Epidemiology of sperm DNA fragmentation in a retrospective cohort of 1191 men.

BACKGROUND: The scientific and clinical communities now recognize that sperm DNA integrity is crucial for successful fertilization, good embryo development, and offspring quality of life. Despite the apparent unanimity, this criterion is rarely evaluated in clinical practice. We evaluated the sperm DNA fragmentation index of nearly 1200 sperm samples and its connections based on the patient's age, body mass index, the season of sperm collection, geographical location, medical history, and addictive behaviors. METHODS: A cohort of 1503 patients who were referred to the Royan Institute between July 2018 and March 2020 was examined. Only 1191 patient records with demographic data, complete semen analysis, and DNA fragmentation index measurements were included in the final cohort. Documents were classified, incorporated into statistical models, and analyzed. RESULTS: The results confirmed previous findings that the sperm DNA fragmentation index was significantly higher in aging men. The sperm DNA fragmentation index and high DNA stainability levels were significantly higher in spring and summer samples than in those of other seasons. No correlation was found between semen DNA fragmentation index and patient body mass index, although the study cohort was significantly overweight. Contrary to what might be expected, we observed that the sperm DNA fragmentation index was higher in rural than in urban patients. Intriguingly, epileptic patients exhibited significantly higher sperm DNA fragmentation index levels. DISCUSSION AND CONCLUSION: Age is the factor that is most strongly associated with sperm DNA fragmentation index levels. Our analysis of 1191 samples indicates that between the ages of 19 and 59, the sperm DNA fragmentation index increases by an average of 2% each year. Intriguingly, from an epidemiological perspective, the warm season (spring/summer) is associated with a higher sperm DNA fragmentation index in the study population, possibly due to the deleterious effect of temperature on sperm quality. Some neurological diseases, such as epilepsy, are associated with decreased sperm DNA integrity. This observation could be related to the iatrogenic effects of associated therapies. In the study cohort, body mass index did not appear to be correlated with the DNA fragmentation index.

Efficacy of adoptively transferred allogeneic CIK cells on colorectal cancer: Augmentative antitumoral effects of GvHD.

OBJECTIVE: A preclinical study was designed to evaluate the effects of adoptively transferred cytokine-induced killer (CIK) cells on colorectal adenocarcinoma. METHODS: Forty NOG mice bearing HT-29 xenograft tumors were developed and equally divided into 2 groups of treatment and control. The mice in the treatment group received cumulatively 40-60 × 106 CIK cells in four divided doses. RESULTS: Median tumor doubling times for HT-29 xenograft tumors in the treatment and control groups were found to be 8.98 and 4.32 days; respectively. The treatment resulted in tumor growth delay (TGD) of 52.5 %. CIK cell-induced log cell kill (LCK) was found to be 0.67, which implies reduction of 78.6 % of neoplastic colorectal cells. Median length of survival in the treated mice was significantly longer than controls (57 (41-63) vs 41 (31-57) days, P < 0.001). Mice in the treatment group experienced graft-versus-host disease (GvHD) from median of day 13th after the cell therapy. LCK and TGD significantly increased after emergence of GvHD. After necropsy, tumors of the treatment group contained high levels of human-originated CD3+, CD4+ and CD8+ cells and showed significantly lower mitotic counts (P < 0.001) and residual tumor scores (P = 0.005) than the controls (entirely negative for the mentioned CD markers). Ninety percent of the treated mice were found to be responding. CONCLUSIONS: Adoptive transfer of allogeneic CIK cells may be an efficient antitumoral therapy for colorectal cancer. Allogeneic CIK cell-mediated GvHD may contribute to amplification of graft-versus-tumor effects of the cellular therapy.

Silibinin exhibits anti-tumor effects in a breast cancer stem cell model by targeting stemness and induction of differentiation and apoptosis.

Introduction: Malignant breast cancer (BC) frequently contains a rare population of cells called cancer stem cells which underlie tumor relapse and metastasis, and targeting these cells may improve treatment options and outcomes for patients with BC. The aim of the present study was to determine the effect of silibinin on the self-renewal capacity, tumorgenicity, and metastatic potential of mammospheres. Methods: The effect of silibinin on viability and proliferation of MCF-7, MDA-MB-231 mammospheres, and MDA-MB-468 cell aggregation was determined after 72-120 hours of treatment. Colony and sphere formation ability, and the expression of stemness, differentiation, and epithelial-mesenchymal-transition (EMT)-associated genes were assessed by reverse transcription-quantitative polymerase chain reaction (qRT-PCR) in mammospheres treated with an IC50 dose of silibinin. Additionally, the antitumor capacity of silibinin was assessed in vivo, in mice. Results: The results of the present study showed that silibinin decreased the viability of all mammospheres derived from MCF-7, MDA-MB-231, and MDA-MB-468 cell aggregation in a dose-dependent manner. Colony and sphere-forming ability, as well as the expression of genes associated with EMT were reduced in mammospheres treated with silibinin. Additionally, the expression of genes associated with stemness and metastasis was also decreased and the expression of genes associated with differentiation were increased. Intra-tumoral injection of 2 mg/kg silibinin decreased tumor volumes in mice by 2.8 fold. Conclusion: The present study demonstrated that silibinin may have exerted its anti-tumor effects in BC by targeting the BC stem cells, reducing the tumorgenicity and metastasis. Therefore, silibinin may be a potential adjuvant for treatment of BC.

The epigenetic alterations of human sperm cells caused by heroin use disorder.

The molecular mechanisms of drug use on sexual health are largely unknown. We investigated, the relationship between heroin use disorder and epigenetic factors influencing histone acetylation in sperm cells. The volunteers included twenty-four 20- to 50-year-old men with a normal spermogram who did not consume any drugs and twenty-four age- to BMI-matched men who consume only the drug heroin for more than last four months. HDAC1 and HDAC11 mRNA expression levels in spermatozoa and miR-34c-5p and miR-125b-5p expression levels in seminal plasma were measured. The heroin-user group showed significantly increased white blood cell counts and decreased sperm motility and survival rates (8.61 ± 1.73, 21.50 ± 3.11, 69.90 ± 4.69 respectively) as compared to the control group (1.49 ± 0.32, 38.82 ± 3.05, 87.50 ± 0.99 respectively) (p ≤ .001). An increase in DNA fragmentation index (DFI) (heroin-user group: 41.93 ± 6.59% and control group: 10.14 ± 1.43%, p = .003), a change in frequency of HDAC1 (heroin-user group: 1.69 ± 0.55 and control group: 0.45 ± 0.14, p = .045) and HDAC11 (heroin-user group: 0.29 ± 0.13 and control group: 2.36 ± 0.76, p = .019) in spermatozoa and a significant decrease in seminal miR-125b-5p abundance (heroin-user group: 0.37 ± 0.11 and control group: 1.59 ± 0.47, p = .028) were reported in heroin consumers. Heroin use can lead to male infertility by causing leukocytospermia, asthenozoospermia, DFI elevation in sperm cells and alterations in seminal RNA profile.

GFc7 as a Smart Growth Nanofactor for ex vivo Expansion and Cryoprotection of Humans' Hematopoietic Stem Cells.

BACKGROUND: Nowadays, smart synthesized nanostructures have attracted wide attention in the field of stem cell nanotechnology due to their effect on different properties of stem cells. METHODS: GFc7 growth nanofactor was synthesized based on nanochelating technology as an iron-containing copper chelator nanocomplex. The effect of this nanocomplex on the expansion and differentiation of hematopoietic stem cells (HSCs) as well as its performance as a cryoprotectant was evaluated in the present study. RESULTS: The results showed that the absolute count of CD34+ and CD34+CD38- cells on days 4, 7, 10 and 13; the percentage of lactate dehydrogenase enzyme on the same days and CD34+CXCR4 population on day 10 were significantly increased when they were treated with GFc7 growth nanofactor in a fetal bovine serum (FBS)-free medium. This medium also led to delayed differentiation in HSCs. One noticeable result was that CD34+CD38- cells cultured in an FBS medium were immediately differentiated into CD34+CD38+ cells, while CD34+CD38- cells treated with GFc7 growth nanofactor in FBS medium did not show such an immediate significant differentiation. De-freezing GFc7-treated CD34+ cells, which were already frozen according to cord blood bank protocols, showed a higher percentage of cell viability and a larger number of colonies according to colony-forming cell assay as compared to control. CONCLUSION: It can be claimed that treating HSCs with GFc7 growth nanofactor leads to quality and quantity improvement of HSCs, both in terms of expansion in vitro and freezing and de-freezing processes.

Suppression of p38-MAPK endows endoderm propensity to human embryonic stem cells.

The ability of human embryonic stem cells (hESCs) to proliferate unlimitedly and give rise to all tissues makes these cells a promising source for cell replacement therapies. To realize the full potential of hESCs in cell therapy, it is necessary to interrogate regulatory pathways that influence hESC maintenance and commitment. Here, we reveal that pharmacological attenuation of p38 mitogen-activated protein kinase (p38-MAPK) in hESCs concomitantly augments some characteristics associated with pluripotency and the expressions of early lineage markers. Moreover, this blockage capacitates hESCs to differentiate towards an endoderm lineage at the expense of other lineages upon spontaneous hESC differentiation. Notably, hESCs pre-treated with p38-MAPK inhibitor exhibit significantly improved pancreatic progenitor directed differentiation. Together, our findings suggest a new approach to the robust endoderm differentiation of hESCs and potentially enables the facile derivation of various endoderm-derived lineages such as pancreatic cells.

Exosomes: Effectual players in rheumatoid arthritis.

Rheumatoid arthritis is a well-known chronic inflammatory joint disorder. It encompasses systemic inflammation, autoimmunity and development of several joint abnormalities leading to the lifelong disability and increased mortality. Exosomes are nano-sized (30-100 nm) mammalian extracellular particles with essential properties to regulate biological processes and cellular signaling by transferring protein and genetic materials. Understanding the diversity in the exosomal contents and their corresponding targets may contribute to better recognition of the processes that are implicated in the development and progression of diseases such as autoimmune disorders. Exosomes may act as a potential biomarker for the diagnosis of autoimmune disorders. In the present review, we aimed to bring together the relevant evidence on the biology of exosomes in rheumatoid arthritis, and also discuss the recent findings regarding the diagnostic, prognostic and therapeutic promise of these nanoparticles.

Suppression of c-Myc using 10058-F4 exerts caspase-3-dependent apoptosis and intensifies the antileukemic effect of vincristine in pre-B acute lymphoblastic leukemia cells.

Despite an old history behind the identification of the leading role of c-Myc in leukemogenesis, the road to constructing a therapeutic perspective for this molecule in acute lymphoblastic leukemia (ALL) is yet mesmerizing. This study was designed to provide a better outlook for the anticancer property of 10058-F4, an appealing inhibitor of c-Myc, in pre-B ALL cell lines either in the context of monotherapy or in combination with chemotherapeutic drugs. Our results declared that abrogation of c-Myc decreased the proliferative capacity of pre-B ALL-derived cells through halting the transition of the cells from G1 phase, and reducing the replicative potential of both REH and Nalm-6 cells, at least partly, through c-Myc-mediated suppression of human telomerase reverse transcriptase. Moreover, 10058-F4 potently induced a caspase-3-dependent apoptosis in pre-B ALL cells via shifting the balance between pro- and anti-apoptotic target genes. Although the inhibition of PI3Kδ using Idelalisib upregulated the messenger RNA expression of autophagy-related genes in 10058-F4-treated cells, treatment with autophagy inhibitor chloroquine decreased viability of the cells, either as a single agent or in combination with Idelalisib and/or 10058-F4; suggesting that the activation of autophagy in pre-B ALL cells could blunt apoptotic events and attenuate anticancer effect of both c-Myc and PI3K inhibitors. Finally, the results of our synergistic experiments delineated that 10058-F4 produced a synergistic effect with vincristine and provided an enhanced therapeutic efficacy in ALL cells, highlighting that c-Myc oncoprotein could be a bona fide target for the treatment of ALL.

Generation of CD19-Targeted Chimeric Antigen Receptor T Cells.

BACKGROUND: Current advancements in the field of chimeric antigen receptor (CAR) therapy, particularly U.S. FDA approval of Kymriah and Yescarta, heralds a new era of cancer treatment. This rapid progress in technology has urged more countries and institutions to keep pace with the fast-growing and developing technology of producing CAR T cell-based therapies in the race to develop new cancer-targeting drugs. Hence, for stepping in line with global advances and to pave the way for subsequent preclinical and clinical studies, we have established a development protocol for a cancer-targeting CAR T cell; we have chosen CD19 CAR T cell as a well-defined model to set-up T cell expansion, activation, and viral transduction as the prerequisites for diverse CAR T cell therapies. METHODS: T cells from peripheral blood mononuclear cells (PBMCs) were activated and expanded. CD19 CAR lentiviral particles were produced in the Lenti-X™ 293T Cell Line using PolyFect Transfection Reagent. RESULTS: Activation protocol resulted in (65 ± 4%; P = 0.046) increase in the rate of activated T cells 24 hours after the initiation of the procedure. The expansion methodology resulted in a high purity of the T cell population (96 ± 3%) in the pool of PBMCs within 14 days of the procedure. Finally, 35 ± 6% of T cells were transduced with CD19 lentivirus with MOI of 3. CONCLUSION: Collectively, the results of this study prove that we have successfully overcome the first hurdle on the road to reach CAR T cell technology which is the prerequisite for developing preclinical and clinical phases of CAR therapy in settings with basic resources.

Differential Frequency of CD8+ T Cell Subsets in Multiple Sclerosis Patients with Various Clinical Patterns.

Recent evidence points to a pathogenic role for CD8+ cytotoxic T (Tc) cells in Multiple sclerosis (MS). Based on cytokine profile, Tc cells can be divided into different subsets: IFN-γ (Tc1), IL-4 (Tc2), IL-10 (Tc10), IL-17 (Tc17), IL-21 (Tc21), IL-22 (Tc22) and TNF-α producing cells. In this study we evaluated the frequency of Tc cell subsets and the serum level of Tc17 differentiation cytokines in MS patients with different clinical patterns. We analyzed Tc cell subsets percentage in peripheral blood of relapsing-remitting (RRMS) (n = 28), secondary-progressive (SPMS) (n = 10) and primary-progressive (PPMS) (n = 4) MS patients in comparison to healthy controls (n = 15) using flow cytometry. Serum level of TGF-ß, IL-6 and IL-23 were measured by ELISA. We showed elevated levels of Tc1 and Tc17 cells in SPMS and RRMS patients in relapse phase, respectively (P = 0.04). Interestingly, the percentage of TNF-α producing CD8+ T cells in relapse and remission phase of RRMS and SPMS patients were higher than controls (P = 0.01, P = 0.004, P = 0.01, respectively) and Tc21 increased in remission phase of RRMS compared to SPMS (P = 0.03). We also found higher frequency of CD8+ IFN-γ+ TNF-α+ IL-17+ T cells in relapse phase of RRMS compared to remission phase, SPMS patients and controls (P = 0.01, P = 0.004 and P = 0.02, respectively). TGF- ß increased in sera of RRMS patients in remission phase (P = 0.03) and SPMS (P = 0.05) compared to healthy subjects. Increased level of Tc17 and CD8+ IFN-γ+ TNF-α+ IL-17+ T cells in relapse phase highlights the critical role of IL-17 in RRMS pathogenesis.

BCc1, the novel antineoplastic nanocomplex, showed potent anticancer effects in vitro and in vivo.

PURPOSE: In spite of all the efforts and researches on anticancer therapeutics, an absolute treatment is still a myth. Therefore, it is necessary to utilize novel technologies in order to synthesize smart multifunctional structures. In this study, for the first time, we have evaluated the anticancer effects of BCc1 nanocomplex by vitro and in vivo studies, which is designed based on the novel nanochelating technology. METHODS: Human breast adenocarcinoma cell line (MCF-7) and mouse embryonic fibroblasts were used for the in vitro study. Antioxidant potential, cell toxicity, apoptosis induction, and CD44 and CD24 protein expression were evaluated after treatment of cells with different concentrations of BCc1 nanocomplex. For the in vivo study, mammary tumor-bearing female Balb/c mice were treated with different doses of BCc1 and their effects on tumor growth rate and survival were evaluated. RESULTS: BCc1 decreased CD44 protein expression and increased CD24 protein expression. It induced MCF-7 cell apoptosis but at the same concentrations did not have negative effects on mouse embryonic fibroblasts viability and protected them against oxidative stress. Treatment with nanocomplex increased survival and reduced the tumor size growth in breast cancer-bearing balb/c mice. CONCLUSION: These results demonstrate that BCc1 has the capacity to be assessed as a new anticancer agent in complementary studies.

Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study.

PURPOSE: The aim of this study was to evaluate the conventional sperm parameters, level of intracellular reactive oxygen species (ROS), DNA fragmentation (DF) and dysfunction of mitochondrial membrane potential (MMP) after semen preparation techniques with flow cytometry. METHODS: Semen samples were obtained from 28 men with normal semen analysis according to WHO (world health organization). Each was divided into three equal parts for processing with routine techniques: conventional swim up (CSW), direct swim up (DSW) and density gradient centrifugation (DGC). The conventional sperm parameters were evaluated with computer-assisted sperm analyzer (CASA) and the level of intracellular ROS, dysfunction of MMP and DF were determined with flow cytometry procedure. RESULTS: Conventional sperm parameters such as motility, progressive motility and normal morphology increased after sperm processing by CSW and DGC compared to DSW. A significant increase in intracellular H2O2 (p < 0.05) was demonstrated in the CSW versus DSW technique, while processed sperm by the DSW procedure showed a significant increase in the percentage of dysfunction of MMP and intracellular O2(•-) (p < 0.05) when compared with CSW and DGC techniques. Additionally, a high mean of DF (p < 0.05) was observed in the DGC technique as compared to CSW. CONCLUSION: Data from flow cytometry study demonstrated that intracellular H2O2 and DF increased after CSW and DGC processing techniques, respectively, whereas the level of intracellular O2(•-) and dysfunction of MMP only increased after the DSW processing technique.

Isolation, characterization, and transplantation of bone marrow-derived cell components with hematopoietic stem cell niche properties.

Although the unique role of hematopoietic stem cell (HSC) niche in hematopoiesis has long been recognized, unsuccessful isolation of intact niche units limited their in vitro study, manipulation, and therapeutic application. Here, we isolated cell complexes based on size fractionation from mouse bone marrow (BM), characterized the derived cells, and transplanted them to irradiated mice. These cell complexes were the origin of both BM mesenchymal stem cells and various hematopoietic lineages when kept in appropriate culture conditions. They also had the potential of recruiting circulating HSC. Intraperitoneal transplantation of these structures into irradiated mice not only showed long-lasting hematopoietic multilineage reconstitution, but also could recover the stromal cells of BM. In conclusion, this study for the first time provides evidences on the feasibility and efficacy of transplantation of HSC in association with their native specialized microenvironment. As the molecular cross-talk between HSC and niche is crucial for their proper function, the proposed method could be considered as a novel hematopoietic transplantation strategy.

Effect of EGF and FGF on the expansion properties of human umbilical cord mesenchymal cells.

Mesenchymal stem cells have been increasingly introduced to have great potential in regenerative medicine, immunotherapy, and gene therapy due to their unique properties of self-renewal and differentiation into multiple cell lineages. Studies have shown that these properties may be limited and changed by senescence-associated growth arrest under different culture conditions. This study aimed to present the ability of some growth factors on human umbilical cord mesenchymal (hUCM) cells expansion and telomerase activity. To optimize hUCM cell growth, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were utilized in culture media, and the ability of these growth factors on the expression of the telomerase reverse transcriptase (TERT) gene and cell cycle phases was investigated. TERT mRNA expression increased in the hUCM cells treated by EGF and FGF. So, the untreated hUCM cells expressed 30.49 ± 7.15% of TERT, while EGF-treated cells expressed 51.82 ± 12.96% and FGF-treated cells expressed 33.77 ± 11.55% of TERT. Exposure of hUCM cells to EGF or FGF also promoted the progression of cells from G1 to S phase of the cell cycle and induced them to decrease the number of cells entering the G2/M phase. Our study showed that EGF and, to a lesser extent, FGF amplify the proliferation and expansion of hUCM cells.

Effect of lithium chloride and antineoplastic drugs on survival and cell cycle of androgen-dependent prostate cancer LNCap cells.

OBJECTIVE: Glycogen synthase kinase-3ß (GSK-3ß) has been reported to be required for androgen receptor (AR) activity. This study sought to determine the usefulness of lithium chloride (LiCl) as a highly selective inhibitor of GSK-3ß to increase the sensitivity of LNCap cells to doxorubicin (Dox), etoposide (Eto), and vinblastine (Vin) drugs. MATERIALS AND METHODS: Thiazolyl Blue Tetrazolium Blue (MTT) assay was used to determine the cytotoxic effect to LiCl alone or in combination with low dose and IC50 doses of drugs. Subsequently, cell cycle analysis was performed by using flow cytometry. RESULTS: LiCl showed cytotoxic effect in a dose- and time-dependent manner (P<0.001). Both Dox (100 or 280 nM) and Vin IC50 (5 nM) doses caused G2/M-phase arrest (P<0.001) compared with control. However, low dose (10 µM) or IC50 (70 µM) Eto doses showed G2/M or S-phase arrests, respectively (P<0.001). Combination of low dose or IC50 dose of Eto with LiCl showed increased apoptosis as revealed by high percent of cells in SubG1 (P<0.05, P<0.01, respectively). Moreover, Eto (10 µM) led to decreased percent of cells in G2/M phase when combined with LiCl (P<0.05). CONCLUSION: This study showed that LiCl increases apoptosis of (LNCap) Lymph Node Carcinoma of the Prostate cells in the presence of Eto, which is S- and G2-phase-specific drug.

A non-rotational, computer-controlled suspension bioreactor for expansion of umbilical cord blood mononuclear cells.

The proliferation and differentiation characteristics of umbilical cord blood mononuclear cells were examined in a non-rotational suspension bioreactor with a fishtail mixer. The system consisted of a glass vessel, a mixer that moved vertically, a data acquisition and control system to continuously monitor pH, temperature and dissolved O(2). The bioreactor provided superior expansion of total HSCs and not total cell number, as well as expression of stemness-related genes which followed with increasing in number of colony-forming cells during 14 days of culture compared to T -lask culture. Vertical agitation thus reduces the total cell number, which may be related to increased shear stress, but has no effect on HSC function.

Effect of culture media on expansion properties of human umbilical cord matrix-derived mesenchymal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been isolated from a number of different tissues, including umbilical cord. Because of the lack of a uniform approach to human umbilical cord matrix-derived mesenchymal (hUCM) cell expansion, we attempted to identify the optimum conditions for the production of a high quantity of hUCM cells by comparing two media. METHODS: We compared the ability of Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and Alpha Minimum Essential Medium (α-MEM) with Glutamax (GL) (α-MEM/GL) to expand hUCM cells. For this purpose, hUCM cells were cultured in plates containing different culture media supplemented with 10% fetal bovine serum (FBS). Culture dishes were left undisturbed for 10-14 days to allow propagation of the newly formed hUCM cells. The expansion properties, CD marker expression, differentiation potential, population doubling time (PDT) and cell activity were compared between the two groups. RESULTS: The hUCM cells harvested from each group were positive for MSC markers, including CD44, CD90 and CD105, while they were negative for the hematopoietic cell surface marker CD34. Differentiation into adipogenic and osteogenic lineages was confirmed for both treatments. Cell activity was higher in the α-MEM/GL group than the DMEM/F12 group. PDT was calculated to be 60 h for the DMEM/F12 group, while for the α-MEM/GL group it was 47 h. CONCLUSIONS: Our data reveal that α-MEM/GL with 10% FBS supports hUCM cell growth more strongly than DMEM/F12 with 10% FBS.

Comparison of different methods for the isolation of mesenchymal stem cells from human umbilical cord Wharton's jelly.

Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.

Expansion of CD4+ CD25+ FoxP3+ regulatory T cells in chronic hepatitis C virus infection.

BACKGROUND: Regulatory T cells (Tregs) have been involved in impaired immunity and may have a pivotal role in persistence of viral infections. OBJECTIVE: To develop a simple and reliable in-house three color flow cytometery of peripheral blood to understand the role of HCV infection in the increase of Tregs. METHODS: The level of naturally occurring CD4+ CD25+ FoxP3+ regulatory T cells (nTregs) in 20 chronically infected with hepatitis C virus (HCV) patients was compared to those of 15 healthy individuals by flowcytometry. In a different approach we performed permeabilization and intracellular staining before surface staining which allows the preservation of the surface molecules in the combined detection process and results in the normal frequency of nTregs in blood. RESULTS: Using the optimized method, it was shown that a significantly higher proportion of nTregs in the total CD4+ T cell population was seen in the peripheral blood of chronic HCV patients (0.83 ± 0.21%, p=0.05) as compared to controls (0.26 ± 0.1, p=0.05). CONCLUSIONS: In accordance with other studies, we showed that HCV infection induces a dramatic increase in Tregs, which might contribute to the immune response failure during HCV infection.

Evaluation of Fas positive sperm and complement mediated lysis in subfertile individuals.

PURPOSE: Evaluation of Fas receptor on surface of sperm, as an apoptotic marker, using flow cytometry and confirming the results using an antibody-antigen complex through the classic complement pathway. MATERIALS AND METHODS: Semen samples were obtained from 10 fertile and 73 infertile individuals with diagnoses of male factor infertility. Expressions of Fas receptor and phosphatidyl serine on sperm were assessed by flow cytometry. Fas expression was further assessed by antibody-antigen complex through the complement pathway. Lysis was detected via PI (propidium iodide) staining. RESULTS: The mean Fas expression was considerably lower than previously reported values. No significant differences in the percentage of PI were detected before and after activation of the classic complement pathway. Annexin V positive samples showed low Fas expression. CONCLUSION: Our results have confirmed the presence of selected apoptotic markers such as Fas or phosphatidyl serine on ejaculate sperm, but suggest that Fas expression is low. Further studies are required to investigate the "abortive apoptosis" mechanism through Fas/Fas L.