Optimization of 3D islet-like cluster derived from human pluripotent stem cells: An efficient in vitro differentiation protocol.

Development of an optimized protocol to produce sufficient functional human insulin-producing islet-like cluster is important as a potential therapeutic strategy for diabetes as well as in vitro studies. Here, we described a stepwise protocol for differentiation of the human induced pluripotent stem cell line (R1-hiPSC1) into the islet-like cluster using several growth factors and small molecules. Therefore, various differentiation steps have been adopted to maximize mimicking of developmental processes in order to form functional islet like cluster. The differentiation protocol enables us to generate 3D islet-like clusters with highly viable cells, which are insulin producer and glucose responsive. Transcriptome analysis of transcription factors and functional genes revealed high coordination between gene expressions and resembling to those reported during natural development of islet cell. This coordination was further confirmed by hierarchical clustering of genes during differentiation. Furthermore, the islet-like clusters were enriched with insulin producing cells and formed glucose responsiveness behavior upon stimulation with glucose. Our protocol provides a robust platform and well-behaved model for additional developmental studies and shed light our clusters as a good candidate for in vitro model. Further studies are needed to assess the hormonal content of this cluster as well as transplantation into the animal model.

CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

Protective Effect of HLA-E∗0101∕∗0103 Genotype in Survival of Patients After Allogeneic Hematopoietic Stem Cell Transplant.

OBJECTIVES: HLA-E is located on the nonclassical major histocompatibility complex class I and acts as the ligand for natural killer cells. Consequently, it has a main role in the regulation of innate immune responses by involving cell identification by natural killer cells. Differences in expression levels among HLA-E alleles have been suggested to affect transplant outcomes. In this study, we evaluated the effects of different HLA-E genotypes on allogeneic hematopoietic stem cell transplant in southern Iran. MATERIALS AND METHODS: We investigated 200 patients (donors and recipients) who underwent allogeneic hematopoietic stem-cell transplant and 100 normal participants (control group) in a case-control study. Detection of HLA-E polymorphisms was performed using a sequence-specific primer polymerase chain reaction method. RESULTS: Statistical analyses indicated that genotypes in the transplant group were not distributed in accordance with Hardy-Weinberg equilibrium (χ²=76.56; P < .001), whereas genotypes in the control group were distributed in accordance with Hardy-Weinberg equilibrium (χ²=0.39; P = .53). No significant differences were observed in cumulative incidence of acute (P = .76; hazard ratio = 0.80; 95% confidence interval, 0.19-3.31) and chronic (P = .75, hazard ratio = 0.048; 95% confidence interval, 0.00) graft-versus-host disease in recipients harboring HLA-E∗0103 allele compared with those homozygous for the HLA-E∗0101 allele. The HLA-E∗0103 allele showed a trend toward lower cumulative incidence of relapse compared with the homozygous HLA-E∗0101 genotype (8% vs 21.5%; P = .37; hazard ratio = 2.50; 95% confidence interval, 0.32-19.20). CONCLUSIONS: Genotypes of the HLA-E molecule did not correlate with acute and chronic graft-versus-host disease in hematopoietic stem cell transplant recipients except for the HLA-E∗0101∗∕∗0103 genotype, which was protective in survival of our study patients.

Optimization of activin-A: a breakthrough in differentiation of human induced pluripotent stem cell into definitive endoderm.

The first step in differentiation of pluripotent stem cell toward endoderm-derived cell/organ is differentiation to definitive endoderm (DE) which is the central issue in developmental biology. Based on several evidences, we hypothesized that activin-A optimization as well as replacement of fetal bovine serum (FBS) with knockout serum replacement (KSR) is important for differentiation of induced pluripotent stem cell (iPSC) line into DE. Therefore, a stepwise differentiation protocol was applied on R1-hiPSC1 cell line. At first, activin-A concentration (30, 50, 70 and 100 ng/ml) was optimized. Then, substitution of FBS with KSR was evaluated across four treatment groups. The amount of differentiation of iPSC toward DE was determined by quantitative gene expression analyses of pluripotency (NANOG and OCT4), definitive endoderm (SOX17 and FOXA2) and endoderm-derived organs (PDX1, NEUROG3, and PAX6). Based on gene expression analyses, the more decrease in concentrations of activin-A can increase the differentiation of iPSC into DE, therefore, 30 ng/ml activin-A was chosen as the best concentration for the differentiation of R1-hiPSC1 line toward endoderm-derived organ. Moreover, complete replacement of FBS with gradually increased KSR improved the differentiation of iPSC toward DE. For this reason, the addition of 0% KSR at day 1, 0.2% at day 2 and 2% for the next 3 days was the best optimal protocol of the differentiation of iPSC toward DE. Overall, our results demonstrate that optimization of activin-A is important for differentiation of iPSC line. Furthermore, the replacement of FBS with KSR can improve the efficiency of iPSC differentiation toward DE.

ROCK Y-27632 Inhibitor, Ascorbic Acid, and Trehalose Increase Survival of Human Wharton Jelly Mesenchymal Stem Cells After Cryopreservation.

OBJECTIVES: Wharton jelly mesenchymal stem cells are good candidates for application in different aspects of regenerative medicine, and their long-time banking is important. In this study, the effects of trehalose, ascorbic acid, and Y-27632 on proliferation and survival rate of these cells after cryopreservation were investigated. MATERIALS AND METHODS: Mesenchymal stem cells were isolated from human umbilical cord Wharton jelly and frozen using a slow-rate cooling process. Different concentrations of trehalose (35, 75, and 125 mM), ascorbic acid (0.06, 0.125, 0.25, and 0.5 mM), and Y-27632 (10 µM) were used to treat culture medium and/or to supplement freezing medium. Assessment of cell viability after thawing was performed using Trypan blue staining, and MTT assay was performed to measure the cell proliferation rate. RESULTS: We observed significantly increased postthaw viability, increased cell proliferation, and decreased doubling time of cells when 75 mM trehalose, 0.25 and 0.5 mM ascorbic acid, and 10 mM Y-27632 were used. In addition, increased viability, proliferation, and attachment were observed after 24 hours of pretreatment with these cryoprotective agents and when they were added to conventional freezing medium. CONCLUSIONS: The use of different cryoprotective agents in culture and freezing media could be useful for long-term storage of Wharton jelly mesenchymal stem cells.

Is There a Correlation between Helicobacter Pylori and Enterohepatic Helicobacter Species and Gallstone Cholecystitis?

BACKGROUND Cholecystitis is a common surgical condition. Recently, several authors have reported that DNA of bile tolerant Helicobacter spp. has been found in the human bile colonizing the biliary tract. The aim of this study was to evaluate the association between the presence of Helicobacter spp. and gallstone cholecystitis. METHODS In this case-control study, gallstones, bile, and gallbladder mucosa were collected from 25 patients without gallstone disease, 24 with acute cholecystitis, and 28 with chronic cholecystitis. The presence of Helicobacter pylori (H. pylori), Helicobacter bilis (H. bilis), Helicobacter hepaticus (H. hepaticus) , and Helicobacter pullorum (H. pullorum) were investigated by polymerase chain reaction (PCR) using species-specific primers. RESULTS In this study, 77 subjects with acute and chronic cholecystitis and control groups with a mean age of 46.85 ± 14.53 years, including 58 (67.25%) women and 19 (32.75%) men were included. DNA of 10 Helicobacter spp. was detected in the bile of the patients with cholecystitis including eight H. pylori and two H. bilis. However, we could not detect H. hepaticus and H. pullorum DNA in the samples. Moreover, there was an association between H. pylori and acute cholecystitis (p = 0.048), which was found to be stronger in 31-40-year-olds group (p = 0.003). CONCLUSION We found an association between the presence of H. pylori DNA and acute gallstone cholecystitis. There is not statistically significant correlation between three enterohepatic Helicobacter spp. ( H. bilis, H. hepaticus , and H. pullorum) and cholelithiasis. Given the low sample size of the patients, more studies are required to clear the clinical role of Helicobacter spp. in the gallstone disease and cholecystitis.

Molecular Epidemiology of ESBL Genes and Multi-Drug Resistance in Diarrheagenic Escherichia Coli Strains Isolated from Adults in Iran.

Resistance to oxyimino cephalosporins antibiotics in Enterobacteriaceae is primarily done by the extended spectrum ß-lactamases (ESBLs). Clear identification of risk factors for ESBLs-producing infections is necessary. Therefore, efficient strategies can be developed to decrease outbreak of these infections. The aim of this study was to determine the antibacterial susceptibility and ESBLs pattern of diarrhogenic Escherichia coli (E. coli) strains isolated from adult patients. In the present study, diarrheogenic E. coli strains were isolated from 54 patients from the University of Medical Sciences hospitals in Shiraz. Antimicrobial susceptibility testing was done by disk diffusion method by CLSI criteria. The presence of bla TEM , bla SHV and bla CTX-M genes was investigated by PCR using designated primers. The prevalence of ESBLs-producer E. coli strains was 12.96%. Antimicrobial resistance testing showed a high resistance to cefexime, trimethoprim-sulfamethoxazole, ampicillin and penicillin. Overall, ß-lactamase genes were identified in 52 (96.30%) isolates which were identified as 45 (83.33%) bla TEM, 17 (31.48%) blaSHV and 11 (20.37%) blaCTX-M. ESBLs-producer E. coli is very prevalent in Diarrheogenic strains isolated from adult patients. Also, this study clearly showed that the bla TEM gene for ESBLs-producer E. coli was widespread in Iran.

The Association of Virulence Determinants of Uropathogenic Escherichia coli With Antibiotic Resistance.

BACKGROUND: The emergence of antimicrobial resistant strains of Escherichia coli has raised considerable interest in understanding the diversity and epidemiology of E. coli infections in humans. Virulence factors of E. coli determine the specific infections caused by this microorganism. OBJECTIVES: This study aimed to determine the prevalence of eight E. coli virulence factors and their association with antimicrobial resistance in bacteria isolated from patients with urinary tract infections (UTI). PATIENTS AND METHODS: One thousand patients with UTI were enrolled in this cross-sectional study. Antimicrobial susceptibility was examined by disc diffusion method according to CLSI guidelines. After DNA extraction, the materials were probed by PCR for eight virulence factors genes, namely fimH, hly, iucC, ibeA, sfa/foc, neuC, papC, and afa genes. RESULTS: The frequency of virulence factors papC, afa, sfa/foc, fimH, hly, neuC, ibeA, and iucC were 53.3%, 51.7%, 53.3%, 56.7%, 23.3%, 31.7%, 20%, and 73.3%, respectively. In addition, there was a high degree resistance to cotrimoxazole and nalidixic acid while a high degree of susceptibility to nitrofurantoin was detected. There was a statistically significant association between fimH gene and resistance to ciprofloxacin (P = 0.006), nalidixic acid (P = 0.025), and cotrimoxazole (P = 0.02). Such associations were found between ibeA gene and amikacin (P = 0.02) and cotrimoxazole (P = 0.02) as well as between afa gene and gentamycin (P = 0.05). CONCLUSIONS: The results showed that E. coli isolated from patients with UTI had eight virulence factors with high frequencies. Moreover, these results alleged a direct connection between virulence factors and antimicrobial resistance in E. coli.

Molecular detection of ESBLs production and antibiotic resistance patterns in Gram negative bacilli isolated from urinary tract infections.

BACKGROUND: ß-lactam resistance is more prevalent in Gram negative bacterial isolates worldwide, particularly in developing countries. In order to provide data relating to antibiotic therapy and resistance control, routine monitoring of corresponding antibiotic resistance genes is necessary. AIMS: The aim of this study was the characterization of ß-lactam resistance genes and its plasmid profile in bacteria isolated from urinary tract infection samples. MATERIALS AND METHODS: In this study, 298 Gram negative bacteria isolated from 6739 urine specimens were identified by biochemical standard tests. Antimicrobial susceptibility testing was performed by the disk diffusion method. Extended-spectrum ß-lactamase (ESBL)-producing strains were also detected by the double-disk synergy test. The presence of blaTEM and blaSHV genes in the strains studied was ascertained by polymerase chain reaction. RESULTS: Of all Gram negative bacteria, Escherichia coli (69.1%) was the most common strain, followed by Klebsiella sp. (12.1%), Enterobacter sp. (8.4%), Proteus sp. (4.4%), Citrobacter (4%) and Pseudomonas sp. (2%). The most antibiotic resistance was shown to tetracycline (95.16%), nalidixic acid (89.78%) and gentamycin (73.20%) antibiotics. Among all the strains tested, 35 isolates (11.75%) expressed ESBL activity. The prevalence of TEM and SHV positivity among these isolates was 34.29%, followed by TEM (31.43%), TEM and SHV negativity (20.0%) and SHV (14.29%), respectively. CONCLUSIONS: Regular monitoring of antimicrobial drug resistance seems necessary to improve our guidelines in the use of the empirical antibiotic therapy.

Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran.

CagA is a major virulence factor of Helicobacter pylori involved in host cell modulation. The C-terminal part of CagA containing the EPIYA motifs is highly variable and is important for the biological activity of the protein. The aim of this study was consideration of the changes in cagA tyrosine phosphorylation motifs (TPMs) of H. pylori. A set of 302 H. pylori DNA samples from the Iranian population from 2006 to 2011 was selected for the proposed study. The cagA gene and its TPMs were assessed by using polymerase chain reaction (PCR) and specific primers. The prevalence of the cagA gene in our study ranged from 91.43% to 97.06% (with an average of 95.03%). Out of the cagA-positive samples, the prevalence of TPMs A and B increased from 12.5% and 23.44% to 71.2% and 63.63%, respectively. Also, the prevalence of samples infected with Western and East Asian types of H. pylori ranged from 64.06% to 5.73% for the Western type and 17.19% to 51.59% for the East Asian type. Overall, our results showed a high prevalence of the cagA gene. Also, it seems that cagA TPMs of H. pylori is undergoing a change from the Western type to the East Asian type in Iran.

Characterization of Shiga-toxin producing E.coli (STEC) and enteropathogenic E.coli (EPEC) using multiplex Real-Time PCR assays for stx1 , stx2 , eaeA.

BACKGROUND AND OBJECTIVE: Diarrheal disease is still a major health problem, especially in developing countries, where it is considered as one of the leading causes of morbidity and mortality especially in children. Studies showed that Diarrheagenic E. coli (DEC) such as STES and EPEC strains are among the most prevalent causative agents in acute diarrhea, particularly in children. Aim of the present study was to investigate the presence and the frequency of STEC and EPEC as etiologic agent of diarrhea in children less than 2 years of age with diarrhea in Shiraz. MATERIALS AND METHODS: A total of 285 stool samples were collected from patients with diarrhea in Shiraz, in 2012. Diarrheagenic E. coli (DEC) strains were isolated by standard biochemical analysis. In this study, we used multiplex Real time PCR and single PCR to detect the presence of indicator genes stx1 , stx2 and eaeA for STEC and EPEC strains, respectively. RESULTS: A total of 285 stool samples were tested in which 49 (17%) were identified as contaminated with E. coli by biochemical tests. Out of total samples, 15 STEC (31%) and 13 EPEC (27%) were identified using multiplex Real-Time PCR assay. Among STEC isolates, 2 strains were stx1 (+), 8 isolates stx2 (+), 3 isolates were stx1 (+) , stx2 (+) and 2 isolates were stx1 (+) , stx2 (+), eaeA (+). CONCLUSION: In this study, we found rather high occurrence of STEC and EPEC virulence genes in children with diarrhea. The results of this study showed that, real time PCR can be used as a replacement for conventional PCR assay in the detecting virulence genes of STEC and EPEC strains. Real-time PCR offers the advantage of being a faster, more robust assay, because it does not require post-PCR procedures to detect amplification products.

Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissue from patients with gastrointestinal disorders.

Helicobacter pylori gram-negative bacteria commonly infect the human gastrointestinal (GI) tract and are readily diagnosed by endoscopy. H. pylori infection causes a broad range of host symptoms from discomfort to significant GI disorders (GIDs). Severity of the clinical manifestations depends mainly upon bacterial load. In this cross-sectional study, we investigated the affects of 23S rRNA point mutations on H. pylori count in naturally infected human GI tissues. Two-hundred H. pylori patients with suspected GIDs were evaluated to determine bacteria concentration and presence of four known 23S rRNA point mutations, causing clarithromycin resistance. Gastric biopsy specimens were examined by rapid urease test and 16S rRNA-targeted PCR to identify H. pylori; then bacterial load was quantified by real-time PCR targeting wild type and known 23S rRNA mutations. Eighty-two percent of the samples were confirmed as H. pylori-positive, having 10(4)-10(12) colony-forming units (CFU)/ml. The 10(6) load was most strongly associated with peptidyltransferase point mutations of the 23S rRNA gene A2144G (p = 0.033), A2143G (p = 0.005), A2143C (p = 0.005), and A2142G (p = 0.015). Thus, our findings indicated that dominant 23S rRNA mutated H. pylori strains have the same growth rate as the wild type in a gastric environment.

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus.

Hepatitis C virus (HCV) is a major public health problem and a leading cause of chronic liver disease. An estimated 180 million people are infected worldwide. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of HCV genomic RNA and compared the sensitivity of LAMP with nested-PCR. A total of 30 blood samples from HCV-infected patients were analyzed with six primers targeting conserved sequences of the HCV 5'UTR within 70 min, under isothermal conditions at 62 °C. Then, visualized by gel electrophoresis with ethidium bromide staining and detected by the naked-eye after adding SYBR Green I. All samples positive for HCV by nested PCR were confirmed by LAMP method. When visualized by gel electrophoresis and ethidium bromide staining, the HCV LAMP assay products appeared in a ladder pattern, with many bands of different sizes. The HCV LAMP product could also be detected by the naked-eye after adding SYBR Green I to the reaction tube and observing a color change from orange to green in positive samples. The HCV LAMP had the same sensitivity as a nested-PCR assay, the detection limit for the both systems were found to be 10 copies/mL of HCV RNA. The LAMP assay reported here is superior for rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of HCV in areas with limited resources, such as developing countries.