Regenerative medicine of pancreatic islets

The pancreas became one of the first objects of regenerative medicine,since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted.The number of people living with diabetes mellitus is currently approaching half a billion,hence the crucial relevance of new methods to stimulate regeneration of the insulin-secreting β-cells of the islets of Langerhans.Natural restrictions on the islet regeneration are very tight;nevertheless,the islets are capable of physiological regeneration via β-cell self-replication,direct differentiation of multipotent progenitor cells and spontaneous α-to or δ-to β-cell conversion(trans-differentiation).The existing preclinical models of β-cell dysfunction or ablation(induced surgically,chemically or genetically) have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation The ultimate goal,sufficient level of functional activity of β-cells or their substitutes can be achieved by two prospective broad strategies β-cell replacement and β-cell regeneration.The "regeneration" strategy aims to maintain a preserved population of β-cells through in situ exposure to biologically active substances that improve β-cell survival,replication and insulin secretion,or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-β-to β-cell conversion.The "replacement" strategy implies transplantation of β-cells(as non-disintegrated pancreatic material or isolated donor islets) or β-like cells obtained ex vivo from progenitors or mature somatic cells(for example,hepatocytes or a-cells) under the action of small-molecule inducers or by genetic modification.We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally activeβ-cells, the innermost hope of millions of people globally.     

Origins of serum semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidases (SSAO) are enzymes that are capable of deaminating primary amines to produce aldehyde, ammonia, and hydrogen peroxide. This activity has been associated with vascular adhesion protein-1 (VAP-1) and is found in the serum, endothelium, adipose, and smooth muscle of mammals. Circulating SSAO activity is increased in congestive heart failure, diabetes, and inflammatory liver diseases. To investigate the origin of circulating SSAO activity, two transgenic mouse models were created with full-length human VAP-1 (hVAP-1) expressed on either endothelial (mTIEhVAP-1) or adipose tissues (aP2hVAP-1), with tie-1 and adipocyte P2 promoters, respectively. Under normal conditions a circulating form of hVAP-1 was found at high levels in the serum of mice with endothelium-specific expression and at low levels in the serum of mice with adipose specific expression. The level of circulating hVAP-1 in the transgenic mice varied with gender, transgene zygosity, diabetes, and fasting. Serum SSAO activity was absent from VAP-1 knockout mice and endothelial cell-specific expression of human VAP-1 restored SSAO activity to the serum of VAP-1 knockout mice. Together, these experiments show that in the mouse VAP-1 is the only source of serum SSAO, that under physiological conditions vascular endothelial cells can be a major source of circulating VAP-1 protein and SSAO, and that serum VAP-1 can originate from both endothelial cells and adipocytes during experimental diabetes. An increased endothelial cell capacity for lymphocyte binding and altered expression of redox-sensitive proteins was also associated with the mTIEhVAP-1 transgene.     

Correlation between oocyte morphology and the embryo aneuploidy rate in IVF cycles

Abstract

Purpose: To evaluate the aneuploidy rates of 13, 18, and 21 and the X and Y chromosomes in embryos from patients with morphologically normal oocytes and different oocyte dysmorphisms.

Methods: This prospective cohort study included 84 patients treated with in vitro fertilization (IVF) at a single academic center. The patients were divided into the following three groups: group 1 – women with cytoplasmic dysmorphisms (n?=?28), group 2 – women with extracytoplasmic dysmorphisms (n?=?28), and group 3 – women with morphologically normal oocytes (n?=?28). One blastomere from each embryo was analyzed for aneuploidies of chromosomes 13, 18, 21, X, and Y.

Results: The highest prevalence of aneuploid embryos was observed in the group 1 (68.4%) followed by the group 2 (38.9%) and the group 3 (31.3%) (р?<?0.0001). The adjusted OR for receiving an aneuploid embryo in the case of cytoplasmic dysmorphism was 3.6 (95% CI?=?1.8; 7.2), in the case of extracytoplasmic dysmorphisms – 1.3 (95% CI?=?0.7; 2.1).

Conclusions: Women with morphological oocyte abnormalities are at risk for developing aneuploid embryos during IVF cycles. We recommend that woman with cytoplasmic oocyte dysmorphisms receive additional genetic counseling to define the indications for the genetic screening of embryos.     

Analysis of granulomonocytic precursor cells from fetal liver used for maintenance therapy in children with Gaucher's disease

Granulocyte-macrophage precursor cells of fetal liver are characterized by high proliferative capacity. Being administered to children with Gaucher's disease, these cells provide a short-term maintenance of the monocyte/macrophage pool with normal level of glucocerebrosidase. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 7, pp. 78–80, July, 1999     

Effect of a BIOcocktail on the immune response at the early postoperative period in mice.

Since human subjects and laboratory animals may develop impaired immune response during surgery and the postoperative period, efforts have been made to preserve normal immune functions following surgery by the administration of nutritional supplements and probiotics. The present study was designed to examine the effect of a new nutritional supplement, BIOcocktail, on immune parameters in mice exposed to surgery. Forty mice were assigned to 4 groups containing 10 animals each. Two control groups (with and without subsequent sham laparotomy) were given tap water for 45 min every day for 2 weeks. The remaining 2 groups, with and without laparotomy, received BIOcocktail given orally for the same period of time. The proliferative response of splenic cells (splenocytes) stimulated with phytohemagglutinin (PHA), concanavalin A (Con A) and lipopolysaccharide (LPS) was determined by [3H]thymidine uptake. Cytokine levels were measured in splenocyte supernatants and sera using enzyme-linked immunosorbent assay (ELISA) kits. Natural killer cell activity of splenocytes was evaluated by 51Cr-release assay. Laparotomy, without BIOcocktail administration, was followed by a decreased proliferative response of splenocytes to PHA, Con A, and LPS and an increase in interleukin (IL)-6 serum level. In addition, a decreased secretion of IL-1beta, IL-12 and tumor necrosis factor (TNF)-alpha by the splenocytes was observed. Mice treated with BIOcocktail before laparotomy maintained a preoperative level of splenocyte proliferative response and serum concentrations of IL-12. It is concluded that BIOcocktail administered to mice for 2 weeks before operation resulted in the preservation of T- and B-cell proliferative response to mitogens and in the prevention of postoperative decrease in IL-12 serum level.     

Application potential of human fetal stem/progenitor cells in cell therapy

We analyzed the possibility of using fetal stem and progenitor cells for the treatment of various pathologies. A comparative characteristic of stem cells from fetuses and adult donors is presented and advantages of the use of fetal biometerial for biotransplantation are described. The main tissue sources of fetal stem cells are characterized and experimental and clinical data on the use of fetal cells for cytotherapy are presented. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 1, pp. 5–13, January, 2007     

Hierarchy of simulation models in predicting structure and energetics of the Src SH2 domain binding to tyrosyl phosphopeptides.

Structure and energetics of the Src Src Homology 2 (SH2) domain binding with the recognition phosphopeptide pYEEI and its mutants are studied by a hierarchical computational approach. The proposed structure prediction strategy includes equilibrium sampling of the peptide conformational space by simulated tempering dynamics with the simplified, knowledge-based energy function, followed by structural clustering of the resulting conformations and binding free energy evaluation of a single representative from each cluster, a cluster center. This protocol is robust in rapid screening of low-energy conformations and recovers the crystal structure of the pYEEI peptide. Thermodynamics of the peptide-SH2 domain binding is analyzed by computing the average energy contributions over conformations from the clusters, structurally similar to the predicted peptide bound structure. Using this approach, the binding thermodynamics for a panel of studied peptides is predicted in a better agreement with the experiment than previously suggested models. However, the overall correlation between computed and experimental binding affinity remains rather modest. The results of this study show that small differences in binding free energies between the Ala and Gly mutants of the pYEEI peptide are considerably more difficult to predict than the structure of the bound peptides, indicating that accurate computational prediction of binding affinities still remains a major methodological and technical challenge.     

Robust Detection of DNA Hypermethylation of ZNF154 as a Pan-Cancer Locus with in Silico Modeling for Blood-Based Diagnostic Development

Sites that display recurrent, aberrant DNA methylation in cancer represent potential biomarkers for screening and diagnostics. Previously, we identified hypermethylation at the ZNF154 CpG island in 15 solid epithelial tumor types from 13 different organs. In this study, we measure the magnitude and pattern of differential methylation of this region across colon, lung, breast, stomach, and endometrial tumor samples using next-generation bisulfite amplicon sequencing. We found that all tumor types and subtypes are hypermethylated at this locus compared with normal tissue. To evaluate this site as a possible pan-cancer marker, we compare the ability of several sequence analysis methods to distinguish the five tumor types (184 tumor samples) from normal tissue samples (n = 34). The classification performance for the strongest method, measured by the area under (the receiver operating characteristic) curve (AUC), is 0.96, close to a perfect value of 1. Furthermore, in a computational simulation of circulating tumor DNA, we were able to detect limited amounts of tumor DNA diluted with normal DNA: 1% tumor DNA in 99% normal DNA yields AUCs of up to 0.79. Our findings suggest that hypermethylation of the ZNF154 CpG island is a relevant biomarker for identifying solid tumor DNA and may have utility as a generalizable biomarker for circulating tumor DNA.CME Accreditation Statement: This activity (“JMD 2016 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2016 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.One in four deaths in the United States is due to cancer, despite an emphasis on prevention, early detection, and treatment that has lowered cancer death rates by 20% in the past two decades.^{1, 2} Further improvements in survival rates are likely to come from improving the limits of detection sensitivity at earlier stages of cancer. Currently, a diagnosis results from a cadre of screening and diagnostic tools that may include physical examination, radiographic imaging, sputum cytologic testing, blood tests, endoscopy, and/or biopsies. However, new approaches that rely heavily on genomic information may change future testing strategies.The future looks bright because minimally invasive sampling techniques coupled with genomic features that distinguish tumor cells from normal cells have the potential to detect cancer at earlier stages. For example, circulating tumor cells or cell-free plasma DNA can be detected in venous peripheral blood and tested for the presence of common mutations.³ Cell-free tumor DNA can also be detected in buccal epithelium, saliva, urine, stools, and bronchial aspirates.⁴ Such DNA has been used to detect mutations in patients with both localized and metastatic cancers.⁵ Moreover, somatic mutations in ovarian and endometrial cancers can potentially be detected using Papanicolaou specimens.⁶In addition to genetic mutations, epigenetic markers are emerging as tools with discriminatory power for disease detection. For example, DNA methylation is a robust epigenetic marker for which a number of commercially available tests have been developed. These tests detect tissue-specific DNA methylation using clinical specimens and are used in colorectal cancer (SEPT9, blood; VIM, stool), lung cancer (SHOX2, bronchial fluid), and brain cancer (MGMT, tumor).⁷ One advantage of this approach is marker stability under common storage conditions.⁴ However, despite DNA methylation''s potential as a diagnostic marker, a general lack of consensus on the methods remains. This is the principal reason for its slow implementation in clinical diagnostics.^{4, 7}Previously, our laboratory reported a pan-cancer hypermethylation signal around a CpG island near human ZNF154.⁸ This signal was initially detected by us in ovarian and endometrial cancers and replicated by us in multiple, independent cohorts from The Cancer Genome Atlas (TCGA), incorporating a total of 15 distinct tumor collections from 13 different organs with almost 6000 samples.⁸ These previous analyses relied on data generated from Illumina Infinium methylation arrays (Illumina Inc, San Diego, CA) to detect the methylation levels at select CpG sites. In this study, we measure the ZNF154 methylation signal across five tumor types using bisulfite amplicon sequencing. With this method, individual sequence reads are used to quantitate methylation levels of all CpGs within the amplicon while providing quantitative data for each DNA molecule in the pooled sample. Furthermore, the approach provides an intrinsic measure of quality control by tracking bisulfite conversion efficiency at cytosines in the non-CpG context wherein extensive amounts of unconverted cytosines signal an incomplete conversion reaction. This procedure is both time efficient and cost-effective because multiple samples can be sequenced in parallel using a 96-well plate and, as we report, generate reproducible measurements when assayed in independent experiments. The amplicon sequencing provides greater resolution of a target region than a methylation array by covering all amplified CpGs, revealing patterns of DNA methylation useful for distinguishing tumor from normal samples.We report that the magnitude and reproducibility of the ZNF154 hypermethylation signal across five solid tumor types reinforces the potential of this site as a biomarker for circulating tumor DNA (ctDNA). Next, we assess the potential application of various computational data classification methods toward cancer screening. By investigating a variety of technical approaches to characterize methylated bases within the sequenced samples, we identify features useful for distinguishing tumor samples from normal samples. Finally, we use a computational simulation to demonstrate the utility of these features in classifying samples as tumor or normal tissue at various abundance levels; here, tumor DNA methylation patterns are compiled into a background of normal DNA methylation patterns, at limiting dilution levels, mimicking the fractions at which ctDNA is recovered from blood.     

Comparative Characteristics of Sialoglycans Expression Disorders in the Placental Barrier Structures in Preeclampsia and Fetal Growth Restriction

Bulletin of Experimental Biology and Medicine - We compared the expression profiles of α2,3- and α2,6-sialoglycans in the glycocalyx of the placental barrier structures in early and late...     

Pathological upstaging of clinical T1 renal cell carcinoma: an analysis of 115,835 patients from National Cancer Data Base, 2004–2013

Purpose

Clinical staging is vital for treatment decision-making by renal cell carcinoma (RCC) patients. Some RCCs clinically appear T1 on CT, but are actually T3a due to extension into fat or renal vein, causing the tumor to be pathologically upstaged. The objective of this study to determine the rate, survival, and predictors of RCC upstaging, for patients with cT1 disease treated surgically.

Methods

Using the National Cancer Data Base Participant User File for RCC from 2004 to 2013, we selected AJCC cT1 patients, who underwent surgical resection and whose AJCC pathological T stage (pT) was available. Upstaging was characterized dichotomously—overall (any pT > T1) and pT3a-specific upstaging. Patient and tumor characteristics of those upstaged and not were compared using Chi-squared analyses. Multivariable logistic regression was used to analyze predictors of upstaging, and Cox proportional hazards regression was used to estimate overall survival hazards ratios.

Results

Overall upstaging (pT > T1) was observed in 8252 (7.1%) patients, and T3a-specific upstaging was observed in 3380 (5.4%) patients. Patients who were older, male, and had comorbidities, and tumors that were cT1b, underwent RN, and had high Fuhrman grade were at a higher risk of pathological upstaging. Upstaging led to a 40% increased risk of death compared to patients who were not upstaged.

Conclusion

The rate of upstaging is not negligible (5–7% of the time), negatively impacts survival, and various patient and tumor characteristics can be used to predict upstaging.