mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans.

AIMS/HYPOTHESIS: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. METHODS: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. RESULTS: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. CONCLUSIONS/INTERPRETATION: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Glucose-stimulated increment in oxygen consumption rate as a standardized test of human islet quality.

Standardized assessment of islet quality is imperative for clinical islet transplantation. We have previously shown that the increment in oxygen consumption rate stimulated by glucose (DeltaOCR(glc)) can predict in vivo efficacy of islet transplantation in mice. To further evaluate the approach, we studied three factors: islet specificity, islet composition and agreement between results obtained by different groups. Equivalent perifusion systems were set up at the City of Hope and the University of Washington and the values of DeltaOCR(glc) obtained at both institutions were compared. Islet specificity was determined by comparing DeltaOCR(glc) in islet and nonislet tissue. The DeltaOCR(glc) ranged from 0.01 to 0.19 nmol/min/100 islets (n = 14), a wide range in islet quality, but the values obtained by the two centers were similar. The contribution from nonislet impurities was negligible (DeltaOCR(glc) was 0.12 nmol/min/100 islets vs. 0.007 nmol/min/100 nonislet clusters). The DeltaOCR(glc) was statistically independent of percent beta cells, demonstrating that DeltaOCR(glc) is governed more by islet quality than by islet composition. The DeltaOCR(glc), but not the absolute level of OCR, was predictive of reversal of hyperglycemia in diabetic mice. These demonstrations lay the foundation for testing DeltaOCR(glc) as a measurement of islet quality for human islet transplantation.

Increased expression of cyclooxygenase-2 in human pancreatic islets treated with high glucose or ligands of the advanced glycation endproduct-specific receptor (AGER), and in islets from diabetic mice.

AIMS/HYPOTHESIS: The cyclooxygenase-2 (PTGS2, previously known as COX2) enzyme and its products, such as prostaglandin E(2) (PGE(2)), have been implicated in the pathogenesis of several inflammatory diseases including islet dysfunction under diabetic conditions. In this study we evaluated whether diabetic conditions in vitro, such as high-glucose (HG) culture or AGE, or in vivo in animal models of diabetes can induce PTGS2 expression and activity in pancreatic islets. MATERIALS AND METHODS: Isolated human pancreatic islets were treated for 24 h with HG (25 mmol/l) or with S100b (5 mg/l), a specific ligand for the AGE-specific receptor. PTGS2 and cyclooxygenase-1 (PTGS1, previously known as COX1) mRNA, protein expression and product PGE(2) were analysed by RT-PCR, Western blots and specific enzyme immunoassay respectively. Islet PTGS2 production in animal models was assessed by immunofluorescence. RESULTS: Treatment of human pancreatic islets with HG and S100b led to a three-five-fold induction of PTGS2 mRNA (p<0.001). PTGS2 protein and its product PGE(2) (351.4+/-13.05 fg/ml vs control 39.4+/-0.11 fg/ml) were also increased (p<0.001). Pretreatment with specific inhibitors demonstrated the involvement of protein kinase C and oxidant stress in S100b- and HG-induced PTGS2 expression. However, insulin secretion was not significantly altered by S100b. Double immunofluorescent staining showed increased PTGS2 production in pancreatic islets from diabetic mice relative to corresponding controls. CONCLUSION/INTERPRETATION: These results show for the first time that diabetes as well as diabetic conditions such as AGE and HG in vitro can directly upregulate the expression of the inflammatory PTGS2 gene in pancreatic islets. This might contribute to the pathogenesis of islet dysfunction in diabetes.

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Identification of dementia cases in the community: a Brazilian experience.

OBJECTIVES: To describe the application in Brazil of a simple, low-cost procedure, developed in India by the 10/66 Dementia Research Group, for the identification of dementia cases in the community. DESIGN: Community-based dementia case-finding method. SETTING: Piraju, São Paulo, Brazil. PARTICIPANTS: Twenty-five community health workers were trained to identify dementia cases in 2,222 people aged 65 and older in Piraju, a Brazilian town with 27,871 inhabitants. MEASUREMENTS: After the training, the health workers prepared a list of possible cases that afterward an experienced psychiatrist clinically evaluated, according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), criteria and the Clinical Dementia Rating. RESULTS: Of the 72 cases that were clinically assessed, 45 met the DSM-IV diagnostic criteria for dementia. Therefore, the positive predictive value of this case finding method was 62.5%; the estimated frequency of dementia was 2%. Most of the confirmed cases met clinical criteria for Alzheimer's disease and vascular dementia. CONCLUSION: This simple method was appropriate to identify cases of dementia in the general population and can possibly be extended to other developing countries with limited resources to be applied in health programs.

Biologic effects of low-level electromagnetic fields: current issues and controversies.

There is uncertainty and controversy about the extent to which low level electromagnetic fields may cause deleterious effects, but even experts who are skeptical about many supposed hazards are willing to agree that electromagnetic fields even weaker than those in the MR environment can have effects under certain conditions. In order that readers can familiarize themselves enough with the subject to make an informed independent assessment, discuss it knowledgeably in public, and have the means with which to evaluate new developments and avoid experimental pitfalls if planning their own research in the area, they are provided with some of the most recent finding of in vitro and in vivo research from outside the MR literature as well as some of the results and controversies coming from recent epidemiological studies.

Effects function analysis of ELF magnetic field exposure in the electric utility work environment.

The incomplete understanding of the relation between power-frequency fields and biological responses raises problems in defining an appropriate metric for exposure assessment and epidemiological studies. Based on evidence from biological experiments, one can define alternative metrics or effects functions that embody the relationship between field exposure patterns and hypothetical health effects. In this paper, we explore the application of the "effects function" approach to occupational exposure data. Our analysis provides examples of exposure assessments based on a range of plausible effects functions. An EMDEX time series data set of ELF frequency (40-800 Hz) magnetic field exposure measurements for electric utility workers was analyzed with several statistical measures and effects functions: average field strength, combination of threshold and exposure duration, and field strength changes. Results were compared for eight job categories: electrician, substation operator, machinist, welder, plant operator, lineman/splicer, meter reader, and clerical. Average field strength yields a different ranking for these job categories than the ranks obtained using other biologically plausible effects functions. Whereas the group of electricians has the highest exposure by average field strength, the group of substation operators has the highest ranking for most of the other effects functions. Plant operators rank highest in the total number of field strength changes greater than 1 microT per hour. The clerical group remains at the lowest end for all of these effects functions. Our analysis suggests that, although average field strength could be used as a surrogate of field exposure for simply classifying exposure into "low" and "high," this summary measure may be misleading in the relative ranking of job categories in which workers are in "high" fields. These results indicate the relevance of metrics other than average field strength in occupational exposure assessment and in the design and analysis of epidemiological studies.

Effects function simulation of residential appliance field exposures.

This paper demonstrates the application of effects function analysis to residential magnetic field exposure, focusing on appliance sources and mitigation choices. Residential field exposure time series were synthesized by using a sample of background household field measurements, a model of average daily appliance use, and a small sample of EMDEX data of field exposure from 12 household strength window, sudden field changes) were simulated by using the synthesized time series data for different exposure situations, such as high and low levels of appliance use, simple avoidance, and use of a set of hypothetical "low field" appliances (50% lower fields). In particular, field exposure from the use of bedside clocks and electric blankets was examined. Results demonstrate that the choice of effects function is critical for the ranks of field sources and exposure reduction choices. For the effects function of average field strength with or without a threshold, exposure from background fields dominated exposure from all appliances except for bedside clocks and electric blankets. In the case of the field strength window effects function, the dominant field sources changed with the width of the window. For the effects function based on rapid field changes, appliance use was the major source of exposure. Because of the small sample of our data set and other simplifications, specific results should be viewed as illustrative.

Review of uses of the laryngeal mask in ENT anaesthesia.

A review of the various uses of the laryngeal mask in otorhinolaryngological surgery is presented. Its primary use is in airway management, especially during recovery from anaesthesia.

Distinguishability of the video display terminal (VDT) as a source of magnetic field exposure.

Magnetic field strength or its surrogate has been used as the measurement of exposure in all studies involving field exposure. Video display terminals (VDTs) are considered a minor source on this scale because its field strengths lie in the low range of magnetic fields. Several experiments show, however, that field strength may not be the only relevant variable in determining exposure. This paper explores the use of a method based on "effects functions" to determine the extent to which the VDT can be distinguished from other sources. It is shown that VDT exposure may be of consequence if exposure depends on certain types of time variation of the field. Because of the incompleteness of science in this area, this is a demonstration of a method rather than an actual demonstration of VDT as a significant source of exposure.

A method for evaluating transmission line magnetic field mitigation strategies that incorporates biological uncertainty.

A method to determine how much reduction in public exposure to power frequency magnetic fields can be obtained for different levels of investment is presented. Which if any "effects function" best describes the relationship between field exposure and biological effect is uncertain at this time. Also, in a particular context such as construction of new transmission lines there are a variety of different technologies which might be used to reduce exposure. We describe and demonstrate a method by which exposure reduction supply curves (i.e., the cost of purchasing different amounts of exposure reduction given various mitigation options) can be estimated parametrically for different exposure conditions and effects functions, and we display illustrative results.

A method for assessing alternative effects functions that uses simulation with EMDEX data.

A method for evaluating a variety of alternative biologically plausible effects functions through the use of simulation studies conducted on personal-monitor exposure data is described. Using magnetic field time series collected with EMDEX instruments, we demonstrate how the method can be used to explore 1) how the outputs from various effects functions simulations compare to the results obtained by assuming that effects are proportional to time average field strength; 2) how the results of epidemiological studies might be used to assess the relative likelihood that each of the alternative effects functions describes biological reality; and 3) how the results might be used to assess possible health risks. Although the available data are sufficient to demonstrate the general method, they are not yet sufficient to support actual discrimination among possible alternatives. The arguments on the use of the method are for illustrative purposes only.

Alternative functional relationships between ELF field exposure and possible health effects: report on an expert workshop.

If exposure to 60 Hz fields poses risks to public health, the relationship between exposure and risk may involve something other than the product of field strength and time. Such alternative possible relations, or "effects functions," are of great interest to epidemiologists, engineers, risk analysts, and regulators. A structured survey and workshop were used to explore whether leading researchers in bioelectromagnetics share similar views about alternative possible effects functions. Substantial agreement was found about several effects functions in a few specific contexts such as calcium-ion efflux and cell signalling, and biosynthesis pathways. No significant agreement emerged in many other contexts. No effects function possibilities were ruled out. Further effort of this sort was judged unlikely to yield greater consensus until more complete scientific understanding becomes available. However, a series of structured workshops on research planning and priority setting were judged to hold great potential for useful results.

Antigenic protein from Mycobacterium leprae released in macrophages in vitro as indicator of viability of bacteria.

Peritoneal macrophages from randombred, Swiss white mice, when cultured and infected with Mycobacterium leprae for 24 hours, are able to show the presence of antigen(s) with binding affinity to antibodies present in the sera of bacteriologically positive, lepromatous leprosy patients. Such antibodies are not seen in sera from normal and healthy persons, tuberculoid leprosy patients, or long-term-treated, bacteriologically negative, lepromatous leprosy patients. The production of the antigen(s) is blocked by the anti-M leprae drug rifampin. Other mycobacteria when incubated with macrophages from mice show very little antigens in the lysate but the antigens have an equal affinity for antibodies in sera from both normal individuals and lepromatous patients. Only the lysates from macrophages exposed to live M. leprae could discriminate and could exhibit differential binding to sera from leprosy patients compared to sera from normal individuals. This antigen(s) does not have any binding ability to the monoclonal antibodies available to the antigens of M. leprae identified at present and shown to be specific to M. leprae. This indicates a separate identity of this product which has potential for further exploitation in exploring host-pathogen interactions related specifically to the leprosy infection and the tolerance of M. leprae inside cells.

Viability of Mycobacterium leprae inside macrophages from different strains of mice and possible genetic control.

Peritoneal macrophages from Swiss white mice in vitro tolerated Mycobacterium leprae and allowed metabolism of the bacteria leading to release of bacteria-specific antigenic protein. This was associated with the maintenance of viability of the bacilli inside the cells. Macrophages from C57BL mice reduced viability of M. leprae after phagocytosis, and this was associated with the production of superoxide. Blockage of superoxide production resulted in maintaining viability of the cells of these mouse strains. Associated with loss of viability of the bacilli is the absence of the production of antigenic protein in the lysate. Interestingly, the maintenance of viability or loss of viability and the factors controlling such viability in the macrophages of Swiss white and C57BL mice, respectively, appeared to be genetically controlled.

Effect of gossypol on pituitary reproductive axis: ultrastructural and biochemical studies.

Oral administration of gossypol induced sterility in male rats by 10 weeks, at a dose of 15 mg/kg body weight/day. The pituitary FSH gonadotroph cells showed dilated endoplasmic reticulum and accumulation of secretory granules in the cytoplasm. LH cells were degranulated. The Leydig cells showed enhanced synthetic activity. There was no change in testis weight and testicular RNA, lipids and cholesterol in the treated group while significant increase was observed in DNA content. Testicular sialic acid content decreased significantly over controls. The Sertoli cells, spermatogonia, spermatocytes and early spermatids were not affected after the treatment. The weights of prostate, seminal vesicle were recorded normal and there were no ultrastructural variations. The levels of acid and alkaline phosphatase and RNA in prostatic tissue were insignificant as compared with controls. However, DNA content of prostate gland showed a significant increase. Sialic acid of seminal vesicle + coagulating gland were within the control range. A marked reduction in fructose values from the same organ was noted.

Lay understanding of low-frequency electric and magnetic fields.

People do not start with a blank slate when they hear risk-communication messages. All such messages are processed through existing knowledge structures and understanding. Hence, to design effective and reliable risk-communication materials one must understand the state of people's knowledge--correct and incorrect--about an issue. We developed a simple "mental model" of what people minimally need to know to make informed decisions about field-related issues. Then we performed studies to explore how and to what extent respondents of various groups understood physical properties of 60-Hz electric and magnetic fields. Actual knowledge of respondents was then compared with the predicates of the model. Electrical engineering juniors and semi-technical employees of utilities displayed a good command of most of the concepts in the simple model, but little awareness of the limits to their knowledge. Lay respondents correctly knew only a few of the simplest elements of the model, but they displayed a much greater awareness of the limits to their knowledge. Both lay and semi-technical respondents were found to share several misconceptions. On average, they correctly rank-ordered some common field-exposure conditions by field strength, but they could not differentiate between electric and magnetic fields and could not differentiate among field strengths associated with different appliances. Most respondents dramatically underestimated the range of actual field strengths. Many respondents understood that field strength decreases with distance from a source, but they underestimated the rate of decrease. In contrast to X-rays and microwaves, which respondents appeared to think about in rather similar terms, 60-Hz fields were not thought of as being highly similar to any other agent, although the closest parallels were found with ultrasound. Changes in mood, thought, and behavior, and the existence of an "electrical aura," were all seen as plausible results of exposure to a 60-Hz field. Although lay respondents displayed a variety of incomplete and confusing ideas, most of these ideas probably do not pose significant obstacles to the learning of a correct, simple, mental model.