Substances Secreted by Starved Human Dermal Fibroblasts Enhancing the Wound Healing Process in Rat without Scar: A Potential Acellular System for Wound Healing

Background Despite being a major cellular component of various engineered skin substituent, underlying mechanism of successful fibroblast transplantation in wound healing is not clear. Here we show that substances derived from starved fibroblast accelerate wound healing process in rat. Material and methods Starved human fibroblast cell culture supernatant (SFS) was prepared and tested for its wound healing capacity on rat skin. Twelve Wistar adult male rats were randomized into four different groups of three. On the back of each rat two wounds were created with area of 452 mm2. Each wounds were treated daily with one milliliter of SFS or cell culture medium (DMEM). The size of the wounds was measured daily until crust formation. The animals were scarified on 4th, 8th, 11th and 15th day of experiment and skin was removed for H&E and trichrome staining. Infiltration of fibroblast and inflammatory cells and collagen formation were analyzed. Results The diameter of the wounds treated the SFS solution was significantly decreased within the first week of treatment, compared with control wound receiving DMEM only (

Interleukin 35 levels in saliva of type 2 diabetic patients with moderate chronic periodontitis

Introduction: Periodontitis is a common disease in patients with diabetes. There is a significant relationship between hyperglycemic degree and severity of periodontitis, but the base of mechanism of this relationship has not been fully defined. Considering the important role of cytokines in periodontal pathogenesis and considering that there has been no study on the comparison of interleukin 35 (IL-35) in these diseases, the aim of this study was to determine the level of this salivary cytokine in patients with type 2 diabetes mellitus with generalized moderate chronic periodontitis. Material & Methods: Totally, 88 subjects (44 female, 44 males) with a mean age of 42.5±10.5 years old participated in this case control study. The subjects were divided into four groups and each group included 22 subjects: Group 1: generalized moderate chronic periodontitis patients with type 2 diabetes, Group 2: generalized moderate chronic periodontitis patients without diabetes, Group 3: diabetic patients with normal periodontium, Group 4: healthy periodontium and non-diabetic group (control) Then saliva were collected and centrifuged, the amount of IL-35 was determined with commercial ELISA kit. Data were analyzed . ANOVA and Tukey post-hoc tests were used to compare the groups. Results: The Mean±SD of IL-35 was significantly higher in the control group (22.59±8.36, p0.05). Conclusion: The salivary IL-35 level is decreased in both periodontitis and type 2 diabetes. However, diabetes mellitus does not exacerbate this decline in patients with periodontitis

Systemic effects of starved fibroblasts culture supernatant on immunosuppressed rats treated with cancer stem cells

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

Ninety-six–hour starved peripheral blood mononuclear cell supernatant inhibited LA7 breast cancer stem cells induced tumor via reduction in angiogenesis and alternations in Gch1 and Spr expressions

Introduction The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats. Method Ninety-six–hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS. Results 96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro ( p < 0.05), whereas stemness gene Oct4 was upregulated ( p < 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group ( p = 0.007). In this group, Gch1 and Spr were significantly downregulated ( p < 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67 + cells) in the 96 h-SPS–treated group was significantly reduced ( p = 0.024). The increased rate of necrosis in this group was statistically significant ( p = 0.04). Last, proteomics analysis revealed candidate effectors’ components of 96 h-SPS solution. Conclusion 96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development

The assessment of cytotoxic T cell and natural killer cells activity in residents of high and ordinary background radiation areas of Ramsar-Iran

The effective radiation dose of human from natural sources is about 2.4 mSv/y and the dose limit for radiation workers is 20 mSv/y. Ramsar, a city in Iran, has been the subject of concern in the last forty years for a high level of radiation measured in some spots as high as 260 mSv/y. Carcinogenesis is one of the most studied effects of radiation especially in high doses. Recent studies showed that the high level of natural radiation received by inhabitants of this area, paradoxically don′t have significant health effect. Natural killer (NK) cells and cytotoxic T cells are the most important cells in tumor immune surveillance and CD107a is a widely expressed intracellular protein located in the lysosomal/endosomal membrane. CD107a transiently located on the cell membrane can be used as a marker of CD8 + T cell degranulation following stimulation. It is also expressed, to a lower extent, on activated NK cells. In this study, 60 healthy people were selected randomly and their consent obtained and confounding factors such as sex, age, life-styles was matched then the count of activated NK and CD8 + cells was compared in high and normal background radiation areas inhabitants of Ramsar. After filling the questionnaire and measurement of background radiation, blood samples of 30 healthy people from each region were analyzed immediately by means of flowcytometry. The leukocytes and their subsets were not significantly different between two groups and the count of active cells was higher in control group. The result shows that the changes in immune system occur due to radiation and maybe it is as a result of higher radiosensitivity of activated cells

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 supernatants in stimulated and unstimulated human fibroblasts to find some proteins indicating the presence of fibroblasts and their activation status. Methods: Dermal 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/Ionomycine 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

Body composition and serum levels of matrix metalloproteinase-9, adiponectin and AMP-activated protein kinase in breast cancer survivors

Background: Available data suggest that obesity is related to changes in the several adipocyte-derived proteins levels, which are involved in cancer recurrence. The purpose of this work was to investigate the correlation between obesity with metalloproteinase-9 (MMP-9), adiponectin and adiponectin and AMP-activated protein kinase (AMPK) levels by comparing serum levels of MMP-9, AMPK in normal weight and obese breast cancer survivors. Materials and Methods: In this cross-sectional study, 30 normal weight breast cancer survivors (body mass index [BMI] 18.5-25 kg/m2) and 30 obese breast cancer survivors (BMI ≥30 kg/m2) were investigated. Anthropometric parameters and serum levels of MMP-9, adiponectin, and AMPK were compared between the two groups. Results: No differences were detected in the serum levels of MMP-9, adiponectin, and AMPK in obese patients and normal weight patients (P > 0.05). There were no correlations between MMP-9, adiponectin, and AMPK levels with anthropometric measurements in two groups (P > 0.05). Conclusion: We found that there was a lack of correlation between obesity measures and serum levels of MMP-9, adiponectin, and AMPK. In breast cancer survivors, it seems that circulating levels of adiponectin, AMPK, and MMP-9 do not change in obesity state

Evaluation of the Level of Salivary sHLA-G in Children Aged 3–5 Years with or without Dental Caries

Aim and Background. Early childhood caries (ECC) is a common type of dental caries affecting children. As dental caries is a bacterial infectious disease, the host immune system parameters including soluble human leukocyte antigen-G (sHLA-G) are essential factors in estimating dental caries. The study aimed to investigate and compare the concentration of sHLA-G in the saliva of children with or without dental caries. Methods and Materials. This analytic cross-sectional study was carried out on 83 healthy children aged 3 to 5 years of both genders, who were divided into three groups based on decayed dental surfaces (ds): group 1, caries-free children (CF, n = 29); group 2, children with 1 ≤ ds ≤ 3, 1 ≤ ds ≤ 4, and 1 ≤ ds ≤ 5 for age 3, 4, and 5 years, respectively (ECC, n = 20); and group 3, children with ds ≥ 4, ds ≥ 5, and ds ≥ 6 for age 3, 4, and 5 years, respectively (S-ECC, n = 34). The unstimulated saliva samples were collected, and the salivary sHLA-G concentration was measured by the ELISA kit. The SPSS Statistics v17.0 software and Mann–Whitney, Kruskal–Wallis, chi-square, and Spearman’s rank correlation tests were used for statistical analysis. The level of significance was considered at p0.05). Spearman’s rank correlation test showed a weak positive correlation (p=0.039, r = 0.22), between the level of salivary sHLA-G and dental caries. Conclusion. The present study provides some preliminary evidences on relationship between sHLA-G and dental caries in children

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&times;106 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&times;105 of PBMCs were isolated from urine and cultured as triplicate in RPMI-1640`supplemented with FBS 10% and&nbsp; 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 while after 60 minutes they exhibited 75.6% of reactivity to PHA versus positive control. 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 alive in urine after 120 minutes, but can also respond to PHA up to 60 minutes at a remarkable level. These data open a new avenue in the designation for cell culture-based techniques in urine cell analysis