Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin.

Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes repair of a subset of DNA DSBs at early time points, which can be rescued by inhibiting transcription globally. An ATM phosphorylation site on BAF180, a PBAF subunit, is required for both processes. Furthermore, we find that subunits of the PRC1 and PRC2 polycomb group complexes are similarly required for DSB-induced silencing and promoting repair. Cancer-associated BAF180 mutants are unable to restore these functions, suggesting PBAF's role in repressing transcription near DSBs may contribute to its tumor suppressor activity.

DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.

A randomised controlled trial evaluating renal protective effects of selenium with vitamins A, C, E, verapamil, and losartan against extracorporeal shockwave lithotripsy-induced renal injury.

OBJECTIVE: To evaluate the protective effects of selenium with vitamins A, C and E (selenium ACE, i.e. antioxidants), verapamil (calcium channel blocker), and losartan (angiotensin receptor blocker) against extracorporeal shockwave lithotripsy (ESWL)-induced renal injury. PATIENTS AND METHODS: A randomised controlled trial was conducted between August 2012 and February 2015. Inclusion criteria were adult patients with a single renal stone (<2 cm) suitable for ESWL. Patients with diabetes, hypertension, congenital renal anomalies, moderate or marked hydronephrosis, or preoperative albuminuria (>300 mg/L) were excluded. ESWL was performed using the electromagnetic DoLiS lithotripter. Eligible patients were randomised into one of four groups using sealed closed envelopes: Group1, control; Group 2, selenium ACE; Group 3, losartan; and Group 4, verapamil. Albuminuria and urinary neutrophil gelatinase-associated lipocalin (uNGAL) were estimated after 2-4 h and 1 week after ESWL. The primary outcome was differences between albuminuria and uNGAL. Dynamic contrast-enhanced magnetic resonance imaging was performed before ESWL, and at 2-4 h and 1 week after ESWL to compare changes in renal perfusion. RESULTS: Of 329 patients assessed for eligibility, the final analysis comprised 160 patients (40 in each group). Losartan was the only medication that showed significantly lower levels of albuminuria after 1 week (P < 0.001). For perfusion changes, there was a statistically significant decrease in the renal perfusion in patients with obstructed kidneys in comparison to before ESWL (P = 0.003). These significant changes were present in the control or antioxidant group, whilst in the losartan and verapamil groups renal perfusion was not significantly decreased. CONCLUSIONS: Losartan was found to protect the kidney against ESWL-induced renal injury by significantly decreasing post-ESWL albuminuria. Verapamil and losartan maintained renal perfusion in patients with post-ESWL renal obstruction.

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases.

Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection.

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using α-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1's BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

Opposing roles for 53BP1 during homologous recombination.

Although DNA non-homologous end-joining repairs most DNA double-strand breaks (DSBs) in G2 phase, late repairing DSBs undergo resection and repair by homologous recombination (HR). Based on parallels to the situation in G1 cells, previous work has suggested that DSBs that undergo repair by HR predominantly localize to regions of heterochromatin (HC). By using H3K9me3 and H4K20me3 to identify HC regions, we substantiate and extend previous evidence, suggesting that HC-DSBs undergo repair by HR. Next, we examine roles for 53BP1 and BRCA1 in this process. Previous studies have shown that 53BP1 is pro-non-homologous end-joining and anti-HR. Surprisingly, we demonstrate that in G2 phase, 53BP1 is required for HR at HC-DSBs with its role being to promote phosphorylated KAP-1 foci formation. BRCA1, in contrast, is dispensable for pKAP-1 foci formation but relieves the barrier caused by 53BP1. As 53BP1 is retained at irradiation-induced foci during HR, we propose that BRCA1 promotes displacement but retention of 53BP1 to allow resection and any necessary HC modifications to complete HR. In contrast to this role for 53BP1 in HR in G2 phase, we show that it is dispensable for HR in S phase, where HC regions are likely relaxed during replication.

Transplantation of insulin-producing cells derived from human mesenchymal stromal/stem cells into diabetic humanized mice.

BACKGROUND: The purpose of this study was to investigate allogenic immune responses following the transplantation of insulin-producing cells (IPCs) differentiated from human adipose tissue-derived stem cells (hAT-MSCs) into humanized mice. METHODS: hAT-MSCs were isolated from liposuction aspirates obtained from HLA-A2-negative healthy donors. These cells were expanded and differentiated into IPCs. HLA-A2-positive humanized mice (NOG-EXL) were divided into 4 groups: diabetic mice transplanted with IPCs, diabetic but nontransplanted mice, nondiabetic mice transplanted with IPCs and normal untreated mice. Three million differentiated cells were transplanted under the renal capsule. Animals were followed-up to determine their weight, glucose levels (2-h postprandial), and human and mouse insulin levels. The mice were euthanized 6-8 weeks posttransplant. The kidneys were explanted for immunohistochemical studies. Blood, spleen and bone marrow samples were obtained to determine the proportion of immune cell subsets (CD4+, CD8+, CD16+, CD19+ and CD69+), and the expression levels of HLA-ABC and HLA-DR. RESULTS: Following STZ induction, blood glucose levels increased sharply and were then normalized within 2 weeks after cell transplantation. In these animals, human insulin levels were measurable while mouse insulin levels were negligible throughout the observation period. Immunostaining of cell-bearing kidneys revealed sparse CD45+ cells. Immunolabeling and flow cytometry of blood, bone marrow and splenic samples obtained from the 3 groups of animals did not reveal a significant difference in the proportions of immune cell subsets or in the expression levels of HLA-ABC and HLA-DR. CONCLUSION: Transplantation of IPCs derived from allogenic hAT-MSCs into humanized mice was followed by a muted allogenic immune response that did not interfere with the functionality of the engrafted cells. Our findings suggest that such allogenic cells could offer an opportunity for cell therapy for insulin-dependent diabetes without immunosuppression, encapsulation or gene manipulations.

Impact of Therapeutic Dose Monitoring of Mycophenolic Acid on the Outcome of Live-Donor Kidney Transplant Recipients - A Prospective Controlled Study.

Immunosuppressive therapy is the backbone to renal transplantation. Although an adequate level of immunosuppression is required to dampen the immune response to the allograft, the level of chronic immunosuppression is slowly decreased over time (as the risk of acute rejection decreases) to help lower the overall risk of infection and malignancy. Several studies have discussed the clinical use of therapeutic drug monitoring of mycophenolic acid (MPA) in kidney transplant recipients. This prospective single-center study included 88 patients with end-stage renal disease who were transplanted in Mansoura Urology and Nephrology Center from living related donors, from the beginning of February 2016 to the end of December 2016. Eight patients were excluded, the remaining 80 patients were divided into two groups; the study group (40 patients) who were followed up using therapeutic trough level monitoring of MPA and, control group (40 patients) who were followed up using the fixed-dose of Mycophenolate according to our local immunosuppressive protocol. These patients were followed up for one year posttransplantation with regard to graft function, rejection episodes, gastrointestinal (GI), and hematological side effects, the incidence of infection or malignancy, patient survival, and graft survival. Fifteen patients from the study group (37.5%) needed dose reduction of MPA, no patients needed to increase the dose. Our study showed insignificant differences regarding the patients' characteristics and demographic data. Significantly higher incidence of GI manifestations was noted in the control group (P = 0.001). Although the higher frequency of incidence of infection, anemia, leukopenia and thrombocytopenia was seen in the fixed- dose group, the difference was statistically insignificant. Regarding proteinuria and post-transplant diabetes mellitus, comparable data were obtained. Significantly higher percentage of recipients in the study group is still having normally functioning grafts (P = 0.02). Furthermore, higher percent of recipients in the control group died with functioning graft after one year of follow-up (P = 0.04). There were insignificant differences as regarding patient and graft survival. The decrease in the dose of MPA reduced the annual cost by around six thousand US dollars. Our results suggest that adopting therapeutic dose monitoring strategy during follow-up of kidney transplant recipients is adequate. Longer-term studies with a larger sample size may be needed to support this policy.

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Immunological Considerations.

Mesenchymal stem cell (MSC)-based therapy for type 1 diabetes mellitus (T1DM) has been the subject matter of many studies over the past few decades. The wide availability, negligible teratogenic risks and differentiation potential of MSCs promise a therapeutic alternative to traditional exogenous insulin injections or pancreatic transplantation. However, conflicting arguments have been reported regarding the immunological profile of MSCs. While some studies support their immune-privileged, immunomodulatory status and successful use in the treatment of several immune-mediated diseases, others maintain that allogeneic MSCs trigger immune responses, especially following differentiation or in vivo transplantation. In this review, the intricate mechanisms by which MSCs exert their immunomodulatory functions and the influencing variables are critically addressed. Furthermore, proposed avenues to enhance these effects, including cytokine pretreatment, coadministration of mTOR inhibitors, the use of Tregs and gene manipulation, are presented. As an alternative, the selection of high-benefit, low-risk donors based on HLA matching, PD-L1 expression and the absence of donor-specific antibodies (DSAs) are also discussed. Finally, the necessity for the transplantation of human MSC (hMSC)-derived insulin-producing cells (IPCs) into humanized mice is highlighted since this strategy may provide further insights into future clinical applications.

Evaluation of serum fatty acid binding protein-4 (FABP-4) as a novel biomarker to predict biopsy outcomes in prostate biopsy naïve patients.

OBJECTIVES: To investigate the role of serum fatty acid-binding protein-4 (FABP-4) as a surrogate of obesity and metabolic syndrome in the prediction of the outcome of prostate biopsy. METHODS: A prospective pilot study was conducted for patients undergoing prostate needle biopsy (PNB) for clinically suspected prostate cancer (PCa) between June 2016 and August 2017. Fifty consecutive patients with biopsy-proven PCa were included as study group and 50 consecutive patients with negative biopsy were included as a control group. Receiver Operating Characteristic (ROC) curve was used to calculate the area under the curve (AUC) to compare the accuracy of the different parameters in the diagnosis as well as the presence of high-grade PCa (Gleason score 8-9) at PNB. Predictors of the outcome were analyzed using univariate and multivariate logistic regression analysis. RESULTS: FABP-4 (AUC: 0.75; P < 0.001) and PSA-density (AUC: 0.84; P < 0.001) were the most accurate to detect PCa at PNB. On multivariate analysis, FABP-4 > 22.5 ng/ml (OR: 16.6; 95% CI 2.8-98; P = 0.002) and PSA-density > 0.38 ng/ml/ml OR: 17.7; 95% CI 5.3-59; P < 0.001) were independent predictors of PCa detection. Regarding high-grade PCa at PNB, FABP-4 (AUC: 0.79; P < 0.001) and %Free-PSA (AUC: 0.75; P < 0.001) were the most accurate. Independent predictors of high-grade PCa were FABP-4 > 32.3 ng/ml OR: 9.2; 95% CI 1.8-45; P = 0.006) and %Free-PSA ≤ 21.9 (OR: 5.5; 95% CI 1.1-27; P = 0.03). CONCLUSIONS: FABP-4 is an independent predictor for both the diagnosis and high-grade Gleason score at PNB. This novel biomarker might have a promising role in optimizing PNB outcomes.

PRDX6 Promotes the Differentiation of Human Mesenchymal Stem (Stromal) Cells to Insulin-Producing Cells.

Mesenchymal stem cells (MSCs) can be differentiated in vitro to form insulin-producing cells (IPCs). However, the proportion of induced cells is modest. Extracts from injured pancreata of rodents promoted this differentiation, and three upregulated proteins were identified in these extracts. The aim of this study was to evaluate the potential benefits of adding these proteins to the differentiation medium alone or in combination. Our results indicate that the proportion of IPCs among the protein(s)-supplemented samples was significantly higher than that in the samples with no added proteins. The yield from samples supplemented with PRDX6 alone was 4-fold higher than that from samples without added protein. These findings were also supported by the results of fluorophotometry. Gene expression profiles revealed higher levels among protein-supplemented samples. Significantly higher levels of GGT, SST, Glut-2, and MafB expression were noted among PRDX6-treated samples. There was a stepwise increase in the release of insulin and c-peptide, as a function of increasing glucose concentrations, indicating that the differentiated cells were glucose sensitive and insulin responsive. PRDX6 exerts its beneficial effects as a result of its biological antioxidant properties. Considering its ease of use as a single protein, PRDX6 is now routinely used in our differentiation protocols.

Safe conversion of mycophenolate mofetil to azathioprine in kidney transplant recipients with sirolimus-based immunosuppression.

AIM: Mycophenolate mofetil (MMF) is a powerful immunosuppressive drug with established efficacy and safety. The long-term use of MMF may bring increased risk of for infection and malignancy and also increased cost of transplantation. The search for minimization of immunosuppressive protocol has led to an open randomized clinical trial of conversion from MMF to azathioprine (AZA). METHODS: A total of 50 kidney allograft recipients treated with prednisone, sirolimus and MMF were randomized into two groups: converted (AZA group) and continuing (MMF group). The average duration of MMF therapy prior to conversion was 43 months in each group. Inclusion criteria included: patients with serum creatinine levels of less than 200 micromol/L; no past history of acute vascular rejection or recent acute rejection 6 months before randomization; and normal liver function tests. RESULTS: Baseline demographics were similar in the two groups. During the 12 month observation period, there were no acute rejection episodes in either group. There were no significant differences in overall patient or graft survival or function. AZA-treated patients had a lower incidence of gastrointestinal complications (P=0.03). Daily cost reduction in the AZA group was more than $US8.79/day per patient. CONCLUSION: In general, replacing MMF with AZA in stable renal transplant recipients is well tolerated and was cost effective with no increased risk of rejection. As the this study was on relatively small samples, larger and longer follow-up studies will be needed to confirm these expected advantages for the long-term outcome and to assess the long-term safety of this minimization of immunosuppressive therapy.

Pretransplant detection of anti-endothelial cell antibodies could predict renal allograft outcome.

OBJECTIVES: Endothelial cells that line the vasculature are targets for immune-mediated assault through anti-endothelial cell antibodies. The aim of this work was to detect anti-endothelial cell antibodies and describe the association with kidney allograft rejection and graft survival. MATERIALS AND METHODS: The study included 60 patients who had undergone live-donor kidney transplant. Inclusion criteria included first kidney transplant, panel reactive antibody titer less than 5%, cause of end-stage renal disease not including vasculitis or systemic lupus erythematosus, and age > 18 years. Patients were classified into 2 groups: 40 patients with anti-endothelial cell antibodies (referred to as the positive group) and 20 patients without anti-endothelial cell antibodies (referred to as the negative group). RESULTS: Serum creatinine level was higher in the positive group at 1 month and 1 year (P = .04). The occurrence of acute rejection was not significantly different in the positive group (18 patients [45.0%]) compared with the negative group (5 patients [25.0%], P = .5). However, the number of acute rejection episodes was higher in the positive group (22 episodes) compared with the negative group (6 episodes, P = .04). In patients who experienced acute rejection, chronic nephropathy was more frequent in the positive group (6 of 18 patients, 33.3%) compared with the negative group (1 of 5 patients, 20.0%) (P = .03). One-year and 5-year graft survival was 91% and 79% in the positive group, and 100% and 91% in the negative group, respectively. The difference at 5 years was significant (P = .04). CONCLUSIONS: The presence of anti-endothelial cell antibodies was associated with a higher number of acute rejection episodes and lower long-term graft survival in kidney transplants. It could be an informative test to identify patients at high risk for immunological graft loss.

Value of donor-specific antibody detection in first-graft renal transplant recipients with a negative complement-dependent cytotoxic crossmatch.

OBJECTIVES: The clinical significance of pretransplant donor specific antihuman leukocyte antigen antibodies that occur despite negative cytotoxicity crossmatches is still unclear. In this study, we assessed the impact of those antibodies on the outcome of renal transplants. MATERIALS AND METHODS: Our study subjects consisted of 153 living-donor kidney transplant recipients whose pretransplant sera were available. All subjects had a negative complement-dependent cytotoxic crossmatch and were retrospectively evaluated for antihuman leukocyte antigen antibodies and their donor specificities by means of LABScan 100 Flow analyzer (Luminex Corporation, Texas, USA). The follow-up data of all subjects were reviewed. RESULTS: Antihuman leukocyte antigen antibodies were detected in 49 patients, donor nonspecific antihuman leukocyte antigen antibodies were found in 33, and donor specific antihuman leukocyte antigen antibodies were identified in 16. There was a trend toward more acute rejection in the patients with antihuman leukocyte antigen antibodies (22%) than in those without antihuman leukocyte antigen antibodies (17%), but that difference had no statistical significance (P = .378). Patients with donor specific antihuman leukocyte antigen antibodies had a significantly higher incidence of acute cellular rejection (19% vs 6%, respectively) and vascular rejection (25% vs 6%, respectively) than did patients with donor nonspecific antihuman leukocyte antigen antibodies (P = .04). CONCLUSIONS: Our results suggest that there is a higher incidence of acute rejection in patients with donor specific antihuman leukocyte antigen antibodies and a negative complement-dependent cytotoxic crossmatch; however, those factors had no statistically significant impact on patient or graft survival.

Serum lipids might improve prostate-specific antigen sensitivity in patients undergoing transrectal ultrasonography-guided biopsy for suspected prostate cancer: A pilot study.

Objectives: To investigate the potential use of body mass index (BMI) and serum lipids in improving prostate-specific antigen (PSA) sensitivity in patients undergoing biopsy for suspicion of prostate cancer, as there is an established relationship between metabolic syndrome, obesity and serum lipids with prostate cancer. Patients and methods: A pilot study was conducted in a tertiary referral centre between June 2016 and August 2017 of patients undergoing transrectal ultrasonography (TRUS)-guided biopsy. After the result of TRUS-biopsy, the first 50 patients diagnosed with prostate cancer (study group) and those with no prostate cancer (control group) were enrolled. BMI, serum PSA level, fasting blood sugar and lipid profile (e.g. cholesterol, triglycerides, low-density lipoprotein [LDL] and high-density lipoprotein [HDL]), were compared between the groups. Results: Higher BMI, cholesterol, LDL and lower HDL together with PSA were significantly associated with a positive biopsy. On multivariate analysis, LDL (odds ratio [OR] 5.3, 95% confidence interval [CI] 1.2-24.9; P = 0.03) and total PSA level (OR 12.9, 95% CI 4.7-35; P < 0.001) were independent predictors of a positive biopsy. A combination of LDL <80 mg/dL and PSA level <26 ng/mL threshold values determined by receiver operating characteristic curve analysis, had a sensitivity and specificity of 94% and 28%, respectively; whilst, the negative (NPV) and positive predictive values were 82.4% and 56.6%, respectively. The sensitivity and NPV of the combination was significantly higher than that of PSA level alone (94% vs 72% and 82.4% vs 75%, respectively; P < 0.001). Conclusions: Serum lipids might have a role in the diagnosis of prostate cancer and could be used as an adjunct to PSA measurement to improve sensitivity and avoid unnecessary biopsies. Abbreviations: AUC: area under the curve; BMI: body mass index; FBS: fasting blood sugar; HDL: high-density lipoprotein; LDL: low-density lipoprotein; LOX-1: lectin-like oxidised LDL receptor-1; OR: odds ratio; ROC: receiver operating characteristic; RP: radical prostatectomy; TG: triglyceride.

Differentiation Potential of Nestin (+) and Nestin (-) Cells Derived from Human Bone Marrow Mesenchymal Stem Cells into Functional Insulin Producing Cells.

The feasibility of isolating and manipulating mesenchymal stem cells (MSCs) from human patients provides hope for curing numerous diseases and disorders. Recent phenotypic analysis has shown heterogeneity of MSCs. Nestin progenitor cell is a subpopulation within MSCs which plays a role in pancreas regeneration during embryogenesis. This study aimed to separate nestin (+) cells from human bone marrow MSCs, and differentiate these cells into functional insulin producing cells (IPCs) compared with nestin (-) cells. Manual magnetic separation was performed to obtain nestin (+) cells from MSCs. Approximately 91±3.3% of nestin (+) cells were positive for anti-nestin antibody. Pluripotent genes were overexpressed in nestin (+) cells compared with nestin (-) cells as revealed by quantitative real time-PCR (qRT-PCR). Following in vitro differentiation, flow cytometric analysis showed that 2.7±0.5% of differentiated nestin (+) cells were positive for anti-insulin antibody in comparison with 0.08±0.02% of nestin (-) cells. QRT-PCR showed higher expression of insulin and other endocrine genes in comparison with nestin (-) cells. While immunofluorescence technique showed the presence of insulin and C-peptide granules in nestin (+) cells. Therefore, our results introduced nestin (+) cells as a pluripotent subpopulation within human MSCs which is capable to differentiate and produce functional IPCs.

Evaluation of a synchronous twin-pulse technique for shock wave lithotripsy: a prospective randomized study of effectiveness and safety in comparison to standard single-pulse technique.

OBJECTIVE: To asses the efficacy and safety of bidirectional synchronous twin-pulse extracorporeal shock wave lithotripsy (ESWL) compared with standard ESWL. PATIENTS AND METHODS: Between March 2003 and December 2006, 240 patients with a radio-opaque single renal stone of 10 mm the rate for the failure of disintegration was 13.3% for the DLS vs 1.4% for the TH (P = 0.009). For stones of 10 mm it was 78.1% and 66.7%, respectively (P = 0.14). The median (range) number of sessions in both groups was 2 (1-5). After ESWL urinary NAG levels were increased significantly in both groups; in the TH group it declined below the level before ESWL after 2 days, while in the DLS group it remained high after 7 days. In the DLS group four patients developed subcapsular or parenchymal haematoma after ESWL, vs none in the TH group. There was loss of corticomedullary differentiation after ESWL in three patients in the DLS group and only one in the TH group. In the DLS group there was a statistically significantly decrease in bilateral renal perfusion after ESWL, but no changes in the TH group. CONCLUSIONS: Synchronous twin-pulse ESWL has clinical advantages over standard ESWL in terms of safety and efficacy.

HLA class I typing in Egyptian childhood minimal change nephrotic syndrome.

BACKGROUND: Reports on the association between human leukocyte antigens (HLAs) and childhood minimal change nephrotic syndrome (MCNS) are variable. The association between HLA class I and MCNS has not been tested among Egyptian children. METHODS: We studied such associations in a group of steroid-responsive MCNS Egyptian children (n=37) in comparison to a group of healthy Egyptians (n=100). Typing was performed using standard lymphocytotoxicity assay. RESULTS: The frequency of HLA-B27 was remarkably higher among MCNS children (18.9%) compared with the control group (1%) (p<0.001, risk ratio [RR] = 3.76, 95% confidence interval [95% CI], 2.5-5.66). Additionally, MCNS children had a significantly higher frequency of HLA-A11 (16.2% vs. 3%) and HLA-B13 (13.5% vs. 2%) (p=0.012 and p=0.016; RR=2.75 and RR=2.9; 95% CI, 1.58-4.79 and 1.66-5.06; respectively). With multivariate analysis, the previous 3 loci were still significant in addition to HLA-B12 (p=0.001, p=0.005, p=0.013 and p=0.033, for HLA-B27, HLA-B13, HLA-A11 and HLA-B12, respectively). The frequency of HLA haplotypes was comparable in both groups. CONCLUSIONS: We concluded that associations of HLA class I with childhood MCNS in Egypt have both similarities and dissimilarities to those in other populations. HLA-B27, which has not been reported before, has the strongest association. However, similar to studies from other populations, HLA-A11, HLA-B13 and HLA-B12 loci were also associated with MCNS.

Cyclosporine therapeutic monitoring with Cmax in kidney transplant recipients: does it fit for all populations?

BACKGROUND: We sought to assess whether the single cyclosporine concentration taken 2 hours after administration (C2) is a good parameter to predict a drug's maximal concentration (Cmax) value in Egyptian kidney transplant recipients. MATERIALS AND METHODS: Fifty stable Egyptian kidney transplant recipients with a previously diagnosed schistosomal infection were compared with 50 Egyptian kidney transplant recipients without a schistosomal infection regarding cyclosporine concentrations at time 0 (trough), and then at 1.5, 2, 2.5, 3, and 3.5 hours after a dose of cyclosporine. We used a linear regression analysis to assess any statistically significant differences between the different cyclosporine time concentrations and drug dosages. RESULTS: Patients in the schistosomal group had significantly lower C2 levels (511 -/+ 118 nmol/L) compared with those in the nonschistosomal (control) group (669 -/+ 213 nmol/L) (P < .05), whereas the C2.5 level was significantly higher (730 -/+ 215 and 527 -/+ 129 nmol/L, respectively; P < .05). A significant linear regression relation was determined for only C2.5 in the schistosomal group with both morning cyclosporine dose and cyclosporine dose expressed as mg/kg/d (P = .0123, r = .573018). CONCLUSIONS: Egyptian patients have special characteristics with regard to drug absorption and metabolism, mostly owing to schistosomal infection, and they may need the use of C2.5 to monitor cyclosporine. If confirmed by subsequent, larger studies, our findings may have a significant effect on our understanding and management of cyclosporine immunosuppression in clinical renal transplants with persons of different ethnicities.