Interaction between cytokines and inflammatory cells in islet dysfunction, insulin resistance and vascular disease.

Inflammation is an established pathogenic player in insulin resistance, islet demise and atherosclerosis. The complex interactions between cytokines, immune cells and affected tissues result in sustained inflammation in diabetes and atherosclerosis. 12- and 15-lipoxygenase (LO), such as 12/15-LO, produces a variety of metabolites through peroxidation of fatty acids and potentially contributes to the complex molecular crosstalk at the site of inflammation. 12- and 15-LO pathways are frequently activated in tissues affected by diabetes and atherosclerosis including adipose tissue (AT), islets and the vasculature. Moreover, mice with whole body and tissue-specific knockout of 12/15-LO are protected against insulin resistance, hyperglycaemia and atherosclerosis supporting functional contribution of 12- and 15-LO pathways in diabetes and atherosclerosis. Recently, it has emerged that there is a temporal regulation of the particular isoforms of 12- and 15-LO in human AT and islets during the development of type 1 and type 2 diabetes and obesity. Analyses of tissues affected by diabetes and atherosclerosis also implied the roles of interleukin (IL)-12 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1) in islets and IL-17A in atherosclerosis. Future studies should aim to test the efficacy of inhibitions of these mediators for treatment of diabetes and atherosclerosis.

Regulation of NOX-1 expression in beta cells: a positive feedback loop involving the Src-kinase signaling pathway.

NADPH oxidase-1 (NOX-1) is upregulated in beta cells in response to pro-inflammatory cytokines. Inhibition of NADPH oxidase activity blocked stimulated NOX-1 expression (p<0.05). Regulation of NOX-1 expression in beta cells followed modulation of cellular reactive oxygen species (ROS); pro-oxidants increased NOX-1 (p<0.001) and anti-oxidants decreased NOX-1 (p<0.05). Activation of Src-kinase followed ROS elevation. Inhibition of Src-kinase decreased NOX-1 expression (p<0.01). Beta cell dysfunction, measured by elevated MCP-1 expression, loss of glucose-sensitive insulin secretion or cell death, was induced by pro-inflammatory cytokine stimulation. Importantly, inhibition of Src-kinase or NOX-1 preserved beta cell function and survival. Collectively, these data indicate that expression of NOX-1 in beta cells is regulated in a feed-forward loop mediated by ROS and Src-kinase. Uncoupling of this feed-forward activation could provide new approaches to preserve and protect beta cells in diabetes.

Production and function of IL-12 in islets and beta cells.

AIMS/HYPOTHESIS: IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. METHODS: In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1ß and IFN-γ). RESULTS: The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. CONCLUSIONS/INTERPRETATION: These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Regional heterogeneity and pharmacodynamics in human solid tumor histoculture.

PURPOSE: Human solid tumor histocultures represent a clinically relevant experimental system for pharmacodynamic study. The evaluation of the drug-induced antiproliferative effect in histocultures is usually performed by manual microscopic scoring of individual cells. This procedure, because of its labor intensive nature, is performed on a single microscopic field, i.e. the field with the highest proliferative activity. Because regional heterogeneity in a solid tumor may result in different drug sensitivities in different parts of a tumor, there is the question as to whether the pharmacodynamic data determined in the most proliferative field is representative of those in the whole tumor. This question was addressed in the present study. METHODS: A recently developed automated image analysis method was used to measure the labeling index of tumor cells. The drug-induced inhibition of DNA precursor incorporation into nuclei of cells in the region with the highest proliferative activity was compared to the inhibition in cells in the entire histoculture. This study was performed in human bladder tumor histocultures treated with several drugs (doxorubicin, mitomycin C, paclitaxel and 5-fluorouridine). A total of 724 pairs of data obtained from untreated and drug-treated histocultures (each data point representing the average of 1 to 6 tumor histocultures) were analyzed. RESULTS: The absolute value of the labeling index in the most proliferative region (LI(one)) was significantly higher than the absolute value of the labeling index in the whole tumor (LI(all)), in both untreated and drug treated samples (mean difference of 18%, range 1-27%). However, when the absolute LI values in drug-treated samples were normalized to the values in untreated controls and expressed as a percentage of control, and used to construct the concentration-response curves, the two curves obtained using LI(one) and LI(all) yielded comparable pharmacodynamic parameters, i.e. curve shape parameters and drug concentrations that produce 30, 50, and 70% inhibition. CONCLUSION: These results indicate comparable pharmacodynamics in the most proliferative region and the whole tumor, and confirm the validity of using the most proliferative field for evaluating chemosensitivity in solid tumor histocultures.

Determinants of paclitaxel penetration and accumulation in human solid tumor.

The present study examined the determinants of the penetration and accumulation of [(3)H]paclitaxel (12-12,000 nM) in three-dimensional histocultures of patient tumors and of a human xenograft tumor in mice. The results showed 1) significant and saturable drug accumulation in tumors, 2) extensive drug retention in tumors, and 3) a slower penetration but a more extensive accumulation in the xenograft tumor compared with patient tumors. Drug penetration was not rate-limited by drug diffusion from medium through the matrix supporting the histocultures. The difference in the expression of the mdr1 P-glycoprotein did not fully account for the difference in the drug accumulation in xenograft and patient tumors. Autoradiography and imaging were used to evaluate the spatial relationship between tumor architecture, tumor cell distribution, and drug distribution as a function of time and initial drug concentration in culture medium. The tumor cell density and the kinetics of drug-induced apoptosis were also evaluated. The results indicate that a high tumor cell density is a barrier to paclitaxel penetration and that the apoptotic effect of paclitaxel enhances its penetration in solid tumor. These factors are responsible for the time- and concentration-dependent drug penetration rate, with drug penetration confined to the periphery until apoptosis and reduction of epithelial cell density occurred at 24 h, after which time paclitaxel penetrated the inner parts of the tumor.

Proliferation indices as molecular pharmacodynamic endpoints in evaluation of anticancer drug effect in human solid tumors.

PURPOSE: The present study compared proliferative indices, i.e. incorporation of DNA precursor (i.e. thymidine or TdR, and bromodeoxyuridine or BrdU) and expression of proliferating cell nuclear antigen (PCNA), as molecular pharmacodynamic endpoints in evaluation of anticancer drug effect in human solid tumors. METHODS: Tumor specimens obtained from patients were grown as histocultures. After treatment with doxorubicin, mitomycin C, and/or paclitaxel, cells labeled by [3H]TdR were identified using autoradiography, and cells labeled by BrdU and PCNA were identified using immunohistochemical techniques. Drug effect was measured as reduction of DNA precursor-labeled cells or PCNA-expressing cells. RESULTS: The results indicate that (a) the two DNA precursors, TdR and BrdU, labeled the same cells and resulted in identical pharmacodynamics, (b) the pharmacodynamics established using inhibition of DNA precursor incorporation were qualitatively and quantitatively different from the pharmacodynamics established using inhibition of PCNA expression, (c) the inhibition of PCNA expression was erratic in some tumors, and (d) the differences in pharmacodynamics established using the two end points are drug-specific, with greater differences for paclitaxel than for mitomycin C. CONCLUSIONS: The erratic results measured by the PCNA labeling method suggest that this method may be less reliable than the conventional DNA precursor labeling method. The finding of identical pharmacodynamics of doxorubicin and paclitaxel established using BrdU and [3H]TdR indicates that the two precursors are interchangeable. Because the methodology for detecting BrdU incorporation requires less time and does not require the use of radioactivity, we conclude that inhibition of BrdU incorporation represents a useful endpoint for evaluating the antiproliferative activity of anticancer drugs in human solid tumors.

Application of automatic thresholding in image analysis scoring of cells in human solid tumors labeled for proliferation markers.

We previously reported an image analysis program that uses four investigator-defined parameters including two thresholds, i.e., gray-level threshold (GLT) and hue threshold (HT), to determine the number of cells (TC) and the proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdUrd) labeling indices (LI) in human solid tumors. The present study investigated if the accuracy and reproducibility of image analysis results can be improved by using computer-defined GLT and HT. Three investigators evaluated 142 images on 3 days, using visual analysis and four image analysis routines, which used different combinations of computer- and investigator-selected GLT and HT. The data show that image analysis using computer-selected GLT and HT yielded (i) LI of PCNA and BrdUrd that were indistinguishable from visual analysis, (ii) equal (BrdUrd LI) or better (TC and PCNA LI) inter-day reproducibility relative to visual analysis results, and (iii) results that were equally (TC) or more accurate (LI of PCNA and BrdUrd) with higher inter-day reproducibility (TC and LI of PCNA and BrdUrd) than image analysis obtained using investigator-defined thresholds. We conclude that the use of computer-defined GLT and HT improved the accuracy and reproducibility of image analysis results.

Comparative scoring by visual and image analysis of cells in human solid tumors labeled for proliferation markers.

This study determined the validity of an image analysis program developed to score individual cells in human solid tumors labeled by proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdUrd). The program used nuclear size, grey level, and perimeter convexity to identify cells, and evaluated labeling by the fraction of nuclear area displaying positive immunostaining (MPB). Total cell number (TC) and BrdUrd or PCNA labeling index (LI) were evaluated in 142 images using visual (TCvisual, LIvisual) and image analysis (TC(IA), LI(IA)). Without the perimeter convexity criterion, image analysis resulted in a) TC(IA) equal to TCvisual in spite of the presence of various non-cellular objects and b) significant correlations between LI(IA) and LIvisual for PCNA and BrdUrd, although for these markers the LI(IA) were 4 and 6% lower than their respective LIvisual. Both visual and image analyses yielded significant inter-investigator variation among three investigators (coefficient of variation between 8.2 and 47.5%) and significant intra-investigator, inter-day variation (coefficients of variation between 3.8 and 51.8%). We conclude that image analysis using size, grey level and MPB is a valid alternative to visual scoring of PCNA and BrdUrd LI in individual cells.

The evolution of duplicate glyceraldehyde-3-phosphate dehydrogenase genes in Drosophila.

In Drosophila melanogaster there are two genes which encode the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Gapdh-43E and Gapdh-13F. We have shown that Gapdh-43E codes for the GAPDH subunit with an apparently larger molecular weight while Gapdh-13F encodes the GAPDH subunit having an apparently smaller molecular weight. Immunoblots of sodium dodecyl sulfate gels were used to survey species from throughout the genus and results indicated that two classes of GAPDH subunits are present only in Drosophila species of the melanogaster and takahashi subgroups of the melanogaster group. Only the smaller subunit is found in species of the obscura group while all other species have only a large subunit. Drosophila hydei was analyzed at the DNA level as a representative species of the subgenus Drosophila. The genome of this species has a single Gapdh gene which is localized at a cytogenetic position likely to be homologous to Gapdh-43 E of D. melanogaster. Comparison of its sequence with the sequence of the D. melanogaster Gapdh genes indicates that the two genes of D. melanogaster are more similar to one another than either is to the gene from D. hydei. The Gapdh gene from D. hydei contains an intron following codon 29. Neither Gapdh gene of D. melanogaster has an intron within the coding region. Southern blots of genomic DNA were used to determine which species have duplicate Gapdh genomic sequences. Gene amplification was used to determine which species have a Gapdh gene that is interrupted by an intron. Species of the subgenus Drosophila have a single Gapdh gene with an intron. Species of the willistoni and saltans groups have a single Gapdh gene that does not contain an intron.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between the width of the mouth, interalar width, bizygomatic width, and interpupillary distance in edentulous patients.

The width of the mouth, interalar width, bizygomatic width, and interpupillary distance were measured in edentulous patients. The widths varied widely, even when the population was separated into groups by sex and/or race. When mean values were studied, black men differed significantly from black women, white women, and white men in interalar and bizygomatic widths; white women differed from the other groups in all widths. No correlation was found between the widths for the population as a whole, nor when the population was further divided into race, sex, or group. When artificial teeth were chosen for eight randomly selected patients using a method recommended for each of the widths, the same mold was dictated by two methods for seven patients, and by three methods for five patients.

Hydroxymethylglutaryl coenzyme A reductase activity of adult Hymenolepis diminuta.

The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestode's cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestode's HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.

A quantitative acoustic microscope with multiple detection modes.

An acoustic microscope that permits operation with both toneburst and impulse excitation of the lens is presented. Either mode can be selected and combined with mechanical scanning in any direction. In the impulse-excited mode, the specular and Rayleigh signals from the sample are resolved in time, and analysis is performed to obtain surface wave propagation parameters. The power of the simultaneous application of these techniques is illustrated by measurements on specimens of intact and fractured glass and duraluminum. Reflection and transmission coefficients for a crack are measured, and conclusions are drawn about V(z) processing. These results are significant because the images of cracks produced by the conventional toneburst scanning acoustic microphone (SAM) tend to be complex. Diffraction from the tips of cracks is observed in the microscope.

A rater training program in removable prosthodontics.

Four faculty members in removable prosthodontics participated in a three-phase rater training program. Nine complete dentures were given an overall rating by three outside experts to obtain an accuracy measure. A three-point rating scale was used: R (cannot be appreciably improved), S (clinically acceptable), and T (clinically unacceptable). During the pretraining phase, the average interrater reliability was .57 as estimated by intraclass correlation, and the mean accuracy correlation was .76. The rater training phase consisted of a four-hour session including presentations and discussions of rating terminology and formats, observer accuracy, and related issues. In the post-training phase, the same nine complete dentures were rated by the four faculty members, using the same three-point rating scale and a five-point scale. The average post-training interrater reliabilities were .56 and .76 on the three- and five-point scales, respectively. The mean accuracy correlation was .78.