PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors.

HOX and TALE transcription factors are important regulators of development and homeostasis in determining cellular identity. Deregulation of this process may drive cancer progression. The aim of this study was to investigate the expression of these transcription factors in the bone marrow derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia (FA) patients, which is a cancer-predisposing disease. Expression levels of HOX and TALE genes in BM-MSCs were obtained from FA patients and healthy donors by RT-qPCR and highly conserved expression levels were observed between patient and donor cells, except PKNOX2, which is a member of TALE class. PKNOX2 was significantly downregulated in FA cells compared to donors (P < 0.05). PKNOX2 expression levels did not change with diepoxybutane (DEB), a DNA crosslinking agent, in either donor or FA cells except one patient's with a truncation mutation of FANCA. A difference of PKNOX2 protein level was not obtained between FA patient and donor BM-MSCs by western blot analysis. When human TGF-ß1 (rTGF-ß1) recombinant protein was provided to the cultures, PKNOX2 as well as TGF-ß1 expression increased both in FA and donor BM-MSCs in a dose dependent manner. 5 ng/mL rTGF-ß stimulation had more dominant effect on the gene expression of donor BM-MSCs compared to FA cells. Decreased PKNOX2 expression in FA BM-MSCs may provide new insights into the molecular pathophysiology of the disease and TGF-ß1 levels of the microenvironment may be the cause of PKNOX2 downregulation.

Dual Effect of Glucocorticoid-Induced Tumor Necrosis Factor-Related Receptor Ligand Carrying Mesenchymal Stromal Cells on Small Cell Lung Cancer: A Preliminary in vitro Study.

BACKGROUND AIMS: TNFR family member glucocorticoid-induced tumor necrosis factor-related receptor (GITR/TNFRSF18) activation by its ligand glucocorticoid-induced TNF-related receptor ligand (GITRL) have important roles in proliferation, death and differentiation of cells. Some types of small cell lung cancers (SCLCs) express GITR. Because mesenchymal stromal cells (MSCs) may target tumor cells, we aimed to investigate the effect of MSCs carrying GITRL overexpressing plasmid on the proliferation and viability of a GITR+ SCLC cell line (SCLC-21H) compared with a GITR- SCLC cell line (NCI-H82). METHODS: Electroporation was used to transfer pGITRL (GITRL gene carrying plasmid) or pCR3 (mock plasmid) into MSCs. Flow cytometry and semi-quantitative polymerase chain reaction were used to characterize the transfected MSCs. Following SCLC-21H or NCI-H82 cell lines were co-cultured with pGITRL-MSCs. RESULTS: Proliferation of NCI-H82 was increased in all types of co-cultures while SCLC-21H cells did not. GITRL expressing MSCs were able to induce cell death of SCLC-21H through the upregulation of SIVA1 apoptosis inducing factor. CONCLUSIONS: The influence of MSCs on SCLC cells can vary according to the cancer cell subtypes as obtained in SCLC-21H and NCI-H82 and enabling GITR-GITRL interaction can induce cell death of SCLC cell lines.

The frequency of Familial Mediterranean fever gene mutations and genotypes at Kirikkale and comparison with the mean of regional MEFV mutation frequency of Turkey.

In this study we have retrospectively analysed the mutation spectrum of the 351 Familial Mediterranean fever patients referred to Kirikkale University Faculty of Medicine, Department of Medical Genetics Laboratory over a period of 5 years and compared them with Turkey's mean. We have found 11 different mutations, including rare mutations such as F479L, K695R, M680I(G/A) and 45 different genotypes showing the heterogeneity of MEFV mutations in Central Anatolia. The most three prevalent mutations were M694V (14.8%), E148Q (7.1%) and M680I(G/C) (4.1%) in accordance with the literature. We have also investigated R202Q in our routine molecular diagnosis. Mutation causing R202Q (c.605G > A) change was described as a frequent polymorphism and G allele was found in linkage disequilibrium (LD) with M694V. There are limited number of studies investigating R202Q, some of them implicate that its homozygote state is disease causing. We showed the high frequency of R202Q (23.7%) with and without M694V in all the groups analysed and its high LD rate with M694V in the diagnosed group. Our study is reflecting the mutational heterogeneity of MEFV and summarize mutational spectrum of Turkey's geographical regions and overall Turkey.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Hepatocyte Growth Factor and Insulin-like Growth Factor-1 based Cellular Therapies for Oxidative Stress Injury.

Imbalance between free radicals and antioxidants causes oxidative stress by the accumulation of reactive oxygen species (ROS) in the tissues and organs. Oxidative stress occurs in many damaged conditions, and the increase of ROS and reduction of antioxidants enhances inflammation, apoptosis, fibrosis and may worsen the pathology leading to organ failure. The potential therapies aim to increase antioxidants and decrease ROS. Mesenchymal stem cells (MSCs) isolated from the stroma of various tissues are multipotent cells and have beneficial effects on several diseases with their immunomodulation and regeneration capacities. MSCs trigger the proliferation of the cells with various secretory factors, reduce oxidative stress and decrease apoptosis, inflammation, fibrosis and thus, increase regeneration. However, survival, engraftment, and differentiation problems of transplanted MSCs restrict their protective and regenerative effects. Preconditioning of MSCs with several factors, such as cytokines, hypoxia, chemical agents, pharmacological drugs, physical factors and growth factors, enhances their repairing efficacy for injury and disease models. This review is mainly focused on insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF), and discusses the research on MSC priming/induction with IGF-1 and HGF stimulation either by supplementation or overexpression that can enhance the regenerative potential of MSCs on various oxidative stress conditions such as acute/chronic kidney diseases, lung injury, cancer, metabolic and cardiovascular diseases.

Development, characterization, and hematopoietic differentiation of Griscelli syndrome type 2 induced pluripotent stem cells.

BACKGROUND: Griscelli syndrome type 2 (GS-2) is a rare, autosomal recessive immune deficiency syndrome caused by a mutation in the RAB27A gene, which results in the absence of a protein involved in vesicle trafficking and consequent loss of function of in particular cytotoxic T and NK cells. Induced pluripotent stem cells (iPSC) express genes associated with pluripotency, have the capacity for infinite expansion, and can differentiate into cells from all three germ layers. They can be induced using integrative or non-integrative systems for transfer of the Oct4, Sox2, Klf4, and cMyc (OSKM) transcription factors. To better understand the pathophysiology of GS-2 and to test novel treatment options, there is a need for an in vitro model of GS-2. METHODS: Here, we generated iPSCs from 3 different GS-2 patients using lentiviral vectors. The iPSCs were characterized using flow cytometry and RT-PCR and tested for the expression of pluripotency markers. In vivo differentiation to cells from all three germlines was tested using a teratoma assay. In vitro differentiation of GS-2 iPSCs into hematopoietic stem and progenitor cells was done using Op9 feeder layers and specified media. RESULTS: All GS-2 iPSC clones displayed a normal karyotype (46XX or 46XY) and were shown to express the same RAB27A gene mutation that was present in the original somatic donor cells. GS-2 iPSCs expressed SSEA1, SSEA4, TRA-1-60, TRA-1-81, and OCT4 proteins, and SOX2, NANOG, and OCT4 expression were confirmed by RT-PCR. Differentiation capacity into cells from all three germ layers was confirmed using the teratoma assay. GS-2 iPSCs showed the capacity to differentiate into cells of the hematopoietic lineage. CONCLUSIONS: Using the lentiviral transfer of OSKM, we were able to generate different iPSC clones from 3 GS-2 patients. These cells can be used in future studies for the development of novel treatment options and to study the pathophysiology of GS-2 disease.

Hereditary haemochromatosis gene (HFE) H63D mutation shows an association with abnormal sperm motility.

The aim of this study was to screen infertile men for HFE H63D mutation in correlation with clinical characteristics of infertile men (sperm concentration, sperm motility, morphology, testicular volume, Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and total Testosterone levels) and find out if the HFE H63D mutation has an effect on male infertility. After excluding hormonal treatment, any scrotal pathology, having any systemic diseases such as diabetes mellitus, sickle cell anemia and microdeletions of the Y chromosome, a total of 148 infertile men with age range between 17 and 52-years-old (average age 29.6 +/- 7.2) were enrolled into the study. Our analysis indicates that the mean FSH levels are significantly higher (6.3 +/- 4.6 mIU/ml, P = 0.03), whereas sperm motility is significantly lower (36.6 +/- 28.1%, P = 0.01) in the infertile men with the HFE H63D mutation compared with subjects lacking this mutation. Comparison of allele frequencies of the infertile men with Ts < 50% versus the infertile men with Ts > 50% revealed a significant difference as expected (P = 0.001, OR = 0.14, %95 CI = 0.04-0.44). Comparison of allele frequencies of infertile men with abnormal sperm motility versus infertile men with normal sperm motility revealed a highly significant difference (P = 0.005, OR = 3.11, %95 CI = 1.41-6.86). Thus, the HFE H63D mutation seems to be an important risk factor for impaired sperm motility and is clinically associated with male infertility.

Bone Marrow Mesenchymal Stem Cells Carrying FANCD2 Mutation Differ from the Other Fanconi Anemia Complementation Groups in Terms of TGF-ß1 Production.

Transforming growth factor beta (TGF-ß) secretion from cells in the bone marrow (BM) niche affects hematopoietic stem cell (HSC) fate and has a cardinal role in HSC quiescence. BM mesenchymal stem cells (BM-MSCs), a component of the BM niche, may produce abnormal levels of TGF-ß in Fanconi anemia (FA) and may play a role in bone marrow failure. Here, we molecularly and cellularly characterized FA BM-MSCs by addressing their immunophenotype, proliferation- and differentiation- capacity, reactive oxygen species (ROS) production, senescence activity as well as expression and secretion levels of TGF-ß isoforms. In ten FA patients, mutations were detected in FANCA (n = 7), FANCG (n = 1) and FANCD2 (n = 2) genes. The immunophenotype, with the exception of CD29, and differentiation capacity of FA BM-MSCs were similar to healthy donors. FA BM-MSCs showed decreased proliferation, increased ROS level and an arrest in G2 following DEB treatment. ß-galactosidase staining indicated elevated senescence of FANCD2-deficient cells. FA BM-MSCs displayed TGF-ß1 mRNA levels similar to donor BM-MSCs, and was not affected by DEB treatment. However, secretion of TGF-ß was absent in FA-D2 BM-MSCs. Absence of TGF-ß secretion may be related to early onset of senescence of the FANCD2-deficient BM-MSCs. The proliferative response of FA-D2 BM-MSCs to rTGF-ß1 was not different from FANCA-deficient and donor cells and raises the possibility that rTGF-ß1 may reverse the senescence of the FANCD2-deficient BM-MSCs which needs to be investigated further.

From Stem Cell Biology to The Treatment of Lung Diseases.

BACKGROUND: The exposure of lung to noxious agents or gasses leads to injury, which further enhances repair mechanisms by promoting the proliferation and differentiation of lung stem cells. These cells could help preserve the anatomical structure and the function of the organ. Unfortunately in many lung diseases, 'this scenario' is changed and injury progresses despite repair mechanisms or conventional treatment. OBJECTIVE: This review summarizes the research on lung stem cells by giving an overview of the biology, function, niches and signaling that play role in lung stem cells and further of the regeneration of the lung. It also highlights the most common lung pathologies thought to be a result of a defective remodeling and overviews the clinical trials having results or publications, which are performed on the field. CONCLUSION: Though not yet approved for clinical usage, the application of stem cell therapies shown to be safe and with minimal adverse effects could be an alternative treatment to many lung diseases giving a hope for the future of severely ill patients refractory to the current therapies.

HFE H63D mutation frequency shows an increase in Turkish women with breast cancer.

BACKGROUND: The hereditary hemochromatosis gene HFE plays a pivotal role in iron homeostasis. The association between cancer and HFE hetero- or homozygosity has previously been shown including hepatocellular and nonhepatocellular malignancies. This study was performed to compare frequencies of HFE C282Y and H63D variants in Turkish women with breast cancer and healthy controls. METHODS: Archived DNA samples of Hacettepe University Oncology Institute were used in this study. The HFE gene was investigated by PCR-RFLP. RESULTS: All subjects studied were free from C282Y mutation. Thirty-nine patients had H63D mutation and were all heterozygous. H63D allele frequency was 22.2% (39/176) in the breast cancer patients, and 14% (28/200) in the healthy volunteers. Statistical analysis of cases with HFE H63D phenotype showed significant difference between breast cancer and healthy volunteers (P = 0.02). CONCLUSION: Our results suggest that HFE H63D mutation frequencies were increased in the breast cancer patients in comparison to those in the general population. Also, odds ratios (odds ratio = 2.05) computed in this study suggest that H63D has a positive association with breast cancer.

Tenascin-R: role in the central nervous system.

Tenascin-R (TN-R), a member of the tenascin family of extracellular matrix glycoproteins, is exclusive to the nervous system. It affects cell migration, adhesion and differentiation, although no remarkable clinical consequences have been shown in knock-out animal models. TN-R's expression pattern suggests a possible primary or secondary role in certain neurological problems including malformations, tumors and neurodegenerative disorders. This review summarizes the structure and molecular interactions of this molecule and discusses its function and possible roles in the central nervous system.

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene.

Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476-G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.

Primary tumor cells obtained from MNU-induced mammary carcinomas show immune heterogeneity which can be modulated by low-efficiency transfection of CD40L gene.

The presence of CD40 on carcinoma cells is an important factor for the generation of tumor-specific responses induced by CD40 ligation. In an N-methyl-N-nitrosourea (MNU)-induced autochthonous mammary tumor model, we analyzed the immune features of primary tumor cells. Here, CD40 was frequently detected on the primary tumor cultures and selectively expressed on the malignant mammary tissue in vivo. On the other hand, every mammary tumor cell culture had a heterogeneous and reduced expression of proinflammatory TNFalpha, IL-1beta, IL-6 and CXCL1 cytokines compared to normal mammary epithelial cells. Low-efficiency transfection of CD40 ligand (CD40L) gene enhanced the expression of proinflammatory cytokines in the tumor cells and strengthened allogeneic immune reactions and costimulatory activity which may help overwhelming suppressive features of the tumor.

The spectrum of FMF mutations and genotypes in the referrals to molecular genetic laboratory at Kirikkale University in Turkey.

Familial Mediterranean Fever (FMF) is an autosomal recessive genetic disorder characterised by recurrent and self-limited abdominal pain, synovitis and pleuritis. MEFV gene mutations are responsible from the disease and its protein product, pyrin or marenostrin, plays an essential role in the regulation of the inflammatory reactions. MEFV gene contains 10 exons and most of the mutations have been found on the last exon. Up to date, 152 mutations and polymorpisms have been reported inwhere V726A, M694V, M694I, M680I and E148Q are the most common mutations. In this study, MEFV allele frequencies of 136 individuals (60 from Pediatry, 76 from Internal Medicine) have been evaluated, and compared with each other. Asymptomatic individuals with FMF family history (4 from Pediatry, 6 from Internal Medicine) were excluded from the analysis. The prominent mutations indicated in the Pediatry group are V726A, M694V and M680I (G/C) and with the allele frequency of 0.06, 0.05 and 0.04 respectively while they were E148Q, M694V, M680I (G/C) in the Internal Medicine group with the allele frequency of 0.12, 0.08 and 0.04. The E148Q mutation is significantly overrepresented in the adult referrals (P = 0.02). Mutation on both alleles was observed in only 12% of cases. Overall mutation frequency was low, seen in 26.2% (66/252). However, when only diagnosed patients were analyzed it is 72.7% (16/22). It is also interesting that 63% of individuals are female that there may be sex influence on FMF phenotype.