. On the 19th day of pregnancy, the blood samples and fetal rats of all groups were collected, fetal rats weight and placental weight was recorded and the serum glucose, lipids, serum insulin and C-peptide of pregnant rats before the delivery were determined. RESULTS: The pregnant rats weight before the delivery, fetal rats weight and placental weight of GDM group were lower than control group and intervention group (P < 0.05). After the treatment of okra extract, serum glucose and lipids levels of intervention group were both improved significantly (P < 0.05), especially, the FBG, HDL, FINS, serum m insulin and hepatic glycogen levels were equivalent to control group (P > 0.05). Antioxidant enzymes levels of GDM group in liver and pancreas tissues were lower than the other groups, and after treatment of okra extract, antioxidant enzymes levels in liver and pancreas tissues were equivalent to control group (P > 0.05). CONCLUSIONS: Okra extract, rich in antioxidant substances, could avoid the excessive consuming of antioxidant enzymes, then, suppresses the oxidative stress and insulin resistance, thereby improving blood glucose level of GDM rats.

Studies on the Method for the Determination of beta1-4 Galactosyltransferase and Its Kinetics.

A fluorescence assay method for beta1-4galactosyltransferase (beta1-4GT) has been developed involving a pyridylaminated sugar as an acceptor substrate, a fluorescent sugar chain, with the reducing end of the Gnbeta1 - 2Malpha1 - 6(Gnbeta1-2Malpha1-3)Mbeta1 - 4Gnbeta1 - 4Gn - PA aminated with 2-aminopyridine. Microsome was prepared from the liver of normal male rats as an enzyme sample. Then the fluorescent reaction product was separated by reverse-phase HPLC. The kinetic experiments were carried out using crude enzyme extracts of the Golgi complex from the rat liver. The enzyme has a pH optimum of 6.5,and optimal concentration of Triton X-100 of 0.5%. The K(m) and V(max) values for the sugar acceptor substrates were found to be 2.31x10(-3)M(-1) and 5.75x10(-2) &mgr;mol/(min.mg) respectively. Furthermore, our research revealed that beta1-4GT had branch specificity. The Gn of alpha1-3 mannose branch of the acceptor substrate was more susceptible to galactosylation than that of the alpha1-6 branch by 2.10 times.

Effects of oxidative stress on immunosuppression induced by selenium deficiency in chickens.

Selenium (Se) is an important nutritional trace element possessing immune-stimulatory properties. The aim of this 75-day study was to investigate effect of oxidative stress on immunosuppression induced by selenium deficiency by determining antioxidative function, morphological changes, DNA damage, and immune function in immune organ of chickens. One hundred sixty 1-day-old chickens (egg-type birds) were randomly assigned to two groups of 80 each and were fed on a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se, sodium selenite), respectively. Se contents in blood and immune organ (thymus, spleen, bursa of Fabricius) were determined on days 30, 45, 60, and 75, respectively. Antioxidative function was examined by total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and xanthine oxidase (XOD), and oxidative damage was examined by malondialdehyde (MDA) detection. DNA damage was measured by comet assay, and immune function was examined by determining serum interleukin-1ß (IL-1ß), interleukin-2 (IL-2), and tumor necrosis factor (TNF) contents. The results showed that Se concentrations in the low-Se group were significantly lower (P < 0.05) than in the control group. Low-Se diet caused a decrease in the activities of T-AOC, SOD, GSH-Px, and an increase in XOD activity and MDA content. Pathological lesions and DNA damage of immune tissues were observed in low-Se group, while the serum IL-1ß and IL-2 contents decreased, and TNF content increased. The present study demonstrated that chickens fed deficient in Se diets exhibited lesions in immune organs, decreased serum IL-1ß, IL-2 content, and serum TNF content, indicating that oxidative stress inhibited the development of immune organs and finally impaired the immune function of chickens.