Surgical treatment of biliary fistulas

The methods of diagnosis and operative treatment of internal (in 19 patients) and external (in 18) biliary fistulas, which appeared due to destructive and complicated forms of cholecystitis were analyzed.     

The podocyte and diabetes mellitus: is the podocyte the key to the origins of diabetic nephropathy?

PURPOSE OF REVIEW: Podocyte injury plays a key role in the development of diabetic nephropathy. This review discusses recent advances in our understanding of mechanisms of podocyte injury in diabetes mellitus and the associated alterations in the function of the glomerular filtration barrier. RECENT FINDINGS: The effects of hyperglycemia on critical podocyte parameters including cell-cell interactions, attachment to the glomerular basement membrane, and podocyte apoptosis have been determined in both cell culture and in-vivo models of diabetes mellitus. The podocyte has also been identified as a target of action for insulin and growth hormone, hormones with significant roles in the altered homeostasis of diabetes mellitus. SUMMARY: Understanding the cellular and molecular basis for changes in podocyte structure and function in diabetes mellitus may lead to novel diagnostic tools and treatment strategies for diabetic nephropathy.     

Ethical, legal, and social dimensions of epilepsy genetics

PURPOSE: Emerging genetic information and the availability of genetic testing has the potential to increase understanding of the disease and improve clinical management of some types of epilepsy. However, genetic testing is also likely to raise significant ethical, legal, and social issues for people with epilepsy, their family members, and their health care providers. We review the genetic and social dimensions of epilepsy relevant to understanding the complex questions raised by epilepsy genetics. METHODS: We reviewed two literatures: (a) research on the genetics of epilepsy, and (b) social science research on the social experience and social consequences of epilepsy. For each, we note key empiric findings and discuss their implications with regard to the consequences of emerging genetic information about epilepsy. We also briefly review available principles and guidelines from professional and advocacy groups that might help to direct efforts to ascertain and address the ethical, legal, and social dimensions of genetic testing for epilepsy. RESULTS: Genetic information about epilepsy may pose significant challenges for people with epilepsy and their family members. Although some general resources are available for navigating this complex new terrain, no guidelines specific to epilepsy have yet been developed to assist people with epilepsy, their family members, or their health care providers. CONCLUSIONS: Research is needed on the ethical, legal, and social concerns raised by genetic research on epilepsy and the advent of genetic testing. This research should include the perspectives of people with epilepsy and their family members, as well as those of health care professionals, policymakers, and bioethicists.     

Conditional Gene Targeting in Mouse Pancreatic ��-Cells: Analysis of Ectopic Cre Transgene Expression in the Brain

OBJECTIVE

Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain.

RESEARCH DESIGN AND METHODS

Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1^tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry.

RESULTS

All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)^89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)^1Lphi mice were the only line that lacked Cre activity in the brain.

CONCLUSIONS

Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.In vivo analysis of gene function in the pancreas and β-cells has benefited from the development of mouse lines expressing Cre in all pancreatic compartments or restricted to the islet β-cells. The choice of promoter to drive recombinase expression is critical for controlling the location and timing of gene activity. In addition, inducible versions of Cre recombinase, e.g., CreER, allow temporal control to the manipulation of gene activity, which becomes important when analyzing gene function at specific embryonic and adult stages (1,2). Promoters of the pancreas duodenal homeobox 1 (Pdx1) (3,4) and insulin (Ins1 and Ins2) (5–8) genes have been well characterized to allow the use of regulatory sequences for directing Cre expression to specific pancreatic cell populations. Commonly used transgenic mouse lines that employ rat Ins2 gene promoter sequences to drive Cre expression within the β-cell population include Ins2-Cre/RIP-Cre [Mouse Genome Informatics (MGI): Tg(Ins2-cre)^25Mgn and Tg(Ins2-cre)^1Herr] (9–11) and RIP-CreER [MGI: Tg(Ins2-cre/Esr1)^1Dam] (12). Pdx1 gene promoter sequences have proven useful for directing Cre expression throughout the early pancreatic epithelium (4,10,13,14) and to the endocrine cells of the pancreas (15). The Pdx1 gene is expressed early in pancreas development throughout the endoderm of the dorsal and ventral buds, but expression becomes restricted during development such that high levels of Pdx1 are maintained in the insulin-producing β-cells with lower levels in subpopulations of acinar cells (8,16). Examples of Pdx1-Cre transgenic lines include Pdx1-Cre^early [MGI: Tg(Pdx1-cre)^89.1Dam] (13), Pdx1-Cre^late [MGI: Tg(Ipf1-cre/Esr1)^1Dam/Mmcd] (10), Pdx1-Cre [MGI: Tg(Ipf1-cre)^1Tuv] (14), and Pdx1-CreER [MGI: Tg(Pdx1-cre/ERT)^1Mga] (15).To assess the specificity of recombination and perform lineage tracing analysis, reporter lines such as the ROSA26-stop-lacZ [MGI: Gt(ROSA)26Sor^tm1Sho], also known as R26R (17), or the ROSA26-stop-YFP [MGI: Gt(ROSA)26Sor^tm1(EYFP)Cos] (18) mice have been developed. Upon Cre-mediated recombination, these reporter lines activate expression of a β-galactosidase (β-gal) or a yellow fluorescent protein (YFP) reporter under the control of the ubiquitously active ROSA26 promoter, resulting in expression that is stably inherited by all cell progeny regardless of their differentiation fate.Here we show that most Cre lines currently being used to mediate pancreas or β-cell recombination also direct Cre expression to areas of the brain, and this may lead to altered gene expression in nutrient-sensing neurons that affects nutrient homeostasis.     

Research into the Strength of an Open Wagon with Double Sidewalls Filled with Aluminium Foam

The research is concerned with the use of double walls filled with aluminium foam for an open wagon in order to decrease the dynamic stresses during the operational modes. The research presents the strength calculation for the bearing structure of an open wagon with consideration of the engineering solutions proposed. It was found that the maximum equivalent stresses appeared in the bottom section of the centre sill behind the back support; they amounted to about 315 MPa and did not exceed the allowable values. The maximum displacements were detected in the middle section of the centre sill and amounted to 9.6 mm. The maximum deformations were 1.17 × 10⁻². The research also presents the strength calculation for a weld joint in the maximum loaded zones of the bearing structure of an open wagon and gives the results of a modal analysis of the bearing structure of the improved open wagon. It was found that the critical oscillation frequencies did not exceed the allowable values. The results of the research may be useful for those who are concerned about designing innovative rolling stock units and improving the operational efficiency of railway transport.     

Effect of hyperosmolality upon the mesothelial monolayer exposed in vivo and in situ to a mannitol-enriched dialysis solution

Studies done using the in vivo mouse model of population analysis of mesothelium showed that dialysis solutions containing high concentrations of glucose induced the development of a hypertrophic phenotype. Since these changes were neither related to the low pH nor to the presence of lactate buffer, we hypothesized that the presence of glucose was at the origin of the observed alterations. Theoretical analysis of the problem points to three possible mechanisms: hyperosmolality; metabolic changes derived from the high-glucose concentration itself, and/or the presence of products derived from the nonenzymatic degradation of glucose. The present study was designed to demonstrate or rule out the eventual effect of hyperosmolality upon the monolayer, applying the in vivo mouse model of population analysis of mesothelium. For this purpose, morphometric observations made in mice injected once a day during 30 consecutive days with a filter-sterilized 4.25% solution of mannitol (233.29 mM) were compared with those seen in intact mice and in a previously reported group of animals exposed to heat-sterilized fluid, having an equimolar concentration of glucose (235.9 mM), and the same osmolality (486 mosm/l) and electrolyte concentrations. The main findings observed in the mannitol-treated mice during the period of exposure included increased cell size and cytoplasmic surface area, as well as decreased cell viability. The regenerative capabilities of the exposed mesothelium remained intact. After a recovery period of 7 days, the aforementioned parameters reverted to normal values. This pattern is significantly different from the hypertrophic, senescent and low regenerative phenotype observed in mice treated with the high-glucose concentration solution. We conclude that, at least in the in vivo and in situ setup, the detrimental effects of hyperosmolality alone upon the exposed mesothelium are quite limited and fully reversible within a recovery period of 7 days.     

A novel cytotoxicity assay to evaluate antigen-specific CTL responses using a colorimetric substrate for Granzyme B

We have utilized the unique enzymatic properties of a key cytotoxic mediator in target cell destruction, Granzyme B (GrB), to establish an attractive alternative to 51Cr-release assays for the assessment of antigen-specific CTL responses. A number of potential colorimetric peptide substrates were compared to evaluate levels of GrB activity in cytolytic cells. The most specific and sensitive substrate for GrB was Ac-IEPD-pNA, as shown by the minimal enzymatic hydrolysis in apoptotic Jurkat cells and strong hydrolysis in human NK cells. When human peripheral blood lymphocytes were stimulated in vitro, elevated GrB levels were detected by both Ac-IEPD-pNA and a GrB ELISA. Analysis of allo-antigen-specific murine CTLs revealed that GrB exocytosis was only detectable upon challenge with appropriate allogeneic target cells and strongly correlated to 51Cr-release data. The validity of using Ac-IEPD-pNA in vaccine trials was demonstrated in mice immunized with allogeneic P815 cells, where GrB enzymatic activity was measurable in ex vivo splenocytes cell cultures only upon co-incubation with P815 targets. Additionally, influenza-infected mice were also assessed for GrB activity following in vitro peptide-stimulation of splenocytes and strongly reflected both peptide-specific tetramer staining and 51Cr-release results. The novel cytotoxic assay presented here should give investigators a sensitive, cross-species, nonradioactive alternative to 51Cr-release assays as a means to assess antigen-specific CTL responses in vaccine trials.