Hypothyroidism and Fertility: An Animal Model follows up in The Second-Generation.

Objective: Hypothyroidism is known as the most common endocrine disorder. The prevalence of hypothyroidism in the female and male population is 2% and 0.2%, respectively. Maternal hypothyroidism is a defect in the thyroid hormones transition from the mother to the fetus. The present study was conducted to find whether maternal hypothyroidism affects the fertility of the second generation. Materials and Methods: In this experimental study, twelve adult female rats weighting 180-220 g were randomly divided into case and control groups. Hypothyroidism was induced by dissolving 0.1 g/L of 6-n-propyl-2-thiouracil in drinking water toward the end of pregnancy and lactation. At the end of the breastfeeding period, the blood samples of female children were collected. Six healthy, mature, female rats were selected and kept until they reached maturity, and were then mated with male rats. After observing the female rats' delivery, blood samples were collected from their male and female newborns and the healthy rats were selected. Results: There was a significant difference in the volume and size of ovarian as well as in the number of secondary follicles in comparison with the control group (P=0.025). However, there were no significant changes in the other parameters including the number of primary follicles, the number of Graafian follicles and sperm parameters. There was no significant decrease in the testicular volume and size, number of Leydig cells and seminiferous tubules diameter. Conclusion: Maternal hypothyroidism has no significant effects on testicular tissue function, and sperm parameters in the second generation, but can significantly reduce the rate of secondary follicles in the second generation female rats.

The efficacy of melatonin against radiotoxicity of iodine-131 and its response to treatment in hyperthyroid patients: a randomized controlled trial.

BACKGROUND: Since melatonin is a non-toxic compound with proven radioprotective effects, we aimed to investigate its efficacy in an in-vivo setting in hyperthyroid patients who are treated with iodine-131. This double-blind placebo-controlled study was conducted on hyperthyroid patients referred to nuclear medicine centers in Babol, Iran. We excluded patients suffering from hypertension treated with warfarin, autoimmune diseases, genetic diseases, cancers, smokers, chemical wounded, radiology and radiotherapy workers, and those who were treated with chemotherapy agents. Patients were randomly assigned to receive a capsule containing 300 mg of melatonin powder or a placebo. Just before receiving iodine-131, blood samples were taken from individuals. All 52 female patients received 10 to 20 mCi iodine-131 for treating hyperthyroidism. A second blood sample was taken one hour after the administration of iodine-131. MATERIAL AND METHODS: To determine the chromosomal damages before and after receiving radioiodine, we performed the cytokinesis- block micronucleus assay. Also, at phase 2, 6 months follow-up was performed, in which patients' positive responses to treatment were compared. RESULTS: The findings of this study indicate that the difference in micronucleus formation between the placebo and melatonin groups is not significant. However, a significant difference in the 6 months follow-up revealed that 61.5% and 85.7% of patients had a positive response to treatment in the placebo and melatonin groups, respectively. CONCLUSIONS: As one of the first studies dealing with the human in-vivo assessment on the radioprotective effects of melatonin, it was concluded that melatonin has a non-significant positive impact on reducing the rate of chromosomal damages in hyperthyroid patients treated with iodine-131. Nevertheless, the outcome of treatment was significantly higher by melatonin compared to the placebo group.

Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant.

BACKGROUND: Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. METHODS: The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining. RESULTS: All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67+, cytokeratin+, vimentine+, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium. CONCLUSION: This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.

Therapeutic effect of cold atmospheric plasma and its combination with radiation as a novel approach on inhibiting cervical cancer cell growth (HeLa cells).

Cervical cancer is one of the important cancers in women. Research on novel treatment approach can reduce the mortality and burden. Although radiotherapy is a common treatment, its negative side effects have concerned physician. In our study, we studied impact of cold atmospheric pressure plasma on the Hela cancer cells, as an alternative treatment. The effect of three different types of such plasma; dielectric barrier discharge (DBD), plasma jet, and afterglow plasma, on the cancer cells were studied. Moreover, some effective operating parameters such as exposure time, applied voltage, composition of working gas in plasma treatment were investigated on the survival of the afterglow plasma. Finally, treatments by the afterglow plasma, gamma radiation (1 Gy), and combination of both were compared. Analysis showed that DBD and plasma jet (direct exposure) effectively killed the cancer cells, even by a minimum applied voltage. But a fraction of the cells survived after the exposure of indirect diffused afterglow plasma. In the case of this plasma, we realized that higher applied voltage and exposure time led to less cell viability. Fewer fractions of survival cells were detected in the case of argon afterglow plasma comparing to oxygen afterglow. Cold atmospheric plasma and its combination with radiation therapy showed a significant decrease in viability of the cells, comparing to the radiation alone. Our research showed that plasma and its combination with radiation therapy have superiority over radiation therapy.

Antibacterial and Cytotoxic Effects of Cyclodextrin-Triazole-Titanium Based Nanocomposite

Abstract In this paper, the antibacterial activity of triazole functionalized cyclodextrin (CD.Click) and cyclodextrin-triazole-titanium based nanocomposite (CD.COM) was evaluated. The results indicated that CD.Click and CD.COM perform a wide range of antibacterial activity against both gram positive (Staphylococcus aureus and Bacillus subtilis) and gram negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The cytotoxic effect of CD.COM was investigated in vitro on cancerous cell lines (cervical cancer, breast carcinoma and sarcoma osteogenic) and fibroblast cells by MTT assay. The cell viability evaluation confirmed that the growth of cancerous cells is inhibited in a dose and time dependent way without any significant effect on the normal fibroblast cells.

Decrease of intracellular ROS by arbutin is associated with apoptosis induction and downregulation of IL-1ß and TNF-α in LNCaP; prostate cancer.

Increased reactive oxygen species (ROS) along with inflammation are involved in the prostate cancer (PCa). Therefore, this study was conducted to investigate the molecular mechanisms that were affected by arbutin as an antioxidant on prostate cancer cell line; LNCap. The intracellular ROS measurement confirmed that arbutin significantly (p < .05) decreased the ROS levels in a dose-dependent manner. Detection of cell death profile established that 1,000 µM of arbutin could remarkably induced apoptosis (p < .05), while tert-butyl hydroperoxide (tBHP) as ROS inducer prompted necrosis. In addition, 1,000 µM of arbutin successfully decreased expressions of IL-1ß and TNF-α genes (p < .05). Furthermore, evaluation of the IL-1ß protein level showed that arbutin could significantly decrease this cytokine (p < .05). In summary, reduction of ROS along with increasing apoptosis and decreasing expression of pro-inflammatory genes following arbutin treatment can open new visions in the treatment of prostate cancer using complementary medicine. PRACTICAL APPLICATIONS: Nowadays, arbutin as a glycosylated hydroquinone is available commercially in both natural and synthetic forms. Arbutin is of interest because of its skin-lightening effect, and used in cosmetic products for cutaneous hyperpigmentation. Arbutin inhibited tyrosinase in melanocytes competitively. Moreover, arbutin was able to attenuate oxidative stress and, its anti-inflammatory activities has been established. In addition, arbutin has represented useful activities for suppression of malignant melanoma development. In addition, arbutin exhibits several pharmacological effects, including antimicrobial, antihyperlipidemic, antihyperglycemic, and alpha amylase inhibitory effects. In this study, we showed its effect on prostate cancer in vitro. Therefore, it opens new insights in the complementary medicine that can maintain or improve human health.

Systemic effects of starved fibroblast culture supernatant on immunosuppressed rats treated with cancer stem cells (LA7).

BACKGROUND: The present study aimed to investigate and compare the effect of starved fibroblast culture supernatant (SFS), DMEM and normal saline alone or along with LA7 on dexamethasone-treated immunosuppressed Wistar rats. METHODS: After the isolation of fibroblasts from the fresh foreskin of children, it was cultured in serum-free DMEM, and the supernatant collected after 16 hours (16h-SFS). This solution and the other treatments were injected subcutaneously into the rats from each group once daily for 14 days. The liver, intestine and lung histology along with blood cellular and biochemical characteristics were studied. RESULTS: The results showed that dexamethasone as immunosuppressant reduced the body weight. The histological change in the liver was mild fibrosis induced by LA7+16h-SFS. Also, among the different blood cellular and biochemical indices measured, the eosinophil percentage in the 16h-SFS treated rats , glucose levels in the 16h-SFS+LA7 group and triglyceride concentrations in the 16h-SFS group were changed (p<0.05). CONCLUSION: This study showed that the secretions of starved fibroblasts especially that combined with LA7 cancer stem cells could induce some minor histological and biochemical changes in immunosuppressed rats, and also it opened a new window for subsequent investigations on unknown mechanisms related to this work.

Targeting LA7 breast cancer stem cells of rat through repressing the genes of stemness-related transcription factors using three different biological fluids.

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.

A comparative study on immunophenotypic characterization and osteogenic differentiation of human mesenchymal stromal cells derived from periodontal ligament and gingiva.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from periodontal ligament (PDL) and gingiva can be used for the development of cell-based regenerative approaches in dentistry and medicine. The purpose of this investigation was to establish a method for isolation of human stem cells from the PDL and gingiva, multilineage differentiation of those cells, and comparison of periodontal ligament mesenchymal stem cells (PDLMSCs) and gingival mesenchymal stem cells (GMSCs). METHODS: PDL and gingival tissues of third molar teeth were digested enzymatically and the proliferative potential of human PDLMSCs and GMSCs was compared by MTT assay. The expression of cell surface epitopes was analyzed by flow cytometry. To investigate the multilineage differentiation capacity of these stem cells, osteogenic and adipogenic differentiation was achieved. The specific staining of nodules was performed to evaluate differentiation, whereas the expression of alkalin phosphatase (ALP) and collagen A I (COL I) genes was analyzed by quantitative real-time polymerase chain reaction. RESULTS: The outgrown cells derived from PDL and gingival tissues were similar, fibroblast-like, and spindle-shaped. Further, the proliferation potential of GMSCs was greater than PDLMSCs. Both types of stem cells expressed MSC precursor markers, including CD73, CD90, and CD105, whereas they were negative for hematopoietic markers, including CD34 and CD45. PDLMSCs demonstrated more osteogenic potential compared to GMSCs with strong mineral noduls, and significantly greater expression of up-regulated bone-related markers ALP and COL I. CONCLUSION: MSCs derived from PDL and gingiva demonstrated multipotent characteristics, suggesting new therapeutic approaches in tissue engineering and PDLMSCs are more appropriate candidates for this purpose.

Mizaj as an Index in Persian Traditional Medicine Index Could Associate with Sensitivity to the Radiation.

BACKGROUND: The sensitivity to the radiation among human population depends on various parameters. This variation could lead to dissimilar outcome of radiotherapy in similar situations. Mizaj is a well-known term in Persian medicine that present an individualized medicine viewpoint. All of the people will be categorized in cold, moderate, and warm Mizaj. In this study, we aimed to evaluate the possible association between Mizaj and radiosensitivity by comet assay. MATERIALS AND METHODS: Peripheral blood sample of 30 healthy volunteers (10 cold, 11 moderate and nine warm Mizaj) were taken and divided into two identical parts. The first part was exposed to 4 Gy x-rays, and the second part was regarded as the sham control. Then, DNA damages of samples were evaluated by the neutral comet assay. RESULTS: The results showed that the mean percentage of damaged cells, in all of the irradiated groups including A (warm), B (moderate) and C (cold) was significantly higher than the controls (P<0.001). Moreover, DNA damage rate in the irradiated warm Mizaj group was higher than both cold and moderate irradiated groups, but the difference between moderate and cold irradiated groups was not statistically significant. CONCLUSION: The results are indicating that warm Mizaj persons could be more radiosensitive than other groups, which their importance in radiotherapy individualization should be evaluated in more extensive studies.

Anticancer properties of chitosan against osteosarcoma, breast cancer and cervical cancer cell lines.

BACKGROUND: Cancer is still the most common cause of morbidity in the world. Chitosan, a commonly used natural polymer, is consisted of different molecular weight with different biological activities.The purpose of this study was to determine cytotoxicity effect of high molecular weight (HMWC) and low molecular weight of chitosan (LMWC) on three cancerous cell lines MCF-7, HeLa and Saos-2 with different histological origin. METHODS: The anticancer property of two types of chitosan on three cancerous cell lines and human fibroblast as normal cell line, was evaluated by cytotoxic activity including their apoptosis induction properties. Chitosan solutions 2% (w/v) were prepared. The cells were treated by different concentration of chitosan and viability was determined by MTT assay after 24, 48 and 72 h .Also the mode of cell death-apoptosis vs necrosis ,was determined by Annexin V staining assay and analyzed by flow cytometry. RESULTS: While both types of chitosan were effective in inhibiting cell proliferation of three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. Despite of a significant decrease in all 3 cell lines viability, up to 90%, but we didn't see a concentration dependent difference between both types of chitosan (HMWC and LMWC) in their cytotoxic effects. Flow cytometry analysis showed necrosis more observable with MCF7 while the apoptosis pattern of death was more in Saos-2 and HeLa. Also, higher viability with both types of chitosan was seen in fibroblast as normal cells. CONCLUSION: While chitosan is compatible with normal diploid fibroblast cells, it shows anticancerous effect against 3 cancerous cell lines. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

Co-administration of aspirin and adipose-derived stem cell conditioned medium improves the functional recovery of the optic pathway in a lysolecithin-induced demyelination model.

INTRODUCTION: Based on beneficial effects of aspirin and mesenchymal stem cells (MSCs) on myelin repair, in a preset study, effects of co-administration of aspirin and conditioned medium from adipose tissue-derived stem cells (ADSC-CM) on functional recovery of optic pathway, demyelination levels, and astrocytes' activation were evaluated in a lysolecithin (LPC)-induced demyelination model of optic chiasm. METHODS: LPC (1%, 2 µL) was injected into the rat optic chiasm and animals underwent daily intraperitoneal (i.p.) injections of ADSCs-CM and oral gavage of aspirin at a dose of 25 mg/kg for 14 days post LPC injection. The conductivity of visual signals was assessed using visual evoked potential recordings (VEPs) before LPC injection and on days 7 and 14 post lesion. Immunostaining against PDGFRα as oligodendrocyte precursor cells marker, MOG as mature myelin marker, and GFAP as astrocyte marker was performed on brain sections at day 14 post LPC injection. FluoroMyelin staining was also used to measure the extent of demyelination areas. RESULTS: Our results showed that administration of ADSCs-CM and aspirin significantly reduced the latency of VEP waves in LPC receiving animals. In addition, demyelination levels and GFAP expressing cells were attenuated while the number of oligodendrocyte precursor cells significantly increased in rats treated with ADSCs-CM and aspirin. CONCLUSION: Overall, our results suggest that co-administration of ADSCs-CM and aspirin improves the functional recovery of optic pathway through amelioration of astrocyte activation and attenuation of demyelination level.

Antibacterial activity of high-molecular-weight and low-molecular-weight chitosan upon oral pathogens.

CONTEXT: One of the common oral bacterial infectious diseases is dental caries. Control of dental plaque formed by Streptococcus mutans and Streptococcus sobrinus leads to prevention and treatment of caries. Chitosan (1-4, 2-amino-2-deoxy-b-D-glucan), a deacetylated derivative from chitin, is an antimicrobial polysaccharide that exerts broad-spectrum activity against pathogenic bacteria and has been suggested as a preventive and therapeutic material for dental caries. AIM: The aim of this investigation is whether chitosan has effective antimicrobial and antibiofilm properties against common cariogenic microorganisms. MATERIALS AND METHODS: The effect of 0.019-5 mg/ml of high-molecular-weight (HMW) and low-molecular-weight (LMW) chitosan on S. mutans and S. sobrinus was evaluated, and minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) were determined. In addition, the effects of HMW and LMW of chitosan on bacterial adhesion to surfaces and biofilm formation were assayed by tube method. RESULTS: The results showed that chitosan is capable of inhibiting S. mutans and S. sobrinus growth (P = 0.001). MIC of HMW chitosan for S. mutans and S. sobrinus was 0.62 mg/mL and MIC of LMW chitosan for S. mutans and S. sobrinus was 0.62 mg/mL, 1.25 mg/mL, respectively. MBC of HMW chitosan for S. mutans and S. sobrinus was 1.25 mg/mL, respectively, and MBC of LMW chitosan for S. mutans and S. sobrinus was 1.25 and 2.5 mg/ml, respectively. On the other hand, HMW chitosan was more effective than LMW chitosan. In addition, S. mutans showed equal MIC and MBC values for both MWs chitosan, but S. sobrinus was more resistant to LMW chitosan. Regarding biofilm growth, chitosan inhibited S. mutans and S. sobrinus adhesion and biofilm formation. The results of tube test showed weak adherence and biofilm formation in concentration of 0.312 and 0.625 mg/ml, but 1.25 and 2.5 mg/ml concentrations of both MWs could completely inhibit biofilm formation. CONCLUSION: These results display the potential of chitosan to be used as an effective antibacterial and antibiofilm agent for oral hygiene and health care.

Association of interleukin-6 (rs1800796) but not transforming growth factor beta 1 (rs1800469) with serum calcium levels in osteoporotic patients.

BACKGROUND: Osteoporosis is a multifactorial disease with a strong genetic influence. Recent studies have demonstrated that cytokines, such as TGF-ß1 and interleukin 6 (IL-6) play complex roles in the normal bone metabolism and pathophysiology of osteoporosis. Here, we investigated the roles of 2 polymorphisms mapping to the promoters of TGF-ß1and IL-6 genes on the genetic susceptibility to osteoporosis as well as calcium and vitamin D levels. METHODS: A cohort of 297 elderly participants in northern Iran comprising 181 osteoporotic patients (mean age ±â€¯SD, 68.36 ±â€¯7.21 years) and 116 unrelated healthy controls (mean age ±â€¯SD, 64 ±â€¯5.44 years) was studied for TGF-ß1(C-509T) and IL-6 (G-634C) polymorphisms using PCR-RFLP method. RESULTS: A significant relationship was observed between calcium level and IL-6 genotypes in osteoporotic males (P = 0.011) and females (P = 0.020). No significant differences were observed between osteoporotic and control groups with respect to allele frequency or genotype distribution based on the 2 selected polymorphisms under different genetic models. The results remained the same after comparing the BMD values of either the femur neck or lumbar spine with the genotypes of the elderly men and women when analyzed separately. CONCLUSION: IL-6 genotype influences serum calcium levels in osteoporotic patients. The lack of association between the common genetic variations of TGF-ß1 and IL-6 genes, and BMD highlights the complex genetic background of osteoporosis in the north of Iran.

Protection of bone marrow, mononuclear, and CD34+ cells by enclosing within the biochemical compound solution during and after transplantation.

We have chosen collagen, chitosan acetate, hyaluronic acid, and propolis as model biochemical compound solution to determine the influence of cell carrier mechanics on cell viability and functionality during and after transplantation. Suspending of bone marrow (BM), mononuclear (MN), and CD34+ cells into a biochemical compounds solution is an attractive tool to achieve to protect and ensure reproducible deliver. Hyperglycemic rats were randomly divided into 2 groups: to receive no cell treatment or approximately 1 × 105 of BM, MN, and CD34+ cells within the PBS or biochemical compound solution. These cells were infused into the hyperglycemic rats on day 10 and again on day 20. At each time point, the animals were anaesthetized with ether, and 200 µL of blood was drawn from the tail vein. Samples were collected to determine whether BM, MN, and CD34+ cell affected glucose content and insulin production. Our results exhibit the use of biochemical compound solution method to overcome the cell transplantation problem during and after injection of these cells into rats. These findings are supported by resulting in significantly greater insulin production and more decreased glucose content than cells injected in PBS only (P < 0.05). These effects displayed the following hierarchy: hyaluronic acid > chitosan acetate > collagen > propolis solution. Our results showed that these compounds demonstrated a capacity to encapsulate the BM, MN, and CD34+ cells. It is proven by decreasing glucose content and increasing insulin secretion by pancreatic cells. The uniqueness of our study is the improvement of current transplantation efficiency.

Sustained small and intermediate size proteins expression in phorbol 12-myristate 13-acetate/ionomycine prolonged stimulated human fibroblasts

Objective:To compare the protein profile of culture supematants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status.Methods:Dcrmal fibroblasts were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycine for 72 h.MTT assay was done to determine cell viability and A/E fluorescent staining was used to evaluate the cell death pattern.Protein analysis was performed by gradient SDS polyacrylamide gel electrophoresis 8％-16％.Results:The supernatant of 24 h cultured both stimulated and unstimulated fibroblasts showed two bands in SDS-PAGE analysis with relative molecular weights of 8.59 and 78,8 kDa.These bands density was decreased during the next 48 h in unstimulated cells while their expression was continued in PMA or PMA/ionomycine stimulated cells and a new 85.3 kDa band was appeared in unstimulated and 72 h PMA stimulated cells.Moreover,we found another seven small size (10-19.5 kDa) proteins in supernatants of 48 h and 72 h unstimulated but not in PMA or PMA/lonomycine stimulated fibroblasts.Most of these proteins expression were down regulated following fibroblast activation.This down-regulation is consistent with our finding that PMA or PMA/ionomycine stimulated cells exhibited a significant level of apoptosis cell death.ConcLusions:Human fibroblasts produce some small to intermediate sized proteins with specific SDS-PAGE profile upon cell activation.Most of these proteins can be excreted in urine and can be immunogen theoretically so this data provided a reliable clue for fibrosis biomarker screening based on designation of an appropriated immunoassay.

Comparative in vitro study of the effectiveness of Green tea extract and common storage media on periodontal ligament fibroblast viability.

OBJECTIVE: Green tea extract (GTE) was shown to be effective in preserving periodontal ligament fibroblasts (PDLFs) of avulsed teeth. This study aimed at determining the potential of GTE in preserving the viability of PDLFs comparing with different storage media. MATERIALS AND METHODS: Periodontal ligament cells were obtained from freshly extracted healthy impacted third molars and cultured in Dulbecco's Modified Eagle Medium (DMEM). Cell viability was determined by storing the cells in seven media; DMEM, tap water, Hank's balanced salt solution (HBSS), whole milk, hypotonic sucrose solution, GTE, and GTE + sucrose for 1, 2, 4, and 24 h at 37°C using tetrazolium salt-based colorimetric (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay. Statistical analysis was performed by one-way analysis of variance and post hoc tests. RESULTS: GTE showed significantly higher protective effect than HBSS at 2, 4, and 24 h (P = 0.009, P = 0.02, P = 0.016), DMED at 2 h (P = 0.003), and milk at 4 h (P = 0.039). CONCLUSION: Although with undesirable osmolality and pH, GTE had a good ability in preserving the PDLFs comparing with other studied media.

Peripheral blood lymphocytes are able to maintain their viability and basic function in normal urine.

BACKGROUND: Similar to inflammatory cells, peripheral blood mononuclear cells (PBMCs) can also infiltrate in to kidney and urinary tracts and subsequently excreted by urine. In this study we determined the viability rate and response to phytohemagglutinin-A (PHA) of human PBMCs in normal urine. METHODS: A number of 1×10(6) ficoll-hypaque isolated PBMCs were dispensed in 1 ml normal urine and 6 molar urea and RPMI-1640+FBS10 % were considered as negative and positive control, respectively. After 20, 60 and 120 minutes the viability of these cells was measured by trypan blue dye exclusion assay. 1×10(5) of PBMCs were isolated from urine and cultured as triplicate in RPMI-1640`supplemented with FBS 10% and PHA for 96hr. MTT assay was performed to determine the PBMCs response to PHA. These experiments were repeated three times independently. RESULTS: There was no significant difference between the viability rates of the PBMCs incubated in urine and positive control after 20, 60 and 120 minutes. Overall, there was a significant difference in trends of viability rate across the three groups (p<0.05). CONCLUSION: Our results showed that not only PBMCs remained remarkably alive in urine after 120 minutes, but can also respond to PHA up to 60 minutes after incubation in urine. These data open a new avenue in the designation for cell culture-based techniques in urine cell analysis.

Anti-perinuclear Factor as Diagnostic Marker in Rheumatoid Arthritis.

INTRODUCTION: Rheumatoid Arthritis (RA) is a systemic autoimmune disease. It is associated with several auto antibodies which can serve as diagnostic and prognostic markers. AIM: In this study, Anti perinuclear Factor (APF) was evaluated as a biomarker in comparison with Rheumatoid Factor (RF) in Rheumatoid Arthritis. MATERIALS AND METHODS: Fifty two sera of patients with RA (mean age 48±15.8), 23 sera of Patient control group (mean age 32.5 ± 16.9) and 30 sera of Healthy control group (mean age 32.1± 16.9) were analysed. The method is based on the binding of APF to perinuclear keratohyalin granules of buccal mucosal cell and its detection using a fluorescently labeled anti human total antiserum. RESULTS: APF were found in 71.2 %(37/52) of patients with RA. The sensitivity and specificity for APF from 1/5 serum dilution was 71.2% and 94.3% respectively. RF test had higher sensitivity (88.5%) compare to the APF test (71.2%), but its specificity was (86.8%) less than APF (94.3%). There was no significant relationship between the onset of APF and severity of disease but there was significant relationship between the APF titer and severity of disease (p<0.05). CONCLUSION: It is concluded that APF test is a valuable serological tool for the diagnosis of the disease and a useful serological marker to differentiate from the other inflammatory rheumatoid diseases.

RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?

The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. Relatively, comparing and measuring some kinds of buffers (Trizol, TriPure) indicated no difference in RNA extraction quality. The last investigated factor was the effect of b- mercaptoethanol which was used along with TriPure to remove the RNAse. Conclusively, no effective impression was observed.