Effects of 1,25-dihydroxyvitamin D3 on IL-17/IL-23 axis, IFN-γ and IL-4 expression in systemic lupus erythematosus induced mice model.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a multi-factorial autoimmune disease which may be characterized by T lymphocytes dysfunctions. Th17 cells have been identified as new effector cells, which play an important role in the pathogenesis. In recent years, immunomodulatory effect of vitamin D3 has been noticed. In the present experiment, the effect of vitamin D3 on the expression of IL-17, IL-23, IL-4 and IFN-γ were assessed in activated chromatin-induced mouse model for SLE. MATERIALS AND METHODS: Five groups of mice were included in this study; Group one received active chromatin +CFA + PBS; Group 2 received vitamin D3 starting 2 weeks before disease induction; Group 3 received vitamin D3 (50 ng/day) starting with the disease establishment; Group 4 received non active chromatin +CFA + PBS; Group 5 received CFA + PBS. On day 56 splenocytes were isolated and gene expression of interleukin IL-17, IL-23, IL-4 and IFN-γ were analyzed by Real-Time PCR method. Proteinuria and serum anti-dsDNA and Th17 levels were measured using commercial kits. RESULTS: The results showed that IL-17, IL-23, and IFN-γ mRNA expression, and IL-17 titers were decreased remarkably and that of IL-4 increased in mice which received vitamin D3 before SLE induction. Administration of vitamin D3 after the establishment of SLE failed to affect the IL-17 or IL-23 mRNA levels. Lastly, pre-treatment of mice with vitamin D3 decreased the anti-ds DNA antibody titer. CONCLUSION: Our findings showed that vitamin D3 supplementation in lupus induced mice through modulating the expression rate of some inflammatory cytokines diminished the inflammatory conditions in SLE.

Expression of T Helper 17 and Regulatory T Cell Cytokines and Molecules in Glomerulonephritis Class IV Systemic Lupus Erythematosus.

INTRODUCTION: Lupus nephritis is a serious organ involvement with unknown etiology, and glomerulonephritis class IV is one of the most severe forms of the disease which correlates with poor prognosis and death. Immunological abnormalities are implicated in the expression of lupus nephritis. In this study, we examined some T helper 17 and regulatory T-related cytokines and molecules in systemic lupus erythematosus patients with glomerulonephritis class IV. MATERIALS AND METHODS: The study group comprised of 20 glomerulonephritis class IV SLE patients and 20 sex- and age-matched SLE patients without kidney involvement as control group. Blood samples was collected from each participant, lymphocytes were isolated, and RNA was extracted from lymphocytes. Then cDNA was synthesized using reverse transcription enzyme, and finally using specific primers and probes, the expression levels of forkhead box P3 (Foxp3), transforming growth factor (TGF)-ß, interferon (IFN)-γ, interleukin (IL)-6, and IL-17 genes were analyzed by real-time polymerase chain reaction based on the TaqMan method. RESULTS: The expression levels of IL-6, IL-17, IFN-γ, and Foxp3 genes were significantly higher in SLE patients with glomerulonephritis class IV than those with non-nephritis SLE. However, the expression of TGF-ß was not significantly different between the SLE patients with and without glomerulonephritis class IV involvement. CONCLUSIONS: According to our results, it seems that in class IV glomerulonephritis patients, increased Foxp3-producing regulatory T cells has an imperfect capacity to control the pathogenic IL-17- and IFN-γ-producing cells.

DNA flow cytometric analysis in variable types of hydropic placentas.

BACKGROUND: Differential diagnosis between complete hydatidiform mole, partial hydatidiform mole and hydropic abortion, known as hydropic placentas is still a challenge for pathologists but it is very important for patient management. OBJECTIVE: We analyzed the nuclear DNA content of various types of hydropic placentas by flowcytometry. MATERIALS AND METHODS: DNA ploidy analysis was performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions) and 20 molar (complete and partial moles), formalin-fixed, paraffin-embedded tissue samples by flow cytometry. The criteria for selection were based on the histopathologic diagnosis. RESULTS: Of 10 cases histologically diagnosed as complete hydatiform mole, 9 cases yielded diploid histograms, and 1 case was tetraploid. Of 10 partial hydatidiform moles, 8 were triploid and 2 were diploid. All of 20 cases diagnosed as spontaneous abortions (hydropic and non-hydropic) yielded diploid histograms. CONCLUSION: These findings signify the importance of the combined use of conventional histology and ploidy analysis in the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion.

Anti-cancer properties of a sesquiterpene lactone-bearing fraction from Artemisia khorassanica.

BACKGROUND: Artemisia species are important medicinal plants throughout the world. The present in vitro study, using a sesquiterpene lactone-bearing fraction prepared from Artemisia khorassanica (SLAK), sought to investigate anti-cancer properties of this plant and elucidate potential underlying mechanisms for the effects. MATERIALS AND METHODS: Anti-cancer potential was evaluated by toxicity against human melanoma and fibroblast cell lines. To explore the involved pathways, pattern of any cell death was determined using annexin-V/PI staining and also the expression of Bax and cytochrome c was investigated by Western blotting. RESULTS: The results showed that SLAK selectively caused a concentration-related inhibition of proliferation of melanoma cells that was associated with remarkable increase in early events and over-expression of both Bax and cytochrome c. CONCLUSIONS: The current experiment indicates that Artemisia may have anti-cancer activity. We anticipate that the ingredients may be employed as therapeutic candidates for melanoma.

Influence of vitamin D on cell cycle, apoptosis, and some apoptosis related molecules in systemic lupus erythematosus.

OBJECTIVES: Genetic and environmental factors are involved in the pathogenesis of systemic lupus erythematosus (SLE). Autoreactive lymphocytes are cleared through apoptosis and any disturbance in the apoptosis or clearance of apoptotic cells may disturb tolerance and lead to autoimmunity. Vitamin D has anti-proliferative effects and controls cell cycle progression. In this study we investigated the effects of vitamin D on cell cycle and apoptosis induction in lupus patients. MATERIALS AND METHODS: Isolated peripheral blood mononuclear cells (PBMCs) from 25 SLE patients were cultured in the presence of 50 nM of 1,25(OH)2D3; then one part of the cells were stained with FITC labeled Annexin V and PI and were analyzed for apoptosis determination. For gene expression assessment of FasL, Bcl-2 and Bax, RNA was extracted from one another part of the cells, cDNA was synthesized and gene expression analysis was performed using Real time PCR. An additional part of the cells were treated with PI and the cell cycle was analyzed using flowcytometer. RESULTS: The mean number of early apoptotic cells in vitamin D treated cells decreased significantly (18.48±7.9%) compared to untreated cells (22.02±9.4%) (P=0.008). Cell cycle analysis showed a significant increase in G1 phase in vitamin D treated cells (67.33±5.2%) compared to non treated ones (60.77±5.7%) (P =0.02). Vitamin D up-regulated the expression levels of Bcl-2 by (18.87 fold increase), and down-regulated expression of Bax (23%) and FasL (25%). CONCLUSION: Vitamin D has regulatory effects on cell cycle progression, apoptosis and apoptosis related molecules in lupus patients.

Assessment of 1,25-dihydroxyvitamin D3 effects on Treg cells in a mouse model of systemic lupus erythematosus.

CONTEXT: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which is characterized by the presence of auto-reactive T cell and anti-ds DNA antibodies. Treg cells are crucial for maintaining immunologic self-tolerance and are shown to be reduced in SLE patients. 1,25-Dihydroxyvitamin D3 has immunomedulatory effects on the immune system and has recently received substantial attention. OBJECTIVE: In this study we evaluated the effects of 1,25-dihydroxyvitamin D3 on Treg cells and related cytokines in lupus-like induced mice model. MATERIALS AND METHODS: Female Balb/c mice were divided into four groups: Group one: injected with PBS and Freund's adjuvant; Group two: injected with non-activated chromatin; Group three: Lupus-like disease was induced with activated chromatin; Group four: Mice were initially treated for two weeks with 1,25-dihydroxyvitamin D3 and then lupus-like disease was induced. Group five: Four mice from group one were treated with 1,25-dihydroxyvitamin D3 for two weeks after disease establishment. Ten weeks after the last injection the mice were killed and spleens were studied for Treg percentages and expression of cytokine genes. RESULTS: We found that treatment with 1,25-dihydroxyvitamin D3 reduces IL-6 and IL-10 mRNA expression and increases TGF-ß and Foxp3 mRNA expression levels, and also enhances spleen Treg percentage. CONCLUSIONS: The remarkable reduction of IL-6 and IL-10 gene expressions, significant enhancement of TGF-ß and Foxp3 gene expressions, along with an increase in Treg cell population after oral 1,25-dihydroxyvitamin D3 administration suggest a possible role for this vitamin as a prophylactic supplement in SLE.

In vitro immunomodulatory properties of a sesquiterpene lactone-bearing fraction from Artemisia khorassanica.

Artemisia species are important medicinal plants throughout the world. The present in vitro study, using a sesquiterpene lactone-bearing fraction prepared from Artemisia khorassanica (SLAK), sought to investigate immunomodulatory/anti-inflammatory properties of this plant and elucidate potential underlying mechanisms for the actions. Effects of the SLAK on mitogen-induced murine splenocyte proliferation and interleukin (IL)-4 and interferon (IFN)-γ secretion were evaluated. To assess anti-inflammatory activities, levels of inducible of nitric oxide (NO) and prostaglandin E2 (PGE2), as well as expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in peritoneal macrophages was examined. The results showed that SLAK noticeably was capable of suppressing PHA/LPS-stimulated splenocyte proliferation and of up-regulating production of the T-helper (TH)-2 cell cytokine IL-4 while down-regulating formation of TH1 IFNγ. In addition, while SLAK caused negligible proliferation inhibition, peritoneal macrophages displayed considerable decrease in NO and PGE2 production along with iNOS and COX-2 expression. The current experiment shows Artemisia khorasanica - a traditionally used herb - may have immunomodulatory and anti-inflammatory effects. It is anticipated that the ingredients may be employed as therapeutic candidates in the regulation of some immune responses implicated in various conditions and ailments.

Rose Bengal suppresses gastric cancer cell proliferation via apoptosis and inhibits nitric oxide formation in macrophages.

Rose Bengal (RB) has been used as a safe agent in clinical diagnosis. In addition, it is used as a photodynamic sensitizer for removing microorganisms and cancer cells. Recently, its preferential toxicity after direct exposure to cancer cells was proven. The present study focuses on anti-cancer and anti-inflammatory activities of RB. The toxicity of RB against AGS gastric cancer and NIH 3T3 fibroblast cell lines was studied using an MTT assay. Patterns of any cell death among the AGS cells were defined using Annexin-V and PI staining. In addition, the effect of RB on nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production induced by lipopolysaccha-ride in J774A.1 macrophages was determined. Modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions in the macrophages was also evaluated by Western blots. The results showed that AGS cells exhibited significant concentration-dependent decreases in growth in response to RB; these cells showed a greater growth inhibition than did non-malignant 3T3 cells, suggesting that anti-growth activity of RB could be cell-specific. Moreover, AGS cells exposed to RB exhibited a significant increase in apoptosis; only at high RB doses did the cells display significant levels of necrosis. While RB also caused a modest decrease in the growth of J774A.1 macrophages, the cells displayed remarkable decreases in NO production and iNOS expression without significant concurrent modulation in PGE(2) production or COX-2 expression. The data from this study appears to suggest that RB differentially impacts on transformed cell lines, preferentially suppresses growth of a gastric cancer cell line through induction of apoptosis, and induces changes in cells that could reflect potential anti-inflammatory effects that might be induced in situ.

Sex hormones and peripheral white blood cell subsets in systemic lupus erythematosus patients.

BACKGROUND: Systemic Lupus Eyrythematosus (SLE) is an autoimmune disease characterized by antibodies to nuclear antigens, particularly anti-dsDNA. Imbalance between production and destruction of immune cells causes cytopenia. Sex hormones have immunomodulatory effects; estrogen increases the production of autoantibodies in SLE prone NZB/NZW mice. OBJECTIVE: To investigate the relationship between sex hormones, anti-dsDNA, and lymphocyte subsets in Iranian patients with SLE. METHODS: 38 SLE patients (28 females and 10 males) meeting 4 of 11 ACR revised criteria for SLE classification, and 20 age and sex matched healthy individuals (10 females and 10 males) participated in this study. Lymphocyte subsets were analyzed using flow cytometric analysis. Serum anti-dsDNA levels and sex hormones concentrations were determined using commercial ELISA and RIA kits, respectively. RESULTS: The absolute count of white blood cells, lymphocytes, T lymphocytes (CD3+), T helper cells (CD3+CD4+), B cells (CD19+) and Nk cells (CD3- CD16+CD56+) in SLE patients diminished significantly in comparison to control group (p<0.05). IgG anti-dsDNA antibody levels were significantly higher in patients compared to controls as expected (p<0.05). Prolactin increased significantly, while DHEAS showed a significant decrease in SLE patients compared with the controls (p<0.05), however the level of estrogen did not have any significant difference in SLE patients in comparison to controls. CONCLUSION: Increased concentration of prolactin together with a simultaneous decrease in serum DHEAS in SLE patients are associated with anti-dsDNA elevation and a decrease in almost all lymphocyte subsets.