Relationship Between Hyperglycemia and Heart Transplant Rejection.

PURPOSE: Hyperglycemia increases risks of kidney and liver transplant rejection. To determine whether perioperative and subsequent glycemic control was associated with increased risk of heart transplant rejection over the year after transplantation, we performed a retrospective analysis of glycemic control and rejection rates in heart transplantation patients. METHODS: Perioperative glucose levels were analyzed in 157 patients undergoing transplantation at Northwestern Memorial Hospital from June 2005 to December 2012 and compared in patients with and without rejection found on routine follow-up biopsy specimens. RESULTS: Grade ≤1R rejection on biopsy was observed in 116 patients and grade ≥2R rejection (grade requiring increased anti-rejection treatment) in 41 patients. Although no significant differences in the preoperative fasting or inpatient mean glucose levels were found, the mean glucose levels from discharge to 1 year trended higher in those with grade ≥2R compared to grade ≤1R (128.8 ± 40.9 versus 142.2 ± 46.6 mg/dL, P = .084). In a multivariable logistic regression model, neither the lowest nor highest quartile of glucose levels had significantly different odds ratios (ORs) for the development of ≥2R compared to the middle 50% glucose levels. Older age (OR 0.96, P = .020) and higher body mass index levels (OR 0.86, P = .004) were significantly associated with lower odds of developing grade ≥2R. CONCLUSIONS: Although the glucose trend regarding rejection was not statistically significant, we cannot exclude the possibility that much higher glucose levels would influence rejection rates.

Genotoxicity studies of a desealant solvent mixture, SR-51.

The Royal Australian Air Force (RAAF) has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977 to mid-1990s was the cause of health problems, including cancer. Particular concern was directed at SR-51, a desealant chemical mixture containing the following four solvents: aromatic 150 solvent (Aro150), dimethylacetamide, thiophenol (TP), and triethylphosphate. The present study examined the mutagenic potential of SR-51 using a range of well-known mutagen and genotoxin assays. The tests used were i) a modified version of the Ames test, ii) the mouse lymphoma assay, iii) the comet assay (a single-cell gel electrophoresis assay), and iv) a mouse micronucleus test. The modified Ames test used mixed bacterial strains in liquid suspension media. The Ames test results showed that SR-51 (tested up to the cytotoxic concentration of 36 microg/ml, 30 min incubation) in the presence and absence of S9 metabolic activation was not mutagenic. The mouse lymphoma assay used cultured mouse lymphoma cells in a microwell suspension method. The mouse lymphoma assay was also negative with SR-51 (tested up to the cytotoxic concentration of 22.5 microg/ml, 3 h incubation) in the presence and absence of S9 metabolic activation. The Comet assay, using cultured mouse lymphoma cells, showed no evidence of DNA damage in cells exposed up to the cytotoxic concentration of SR-51 at 11.25 microg/ml. The in-vivo mouse micronucleus test was undertaken in wild-type C57Bl6J male mice dosed orally with SR-51for 14 days with a single daily dose up to 360 mg/kg/day (the maximum-tolerated dose). No increases were observed in micronuclei (MN) frequency in bone marrow collected (24 h after final dose) from SR-51-treated mice compared to the number of MN observed in bone marrow collected from untreated mice. Tissues collected from treated mice at necropsy demonstrated a significant increase in spleen weights in the high dose mice. Gas chromatography analysis of SR-51 identified more than 40 individual components and an oxidation product, diphenyldisulfide derived from TP under conditions of mild heating. In conclusion, there was no evidence that SR-51 is mutagenic.

Effects of a desealant formulation, SR-51(®) and its individual components on the oxidative functions of mitochondria.

The Royal Australian Air Force has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977-mid-1990s was the cause of health problems. Particular concern was directed at a desealant chemical mixture known as SR-51(®). The current study, using in vitro submitochondrial assays, was designed to investigate the relative toxicities of the four components of SR-51(®) (Aromatic 150 solvent (Aro150), dimethylacetamide (DMA), thiophenol (TP) and triethylphosphate (TEP)). Based on the EC(50) values, TP and Aro150 were the most toxic components and were markedly more toxic than TEP and DMA.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

The in vitro evaluation of the toxicities of three related herbicide formulations containing ester derivatives of 2,4,5-T and 2,4-D using sub-mitochondrial particles.

The aim of this investigation was to determine the contribution made by the different components of herbicide formulations to the overall toxicity of the formulations. Three related herbicide formulations were chosen. The first, Agent Orange, consisted only of the butyl esters of 2,4,5-T and 2,4-D. The second was Agent Orange diluted with diesel fuel and the third formulation tested was a tree and blackberry killer, which consisted of the butyl ester of 2,4,5-T, the ethyl ester of 2,4-D, diesel fuel and two surfactants. The potential toxic effects of these three formulations were evaluated by determining their inhibitory effects on the oxidative functions of submitochondrial particles prepared from beef heart mitochondria. The effective concentration that caused a 50% inhibition of the activities of the submitochondrial particles was determined for all three formulations. When the toxicity of the individual components of these formulations was evaluated, it was established that the so-called 'inert' components i.e. diesel fuel and surfactants contributed approximately 50% of the overall toxicity of the complete formulations. Hence the results confirm the importance of evaluating the toxicity of complete formulations, rather than only focussing on the active components. While cellular and sub-cellular assays cannot account for pharmacokinetic and pharmacodynamic changes that may affect the toxicity of xenobiotics, the sub-mitochondrial particle test is useful as an initial screening assay.

Effects of a herbicide formulation, Tordon 75D, and its individual components on the oxidative functions of mitochondria.

This investigation evaluates the toxicity of a herbicide formulation, as well as testing its active and other components (other components comprise all components of Tordon 75D excluding the active components: i.e. the solvents, triisopropanolamine and diethyleneglycol monoethyl ether, a silicone defoamer and a proprietary surfactant, polyglycol 26-2). The results showed that Tordon 75D (a mixture of the triisopropanolamine salts of 2,4-dichlorophenoxy acetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram) and its other components) impaired the oxidative functions of submitochondrial particles (SMPs). The effective concentrations that caused 50% inhibition of SMP activity (EC50s) for Tordon 75D were in the low micromolar range for 2,4-D and picloram in the presence of the other components, while in the absence of the other components exposure to 136 times higher concentrations of the triisopropanolamine forms of 2,4-D and picloram administered as a mixture were required to inhibit the oxidative functions of SMPs. Tordon 75D also significantly decreased the respiratory control ratio of intact rat liver mitochondria. The results show that the toxic effects of Tordon 75D on SMPs (at the EC50) and intact rat liver mitochondria were not due to any additive or synergistic actions of a mixture of its active and other components, but rather were caused solely by the proprietary surfactant. Since mitochondria are responsible for over 90% of the energy production in all eukaryotic organisms, the use of the SMP assay provides a convenient in vitro assay for evaluating cellular toxicity and can be regarded as an informative screening assay when designing chemical products which contain mixtures of chemicals.

Prothrombin and PIVKA-II levels in cord blood from newborn exposed to anticonvulsants during pregnancy.

PURPOSE: To determine whether anticonvulsant exposure during human pregnancy caused an increase of the abnormal form of prothrombin, known as PIVKA-II (prothrombin induced by vitamin K absence for factor II), and a decrease in total prothrombin, in the blood of the newborn. METHODS: Cord blood was collected from the placenta at the time of parturition from 12 women who had received anticonvulsant therapy during pregnancy and from 11 control women. RESULTS: PIVKA-II was present in cord blood from control mothers at low or nondetectable levels. In the same samples, total prothrombin concentrations were approximately 50% of adult levels, but there was wide variation between individuals. Exposure to carbamazepine (CBZ) alone during pregnancy was associated with markedly increased PIVKA-II levels in four of six samples and decreased total prothrombin levels for the whole group. High PIVKA-II levels also were recorded in one cord blood sample from a mother who received phenytoin (PHT) and vigabatrin (VGB). Two cases of PHT alone and one of valproic acid (VPA) alone were not associated with increased PIVKA-II levels. CONCLUSIONS: These results are consistent with the hypothesis that some anticonvulsants (particularly CBZ) interfere with vitamin K metabolism during pregnancy and may result in hematologic signs of vitamin K deficiency in the newborn.

Model predicting the teratogenic potential of retinyl palmitate, using a combined in vivo/in vitro approach.

Retinyl palmitate (RP) is a known laboratory animal teratogen inducing abnormalities of the second visceral arch when administered on day 9 of gestation in the rat. However, there are significant problems when attempting to extrapolate this result to the human. A combined in vivo/in vitro model was developed to assist in human risk assessment. The in vitro teratogenic threshold concentration of a number of retinyl palmitate metabolites was established. Serum concentrations of retinyl palmitate metabolites following a single teratogenic dose of RP in the pregnant rat were also measured. These dosed sera were also used to culture rat embryos. Our hypothesis was that malformations would only be induced by the dosed sera in vitro if the threshold concentration(s) of one or more metabolites was exceeded. Using this approach, it was determined that the teratogenicity of the sera were best predicted by serum retinol levels, with some indication that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid could be involved in some cases. The available human data suggest that threshold concentrations of these retinoids were unlikely to be exceeded following vitamin A supplements of 25,000 IU/day. While the proposed model does not take into account species differences, protein binding, and transfer to the embryo, it does have potential for human risk assessment.

Expression of the LFA-1 beta 2 integrin (CD11a/CD18) and ICAM-1 (CD54) in normal and coeliac small bowel mucosa.

The leucocyte adhesion molecules (beta 2 integrins) comprise CD11 alpha-chains and a common beta-chain (CD18). CD11a (leucocyte function-associated antigen 1, LFA-1) is expressed by most T cells, and is involved in antigen presentation by macrophages via its counter-receptor, intercellular adhesion molecule (ICAM-1, CD54). By criteria of double-label immunofluorescence of cryostat tissue sections, virtually all lamina propria T cells of the normal small bowel were found to express LFA-1 strongly. By contrast, only 30-60% of intra-epithelial lymphocytes (IEL) expressed detectable LFA-1, most of which were LFA-1 weak and CD18-. ICAM-1 was expressed strongly only by vascular endothelium. In coeliac disease, there was a modest increase of diffuse ICAM-1 expression in the lamina propria, mainly in the subepithelial zone, where ICAM-1+ macrophages were occasionally seen. There was also a slight overall increase in CD11a expression by IEL, seen predominantly in surface epithelium and mainly by the CD4+ minority subset, but not by CD4-CD8- (TcR gamma delta +) cells. These data suggest that the LFA-1/ICAM-1-dependent antigen presentation pathway is of minor importance to IEL in the normal small bowel, and does not assume a major role in coeliac disease.

Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence and V region gene expression in Heliobacter pylori-associated gastritis, coeliac disease and inflammatory bowel disease.

T cells expressing the gamma delta heterodimer of the T cell receptor (TCR) were studied with respect to their occurrence and expression of gamma delta TCR variable region (V) genes in the normal gastrointestinal mucosa and in a variety of inflammatory conditions. In controls, gamma delta TCR+ cells were a minority population confined to the epithelial compartment of stomach, small bowel and colonic mucosae. Unlike in the periphery, gastro-intestinal gamma delta TCR+ intraepithelial lymphocytes (IEL) were mainly V delta 1+ (89.98 +/- 17.70%); few were V delta 2+ (6.04 +/- 13.8%) or V gamma 9+ (11.38 +/- 10.73%). All gamma delta TCR+ IEL were CD5low; nearly half were CD8+ and the remainder were CD4-CD8- 'double negatives'. There was no significant change from normal in percentages of gamma delta TCR+ IEL in H. pylori-associated gastritis, Crohn's disease and ulcerative colitis. However, in coeliac disease, gamma delta TCR+ IEL were elevated from 2.54% (+/- 1.71) in controls to 29.6% (+/- 16.1) in untreated patients (P less than 0.001) and 18.5% (+/- 7.2) in treated patients (P less than 0.001) and more were CD4-CD8-. Otherwise, gamma delta TCR+ IEL phenotypes were little changed: the majority remained V delta 1+V delta 2-V gamma 9- and all were CD5low. These data suggest that increased gamma delta TCR+ IEL are not a generalized response to intestinal inflammation or to stress proteins, although the typical V delta 1+V delta 2-V gamma 9- CD5low phenotype is retained.

Expression of T-cell receptors TcR1 (gamma/delta) and TcR2 (alpha/beta) in the human intestinal mucosa.

Cryostat sections of normal human adult gastrointestinal mucosae were studied by double-label immunofluorescence with antibodies to CD3, CD4, CD8, CD5 and CD6, in parallel with antibodies beta F1 and TCR delta 1 against beta-chains and delta-chains of the T-cell receptor (TcR) types TcR2 (alpha/beta) and TcR1 (gamma/delta), respectively. Virtually no TcR1+ were found within the lamina propria. In the epithelial compartment, TcR1+ cells were infrequent: in the small bowel, congruent to 2% of T cells were TcR1+. In the colonic epithelium, the percentage of T cells expressing gamma/delta-chains was higher, with a mean value approximating 15-20%, although this apparently large percentage increase compared with small bowel reflects in part a much lower density of colonic IEL, as absolute numbers of TCR delta 1+ cells were comparable. Of the TcR1+ population, about half were CD4- CD8-, 'double negatives' and the remainder were CD8+. TcR1+ cells were also CD5- CD6-, irrespective of expression of CD8. No CD4+ cells expressing TcR1 were observed: essentially all CD4+ cells were beta F1+, with some variability of labelling intensity. Approximately 30-50% of the CD8+ subset expressed the beta F1 antigen strongly. However, in the remaining TcR1- CD8+ cells, which were all of the CD5- CD6- phenotype, expression of the beta F1 antigen was only detectable when streptavidin and biotin conjugates were used for amplification of labelling. Thus, the CD8+ CD5- subset, a prominent population of the epithelial compartment of the small bowel, was either TcR2dull in the majority or TcR1+ in a minority. Our data imply that gamma/delta TcR1 cells may be actively excluded from intestinal lamina propria, and that any preferential localization that does occur is limited and is rather a feature of the colonic mucosa, rather than the small bowel.

Colonic mucosal T lymphocytes in ulcerative colitis: expression of CD7 antigen in relation to MHC class II (HLA-D) antigens.

T-cell subsets and their activation state were examined by double-label immunofluorescence of cryostat tissue sections of the colon from 21 patients with ulcerative colitis (UC) and 30 histologically normal controls. Expression of MHC class I (HLA-A, B, C) and class II (HLA-D) antigens was studied in parallel. In the normal colonic mucosa, the CD4:CD8 ratio in the epithelial compartment approximated 1:1, and in the lamina propria, 2.55:1. Of the CD8+ (cytotoxic/suppressor) subset, approximately half did not express the CD5 "pan-T" marker in either compartment. Virtually no Leu8+ cells were observed, implying that the CD4+ subset consisted of helper, rather than suppressor-inducer cells. Classical markers of T-cell activation (CD25, HLA-D) and proliferation were absent, and strong expression of the CD7 "immunostimulation" marker was approximately equal in both CD4 and CD8 subsets. The epithelium was uniformly negative for class II antigens, but positive for class I. In UC, there were no significant alterations in CD4:CD8 ratios in either compartment, and there were no changes with respect to phenotype of the subsets. In 11 of 19 patients (mainly with total colitis), enterocytes were HLA-D+. In this HLA-D+ group, there was an increase in the percentage of CD4+ cells coexpressing CD7; this difference was significant (P less than 0.02) in the lamina propria. Increased expression of CD7 was also found by the CD6+ T cell subset (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Selective potentiating effect of beta-p-chlorophenylglutamate on responses induced by certain sulphur-containing excitatory amino acids and quisqualate.

In dorsal horn neurones of the cat spinal cord iontophoretically administered (+/-)-beta-p-chlorophenylglutamate (chlorpheg) markedly enhanced the excitatory responses induced by L-homocysteate, L-homocysteine sulphinate, S-sulpho L-cysteine, L-cysteate and quisqualate, while responses to NMDA, kainate, L-glutamate, L-aspartate and L-cysteine sulphinate were generally unaffected. Preliminary data obtained on frog spinal cord in vitro supports the possibility that such selective potentiation may be due to differential inhibition by chlorpheg of amino acid uptake. No potentiating effects of chlorpheg were observed on spinal synaptic excitation.

Urinary pseudouridine/creatinine ratio as an indicator of gastrointestinal cancer.

Urinary pseudouridine/creatinine ratio was determined in 74 patients with gastrointestinal tumours and 34 patients with no known malignant disease. The reproducibility of a single random urine sample was demonstrated. The mean ratio for control patients was 26.9 +/- 7.7 nmol/mumol and no control patient exceeded the mean by 2 standard deviations. There was no difference in the ratio between the sexes. Sixty-five per cent of colon cancer patients and 37.5 per cent of gastric and rectal cancer patients exceeded this upper limit of normality. There was no correlation between pseudouridine/creatinine ratio and histological differentiation, liver involvement or stage in either colorectal or gastric cancer patients. Urinary pseudouridine/creatinine ratio is one of the better non-specific cancer markers and may be particularly useful for detecting colonic cancer.

Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations.

1. The depressant actions of Mg2+ and a range of other divalent ions on synaptic excitation and on responses produced by excitatory amino acids and other putative transmitters have been investigated in hemisected isolated spinal cords of frogs and neonatal rats. Some comparative studies were also made using the rat isolated superior cervical ganglion. 2. At concentrations above 10 microM, Mg2+ selectively antagonized N-methyl-D-aspartate (NMDA)-induced motoneurone depolarization as recorded from ventral roots of tetrodotoxin-blocked spinal cords. Depolarization evoked by quisqualate (unaffected by 20 mM-Mg2+) was resistant to the depressant action of these ions, while depolarizations evoked by other excitant amino acids were depressed to intermediate degrees. 3. Mn2+, Co2+ and Ni2+ had qualitatively similar actions to Mg2+; Mn2+ was somewhat less potent and Co2+ and Ni2+ more potent than Mg2+. The alkaline earth metal ions, Ca2+, Sr2+ and Ba2+, had very weak Mg2+-like actions. Ca2+ and Mg2+ acted additively in depressing amino acid-induced responses. 4. Mg2+ also depressed motoneurone responses evoked by noradrenaline, substance P and carbachol in the neonatal rat isolated spinal cord. However, none of these effects were as marked as the depression of NMDA-induced responses by Mg2+ in this preparation. Mg2+ did not depress motoneurone depolarization produced by 5-HT in the rat spinal cord or the depolarizing action of GABA on primary afferent terminals of the isolated frog spinal cord. 5. At concentrations producing marked depression of NMDA-induced responses, Mg2+ also depressed synaptic transmission in spinal cords in the absence of an effect on ganglionic transmission. At the same concentrations, Mn2+, Co2+ and Ni2+ depressed synaptic transmission in both preparations. 6. From the similarity in action between Mg2+ and the D-alpha-aminoadipate group of NMDA antagonists, it is suggested that the central depressant action of low concentrations of Mg2+ involves predominantly a postsynaptically mediated interference with the action of an excitatory amino acid transmitter.