Seeing double: two different homospermidine oxidases are involved in pyrrolizidine alkaloid biosynthesis in different organs of comfrey (Symphytum officinale).

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper-containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9-mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA-free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species-specific, tight spatio-temporal regulation, and independent evolutionary origins in multiple plant lineages.

Urinary congophilia in pregnancy: a marker of kidney injury rather than preeclampsia.

BACKGROUND: The differentiation between preeclampsia and similarly presenting kidney disease in pregnancy is a diagnostic challenge. Although some laboratory tests have been utilized, globally validated tools are yet needed, particularly in resource-limited settings. Congophilic proteins are abundantly detected in the urine of pregnant women who develop preeclampsia that is thought to be a marker of disease process. The present study aimed to assess the diagnostic and predictive utility of urinary congophilia in pregnant women with hypertensive disorders of pregnancy as well as kidney diseases. METHODS: This cohort study included 157 pregnant women, classified as healthy controls ( n  = 38), preeclampsia/eclampsia ( n  = 45), gestational hypertension ( n  = 9), chronic hypertension ( n  = 8), chronic kidney disease (CKD) ( n  = 27), and pregnancy-related acute kidney injury (PR-AKI) ( n  = 30). Urinary congophilia was assessed by Congo Red Dot Blot assay. RESULTS: Congo red retention (CRR) values were significantly higher in women with preeclampsia/eclampsia ( P  ≤ 0.001), chronic hypertension ( P  = 0.029), gestational hypertension ( P  = 0.017), CKD ( P  ≤ 0.001), PR-AKI secondary to preeclampsia ( P  ≤ 0.001), and PR-AKI secondary to other causes ( P  = 0.001), compared with healthy controls. Women with preeclampsia, CKD, and PR-AKI (non-preeclampsia related) exhibited the highest levels of CRR. CRR positively correlated to proteinuria ( P  = 0.006) and serum creatinine ( P  = 0.027). CRR did not significantly vary between women who presented antepartum and those presented postpartum after removal of the placenta ( P  = 0.707). CRR at a cut-off point of at least 1.272 had 91% specificity and 61.1% sensitivity in predicting renal recovery in PR-AKI patients. CRR had a poor specificity in discriminating preeclampsia from the other clinical presentations. CONCLUSION: Urinary congophilia could not discriminate preeclampsia from similarly presenting kidney diseases in pregnancy. Further studies are needed to improve differentiation of these conditions.

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.

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

One-anastomosis gastric bypass (OAGB) with fixed bypass of the proximal two meters versus tailored bypass of the proximal one-third of small bowel: short-term outcomes.

BACKGROUND: One-anastomosis gastric bypass (OAGB) is an effective bariatric procedure that confers satisfactory weight loss and improvement in comorbidities. The present study aimed to compare OAGB with fixed bypass of the proximal 200 cm of small bowel and tailored bypass of the proximal 1/3 of bowel. METHODS: Patients with class II/III obesity underwent OAGB with either fixed bypass of the proximal two meters or tailored bypass of the proximal 1/3 of bowel. The main outcomes of the study were weight loss, improvement in comorbidities, complications, and changes in nutritional parameters after each technique. RESULTS: The present study included 80 patients (62 female) of a mean age of 41 years and mean body mass index (BMI) of 50.9 kg/m2. The tailored bypass group was followed by a significantly lower BMI and significantly higher excess weight loss and total weight loss at 6 and 12 months postoperatively. There was no significant difference between the two groups in terms of improvement in comorbidities. The fixed bypass group was associated with a significantly higher complication rate than the tailored bypass group (22.5 vs. 5%, P = 0.04). Both groups were associated with similar changes in the nutritional parameters at 12 months postoperatively, except for the higher serum albumin levels after the tailored bypass than the fixed bypass. CONCLUSIONS: OAGB with tailored bypass of the proximal one-third of bowel was associated with greater weight loss and comparable improvement in comorbidities as compared to fixed bypass of the proximal two meters of intestine.

Diacerein ameliorates kidney injury induced by cisplatin in rats by activation of Nrf2/Ho-1 pathway and Bax down-regulation.

Cisplatin is an antineoplastic medicine used for solid tumor treatment. The main side effect that limits its dose is nephrotoxicity. Diacerein has been used for the treatment of joint diseases like osteoarthritis. It also has exhibited analgesic effects and antipyretic activities in animal models so this study targets to indicate the diacerein effect on nephrotoxicity induced by cisplatin in rats. Rats were distributed into four groups: normal healthy control; diacerein, which received diacerein daily by gastric gavage (50 mg/kg/day); cisplatin, which received only one intraperitoneal injection of cisplatin (6 mg/kg) and cisplatin and diacerein, which received diacerein daily after the cisplatin injection till 7th and 12th days, respectively. Diacerein treatment decreased kidney function markers so the cisplatin effect was reversed. Also, diacerein increased the renal antioxidants and decreased oxidative stress. Diacerein up-regulated Ho-1 (heme oxygenase 1), Nrf2 (Nuclear factor erythroid 2-related factor 2) and endothelial nitric oxide synthase (eNOS) genes expression, while down-regulated Bcl-2-associated X protein (Bax) gene expression. Furthermore, the renal transforming growth factor beta-1 (TGF-ß1) decreased by the diacerein effect. Consequently, diacerein has a curative effect against cisplatin due to its anti-inflammatory, antioxidant, and antiapoptotic properties.

Inflammatory markers in chronic kidney disease and end stage renal disease patients.

BACKGROUND: Chronic kidney disease (CKD) is condition characterized by a gradual loss of kidney function, patient with CKD suffering from a variety of immune system defects. METHODS: This study looked at Fas, T cell, BCl2, and P53 activity in people with CKD, end stage renal disease (ESRD), and stable controls. RESULTS: The CD4+ and CD8+ levels in ESRD patients' peripheral blood were slightly lower than those in CKD patients. The CKD and ESRD groups had slightly higher Fas and FasL mRNA expression and slightly lower BCl2 mRNA gene expression than the normal control group (P < 0.05). P53 mRNA gene expression was shown to be higher in the patients than in the controls (P < 0.01). CONCLUSIONS: ESRD patients have a significantly lower number of T-cell subsets than CKD patients this is related to a higher degree of apoptosis in these cells.

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Protective role of zinc oxide nanoparticles in kidney injury induced by cisplatin in rats.

Cisplatin used as chemotherapy for various cancers may leads to accumulation of platinum within the kidney and disturb its function. Zinc oxide nanoparticles (ZnO-NPs) are of low toxicity nanomaterials and have many medical fields so this study aims to indicate ZnO-NPs effect in kidney injury induced by cisplatin. Adult male rats were pre-injected with one dose of ZnO-NPs (5 mg/kg IP) and after 2 h from injection, the rats were injected with also only one dose of cisplatin (6 mg/kg IP) and two additional groups were served as controls treated with either ZnO-NPs or cisplatin only, respectively, and normal control was involved and euthanization occurred after 7 and 12 days. Cisplatin-induced nephropathy increased kidney function parameters; serum creatinine, blood urea nitrogen and microalbuminuria. Conversely, these parameters were down regulated after ZnO-NPs treatment. ZnO-NPs reversed the decrease of renal superoxide dismutase, catalase and glutathione reductase and the increase of renal malondialdehyde induced by cisplatin. In addition, the annexin V demonstrated that the proportion of viable cells was significantly elevated and the proportion of apoptotic and necrotic cells significantly reduced. Also, the level of renal transforming growth factor beta 1 decreased in group pre-treated with ZnO-NPs. The Nuclear factor-E2-related factor, heme oxygenase-1 and endothelial nitric oxide synthase expression genes were up regulated while Bcl-2-associated X protein expression was down regulated in kidney tissue via ZnO-NPs. Histopathological and immunohistochemical observations were context with these findings. In conclusion, ZnO-NPs treatment revealed renoprotective effect against cisplatin drug, probably via its antioxidant, anti-inflammatory and antiapoptotic properties.

Escherichia coli foster bladder cancer cell line progression via epithelial mesenchymal transition, stemness and metabolic reprogramming.

Bacteria is recognized as opportunistic tumor inhabitant, giving rise to an environmental stress that may alter tumor microenvironment, which directs cancer behavior. In vitro infection of the T24 cell line with E. coli was performed to study the bacterial impact on bladder cancer cells. EMT markers were assessed using immunohistochemistry, western blot and RT-PCR. Stemness characteristics were monitored using RT-PCR. Furthermore, the metabolic reprograming was investigated by detection of ROS and metabolic markers. A significant (p ≤ 0.001) upregulation of vimentin as well as downregulation of CK19 transcription and protein levels was reported. A significant increase (p ≤ 0.001) in the expression level of stemness markers (CD44, NANOG, SOX2 and OCT4) was reported. ROS level was elevated, that led to a significant increase (p ≤ 0.001) in UCP2. This enhanced a significant increase (p ≤ 0.001) in PDK1 to significantly downregulate PDH (p ≤ 0.001) in order to block oxidative phosphorylation in favor of glycolysis. This resulted in a significant decrease (p ≤ 0.001) of AMPK, and a significant elevation (p ≤ 0.001) of MCT1 to export the produced lactate to extracellular matrix. Thus, bacteria may induce alteration to the heterogonous tumor cell population through EMT, CSCs and metabolic reprogramming, which may improve cancer cell ability to migrate and self-renew.

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Progress and Challenges.

Mesenchymal stromal cells (MSCs) are an attractive option for cell therapy for type 1 diabetes mellitus (DM). These cells can be obtained from many sources, but bone marrow and adipose tissue are the most studied. MSCs have distinct advantages since they are nonteratogenic, nonimmunogenic and have immunomodulatory functions. Insulin-producing cells (IPCs) can be generated from MSCs by gene transfection, gene editing or directed differentiation. For directed differentiation, MSCs are usually cultured in a glucose-rich medium with various growth and activation factors. The resulting IPCs can control chemically-induced diabetes in immune-deficient mice. These findings are comparable to those obtained from pluripotent cells. PD-L1 and PD-L2 expression by MSCs is upregulated under inflammatory conditions. Immunomodulation occurs due to the interaction between these ligands and PD-1 receptors on T lymphocytes. If this function is maintained after differentiation, life-long immunosuppression or encapsulation could be avoided. In the clinical setting, two sites can be used for transplantation of IPCs: the subcutaneous tissue and the omentum. A 2-stage procedure is required for the former and a laparoscopic procedure for the latter. For either site, cells should be transplanted within a scaffold, preferably one from fibrin. Several questions remain unanswered. Will the transplanted cells be affected by the antibodies involved in the pathogenesis of type 1 DM? What is the functional longevity of these cells following their transplantation? These issues have to be addressed before clinical translation is attempted. Graphical Abstract Bone marrow MSCs are isolated from the long bone of SD rats. Then they are expanded and through directed differentiation insulin-producing cells are formed. The differentiated cells are loaded onto a collagen scaffold. If one-stage transplantation is planned, a drug delivery system must be incorporated to ensure immediate oxygenation, promote vascularization and provide some growth factors. Some mechanisms involved in the immunomodulatory function of MSCs. These are implemented either by cell to cell contact or by the release of soluble factors. Collectively, these pathways results in an increase in T-regulatory cells.

Laparoscopic Sleeve Gastrectomy with T-Shaped Omentoplasty: Impact on the Early Postoperative Outcomes.

PURPOSE: Laparoscopic sleeve gastrectomy (LSG) is an effective bariatric procedure, yet can be followed by complications such as staple line leak and bleeding, vomiting, and gastroesophageal reflux disease (GERD). Various attempts have been described in the literature to improve the early outcome of LSG through various measures. This study aimed to assess the impact of adding T-shaped omentoplasty to LSG on the short-term outcome of the procedure. METHODS: This was a retrospective cohort study on patients with morbid obesity who underwent LSG in the period of November 2015 to November 2018. The outcome of patients with morbid obesity who underwent LSG combined with T-shaped omentoplasty (group I) was compared with that of a similar number of patients who underwent classical LSG without staple line fixation (group II). The main outcome measures were the rates of staple line bleeding and leak, postoperative nausea and vomiting (PONV), GERD, gastric axial rotation, other complications, and weight loss. RESULTS: The study included 106 patients of a mean BMI of 49.8 kg/m2. Group II had significantly higher PONV) scale at 1 week and 1 month than group I. Group I had significantly lower rates of staple line bleeding (0 vs 9.6%, p = 0.02) and GERD (3.7% vs 17.3%, p = 0.02) than group II. Both groups had similar rates of staple line leak and comparable operation time. CONCLUSION: Staple line fixation using the T-shaped omentoplasty technique was associated with lower incidence of significant PONV, staple line bleeding, and GERD as compared with classical LSG.

Subcutaneous transplantation of bone marrow derived stem cells in macroencapsulation device for treating diabetic rats; clinically transplantable site.

BACKGROUND/AIM: Diabetes mellitus (DM) is a serious, chronic and epidemic disease. Its effective therapy with exogenous insulin places an overwhelming burden on the patient's lifestyle. Moreover, pancreatic islet transplantation is limited by the scarceness of donors and the need for chronic immunosuppression. Cell-based therapy is considered an alternative source of insulin-producing cells (IPCs); encapsulating such cellular grafts in immunoisolating devices would protect the graft from immune attack without the need for immunosuppression. Herein, we investigate the ability of TheraCyte capsule as an immunoisolating device to promote the maturation of differentiated rat bone marrow derived mesenchymal stem cells (BM-MSCs), transplanted subcutaneously to treat diabetic rats in comparison with intratesticular transplantation. MAIN METHODS: Rat BM-MSC were differentiated into IPCs, and either encapsulated in TheraCyte capsules for subcutaneous transplantation or transplanted intratesticular into diabetic rats. Serum insulin, C-peptide & blood glucose levels of transplanted animals were monitored. Retrieved cells were further characterized by immunofluorescence staining and gene expression analysis. KEY FINDINGS: Differentiated rat BM-MSC were able to produce insulin in vitro, ameliorate hyperglycemia in vivo and survive for 6 months post transplantation. Transplanted cells induced higher levels of insulin and C-peptide, lower levels of blood glucose in the cured animals of both experimental groups. Gene expression revealed a further in vivo maturation of the implanted cells. SIGNIFICANCE: These data suggest that TheraCyte encapsulation of allogeneic differentiated stem cells are capable of reversing hyperglycemia, which holds a great promise as a new cell based, clinically applicable therapies for diabetes.

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.

Xeno-free trans-differentiation of adipose tissue-derived mesenchymal stem cells into glial and neuronal cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells that have the ability of self-renewal and trans-differentiation into other cell types. They hold out hope for finding a cure for many diseases. Nevertheless, there are still some obstacles that limit their clinical transplantation. One of these obstacles are the xenogeneic substances added in either proliferation or differentiation media with subsequent immunogenic and infectious transmission problems. In this study, we aimed to replace fetal bovine serum (FBS), the main nutrient source for MSC proliferation with xeno-free blood derivatives. We tested the effect of human activated pure platelet-rich plasma (P-PRP) and advanced platelet-rich fibrin (A-PRF) on the proliferation of human adipose derived-MSCs (AD-MSCs) at different concentrations. For the induction of MSC neural differentiation, we used human cerebrospinal fluid (CSF) at different concentrations in combination with P-PRP to effect xeno-free/species-specific neuronal/glial differentiation and we found that media with 10% CSF and 10% PRP promoted glial differentiation, while media with only 10% PRP induced a neuron-like phenotype.

Ameliorative effects of silymarin on HCl-induced acute lung injury in rats; role of the Nrf-2/HO-1 pathway.

OBJECTIVES: Aspiration is a common cause of acute lung injury (ALI), which lacks an effective treatment. Inflammation and oxidative stress play key roles in ALI development. Silymarin is an active extract of Silybum marianum plant seeds (milk thistle). Silymarin has potent anti-inflammatory and antioxidant effects; however its role in aspiration induced ALI has not been investigated. The aim of this study is to investigate the role of silymarin in the treatment of hydrochloric acid (HCl) aspiration induced ALI and explores its mechanisms of action. MATERIALS AND METHODS: The study included three groups of rats: Control non-treated group, ALI group (intra-tracheal HCl injected), and silymarin treated ALI group. White blood cells (WBCs) with differential count, oxidative stress parameters, B-cell lymphoma 2 (Bcl-2), transforming growth factor-beta (TGF-ß), cyclooxygenase 2 (COX-2), nuclear factor erythroid 2-related factor-2 (Nrf-2), and heme oxygenase-1 (HO-1) were investigated. Lung tissue histopathology and immunohistochemical expression of survivin and proliferating cell nuclear antigen (PCNA) were also examined. RESULTS: The results of the study showed that HCL caused histopathological changes in ALI with leukocytopenia and increased oxidative stress biomarkers. It increased TGF-ß, up-regulated mRNA expression of COX-2, Nrf-2, and HO-1 and increased survivin and PCNA but decreased Bcl-2. Silymarin ameliorated the histopathological lung injury with further up-regulation of Nrf-2 and HO-1 mRNA and decreased the inflammatory and fibrotic parameters together with up-regulation of the anti-apoptotic and the proliferation parameters. CONCLUSION: The protective effect of silymarin against ALI is mediated by Nrf-2/HO-1 pathway with subsequent antioxidant, anti-inflammatory, antiapoptotic, and proliferating activities.

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.

Laparoscopic Roux-en-Y Gastric Bypass for Failed Vertical Banded Gastroplasty.

BACKGROUND: Although the vertical banded gastroplasty (VBG) is effective in achieving weight loss without metabolic side effects during the first few years, late failures cause weight regain in about 20% of the patients. The laparoscopic Roux-en-Y gastric bypass (LRYGB) is the procedure of choice to convert a failed VBG. MATERIALS AND METHODS: Between August 2014 and June 2016, 37 patients (23 females and 14 males) underwent revisional LRYGBs after failed VBGs. Thirty-four patients had open VBGs and two patients had laparoscopic butterfly gastroplasties. These patients completed follow-ups of at least 1 year. RESULTS: The median operating time was 164 min (range 132-246), and the mean length of the hospital stay was 2.4 days (range 2-7). The mean body mass index (BMI) of the study group was 42.5 kg/m2, while the mean BMI and the percentage of excess body weight loss (%EBWL) for the patients 6 months after the operation were 31.7 kg/m2 and 52.1%, respectively. The mean BMI and %EBWL for the patients 1 year after the operation were 29.5 kg/m2 and 67.7%, respectively. There were neither intraoperative complications nor postoperative mortalities, but the overall complication rate was 22% (eight patients). CONCLUSION: An LRYGB following a VBG is technically challenging, but safe, with low morbidity and mortality rates. Moreover, a revisional LRYGB provides excellent weight loss results.

Insulin-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study.

Ten mongrel dogs were used in this study. Diabetes was chemically induced in 7 dogs, and 3 dogs served as normal controls. For each diabetic dog, 5 million human bone marrow-derived mesenchymal stem cells/kg were differentiated to form insulin-producing cells using a trichostatin-based protocol. Cells were then loaded in 2 TheraCyte capsules which were transplanted under the rectus sheath. One dog died 4 d postoperatively from pneumonia. Six dogs were followed up with for 6 to 18 mo. Euglycemia was achieved in 4 dogs. Their glucose tolerance curves exhibited a normal pattern demonstrating that the encapsulated cells were glucose sensitive and insulin responsive. In the remaining 2 dogs, the fasting blood sugar levels were reduced but did not reach normal values. The sera of all transplanted dogs contained human insulin and C-peptide with a negligible amount of canine insulin. Removal of the transplanted capsules was followed by prompt return of diabetes. Intracytoplasmic insulin granules were seen by immunofluorescence in cells from the harvested capsules. Furthermore, all pancreatic endocrine genes were expressed. This study demonstrated that the TheraCyte capsule or a similar device can provide adequate immunoisolation, an important issue when stem cells are considered for the treatment of type 1 diabetes mellitus.