Hypoxia reduces expression and function of system A amino acid transporters in cultured term human trophoblasts.

We tested the hypothesis that hypoxia diminishes the expression and transport of neutral amino acids by system A in full-term human trophoblasts. Cytotrophoblasts from normal human placentas were cultured in standard conditions of 20% O(2) or in 1% and 3% O(2) for 24 h before assay. Neutral amino acid transport for systems A, ASC, and L was assayed at 24 and 72 h by the cluster-tray technique. Hypoxia during the initial 24 h of culture reduced system A transport by 82% in 1% O(2) and by 37% in 3% O(2) (P < 0.01) compared with standard conditions. Hypoxia during the latter 24 h of the 72 h in culture reduced system A transport by 55% in 1% O(2) and by 20% in 3% O(2) (P < 0.05) compared with standard conditions at 72 h. Hypoxia (1% O(2)) also reduced total amino acid transport by 40% in the more differentiated syncytiotrophoblasts present at 72 h. Northern analysis of trophoblasts in standard conditions showed that subtypes of human amino acid transporter A (hATA1 and hATA2) were each expressed in cytotrophoblasts and syncytiotrophoblasts. Hypoxia decreased expression of hATA1 and hATA2 in both trophoblast phenotypes. We conclude that hypoxia downregulates system A transporter expression and activity in cultured human trophoblasts.

Evaluation of the TAS analyzer and the low-range heparin management test in patients undergoing extracorporeal membrane oxygenation.

BACKGROUND: The new Low-Range Heparin Management Test (LHMT), a method for point-of-care testing (POCT) of heparinization, has been designed to function at the low to moderate heparin concentrations typically found in patients undergoing extracorporeal membrane oxygenation (ECMO). In this study, the new method is compared with two POCT methods and a laboratory-based anti-Xa assay. METHODS: We obtained 760 whole blood samples from 13 patients undergoing ECMO. All samples were tested immediately by the LHMT, the Activated Clotting Time (ACT) test, and its low-range counterpart (ACT-LR). Aliquots from the same blood draw were frozen for later anti-Xa analysis using the Diagnostica Stago method on the Roche Cobas Fara-II. RESULTS: The precision was best for duplicate citrated LHMT samples (CV = 3.1%). LHMT clotting times (overall median, 162 s) were typically shorter than ACT or ACT-LR times (247 and 235 s, respectively). The relationship between the LHMT and the other POCT methods differed significantly from patient to patient (P <0.0001), and a meaningful single relationship between the methods could not be obtained. The overall correlation coefficient between clotting time values and actual heparin concentrations was < or = 0.48 for each of the instruments tested, although time plots of each analyzer's data suggested that they detected heparin dosage changes within single patients. CONCLUSIONS: The performance of the LHMT on the TAS Analyzer is equivalent to that of currently commercially available POCT methods. The lack of agreement between absolute clotting time values and heparin concentrations suggests the need for reexamination of current ECMO patient management strategy.

Effect of NGF and neurotrophin-3 treatment on experimental diabetic autonomic neuropathy.

Peripheral neuropathy is a significant complication of diabetes resulting in increased patient morbidity and mortality. Deficiencies of neurotrophic substances (e.g. NGE NT-3, and IGF-I) have been proposed as pathogenetic mechanisms in the development of distal symmetrical sensory diabetic polyneuropathy, and salutary effects of exogenous NGF administration have been reported in animal models. In comparison, relatively little is known concerning the effect of NGF on experimental diabetic sympathetic autonomic neuropathy. We have developed an experimental animal model of diabetic autonomic neuropathy characterized by the regular occurrence of pathologically distinctive dystrophic axons in prevertebral sympathetic ganglia and ileal mesenteric nerves of rats with chronic streptozotocin (STZ)-induced diabetes. Treatment of STZ-diabetic rats for 2-3 months with pharmacologic doses of NGF or NT-3, neurotrophic substances with known effects on the adult sympathetic nervous system, did not normalize established neuroaxonal dystrophy (NAD) in diabetic rats in the prevertebral superior mesenteric ganglia (SMG) and ileal mesenteric nerves as had pancreatic islet transplantation and IGF-I in earlier experiments. NGF treatment of control animals actually increased the frequency of NAD in the SMG. New data suggests that, in adult sympathetic ganglia. NGF may contribute to the pathogenesis of NAD rather than its amelioration, perhaps as the result of inducing intraganglionic axonal sprouts in which dystrophic changes are superimposed. NT-3 administration did not alter the frequency of NAD in diabetic animals, although it resulted in a significant decrease in NAD in control SMG. Although deficiencies of neurotrophic substances may represent the underlying pathogenesis of a variety of experimental neuropathies, delivery of excessive levels of selected substances may produce untoward effects.

Inhibition of sorbitol dehydrogenase exacerbates autonomic neuropathy in rats with streptozotocin-induced diabetes.

We have developed an animal model of diabetic autonomic neuropathy that is characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin (STZ)-diabetic rats. Studies with the sorbitol dehydrogenase inhibitor SDI-158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the sorbitol pathway), have shown a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG of SDI-treated versus untreated diabetics. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics, their distinctive ultrastructural appearance was identical to that previously reported in long-term untreated diabetics. An SDI effect was first demonstrated in the SMG of rats that were diabetic for as little as 5 wk and was maintained for at least 7.5 months. As in untreated diabetic rats, rats treated with SDI i) showed involvement of lengthy ileal, but not shorter, jejunal mesenteric nerves; ii) demonstrated NAD in paravascular mesenteric nerves distributed to myenteric ganglia while sparing adjacent perivascular axons ramifying within the vascular adventitia; and, iii) failed to develop NAD in the superior cervical ganglia (SCG). After only 2 months of SDI-treatment, tyrosine hydroxylase immunolocalization demonstrated marked dilatation of postganglionic noradrenergic axons in paravascular ileal mesenteric nerves and within the gut wall versus those innervating extramural mesenteric vasculature. The effect of SDI on diabetic NAD in SMG was completely prevented by concomitant administration of the aldose reductase inhibitor Sorbinil. Treatment of diabetic rats with Sorbinil also prevented NAD in diabetic rats not treated with SDI. These findings indicate that sorbitol pathway-linked metabolic imbalances play a critical role in the development of NAD in this model of diabetic sympathetic autonomic neuropathy.

Effect of IGF-I and neurotrophin-3 on gracile neuroaxonal dystrophy in diabetic and aging rats.

Neuroaxonal dystrophy (NAD), a distinctive axonopathy characterized by dramatic swelling of preterminal axons and nerve terminals by the accumulation of a variety of subcellular organelles, develops in the central projections of sensory neurons to medullary gracile nuclei in aged animals and man, and in a number of diseases and experimental conditions. Although its pathogenesis is unknown, proposed mechanisms include abnormalities of axonal regeneration, collateral sprouting and synaptic plasticity which may reflect alteration in neurotrophic support. In the current study, we have demonstrated quantitatively that aging causes the expected marked increase in the frequency of gracile NAD; however, substantial numbers of dystrophic axons develop between 6 and 10 months of age, earlier than expected. Although diabetes has been reported to increase the frequency of NAD in the central processes of sensory neurons in the gracile fasciculus of genetically diabetic BB rats, we have found that 8-10 months of streptozotocin-induced diabetes results in fewer dystrophic axons in the gracile nucleus than in age-matched controls. Administration of neurotrophin-3 (NT-3) and insulin-like growth factor-I (IGF-I), which have been shown to affect synaptic plasticity (implicated in the pathogenesis of NAD), for the last two months before sacrifice did not affect the frequency of gracile NAD in controls or diabetics. The sensory terminals in the gracile nuclei provide a simple, well-characterized experimental system in which questions of pathogenesis and prevention of neuroaxonal dystrophy can be addressed.

Leptin in cerebrospinal fluid from children: correlation with plasma leptin, sexual dimorphism, and lack of protein binding.

BACKGROUND: Previous studies in adults have established that leptin is present at very low concentrations in cerebrospinal fluid (CSF), but few data exist concerning CSF leptin in children. Current evidence suggests that CSF leptin concentrations interact with hypothalamic centers controlling food intake. Serum leptin concentrations manifest a sexual dimorphism that arises during puberty. METHODS: Leptin concentrations were determined in CSF from 42 pre- and postpubertal children who had been objectively classified into non-neurological disease or aseptic meningitis groups. Multivariate analysis of the dependence of CSF leptin on gender, pubertal state, body mass index (BMI), presence of aseptic meningitis, and CSF protein concentration was performed. RESULTS: CSF leptin concentrations correlated with log-transformed plasma leptin concentrations in concomitantly collected samples (r = 0.582; P = 0.029). BMI and gender were significant determinants of CSF leptin in postpubertal children, but only BMI was significant in prepubertal children. Analysis with HPLC to separate protein-bound and free forms of leptin found only free leptin in CSF. CONCLUSIONS: CSF leptin concentrations in children reflect plasma leptin concentrations, including the advent of sexual dimorphism at puberty. Only free leptin is detectable in CSF, suggesting that it is the biologically active form.

Effect of streptozotocin-induced diabetes on NGF, P75(NTR) and TrkA content of prevertebral and paravertebral rat sympathetic ganglia.

Diabetic autonomic neuropathy results in significant morbidity and mortality. Both diabetic humans and experimental animals show neuroaxonal dystrophy of autonomic nerve terminals, particularly in the prevertebral superior mesenteric ganglia (SMG) and celiac ganglia (CG) which innervate the hyperplastic/hypertrophic diabetic small intestine. Previously, investigators suggested that disturbances in ganglionic nerve growth factor (NGF) content or transport might play a pathogenetic role in diabetic autonomic pathology. To test this hypothesis, we measured NGF content and NGF receptor expression, p75(NTR) (low affinity neurotrophin receptor) and trkA (high affinity NGF receptor), in control and diabetic rat SMG, CG and superior cervical ganglia (SCG). Surprisingly, rather than a decrease, we observed an approximate doubling of NGF content in the diabetic SMG and CG, a result which reflects increased NGF content in the hyperplastic diabetic alimentary tract. No change in NGF content was detected in the diabetic SCG which is relatively spared in experimental diabetic autonomic neuropathy. NGF receptor expression was not consistently altered in any of the autonomic ganglia. These observations suggest that increased NGF content in sympathetic ganglia innervating the diabetic alimentary tract coupled with intact receptor expression may produce aberrant axonal sprouting and neuroaxonal dystrophy.

Performance evaluation and planning for patient-based quality control procedures.

In a recent publication, the effect of the length of time between the routine testing of quality-control (QC) samples on QC performance was studied using a newly proposed performance measure: the average number of patient samples that contain unacceptable analytic error due to an out-of-control error condition (ANPTE). We show that ANPTE also perfectly suits the evaluation of patient-based QC procedures. We used ANPTE to study the effect of the number of patient results averaged and the width of the truncation limits on an average of normals QC procedure. Estimates of ANPTE were obtained by using computer simulations that use actual patient result distributions. Based on ANPTE, the conclusions about the effects of truncation limits and number of patient results averaged on the performance of an average of normals QC procedure are substantially different from those described by others using the probability of error detection to characterize QC performance.

Insulin-like growth factor I reverses experimental diabetic autonomic neuropathy.

Recent studies have suggested a role for neurotrophic substances in the pathogenesis and treatment of diabetic neuropathy. In this study, the effect of insulin-like growth factor I (IGF-I) on diabetic sympathetic autonomic neuropathy was examined in an experimental streptozotocin-induced diabetic rat model. Two months of IGF-I treatment of chronically diabetic rats with established neuroaxonal dystrophy (the neuropathological hallmark of the disease) involving the superior mesenteric ganglion and ileal mesenteric nerves resulted in nearly complete normalization of the frequency of neuroaxonal dystrophy in both sites without altering the severity of diabetes. Treatment with low-dose insulin (to control for the transient glucose-lowering effects of IGF-I) failed to affect the frequency of ganglionic or mesenteric nerve neuroaxonal dystrophy or the severity of diabetes. The striking improvement in the severity of diabetic autonomic neuropathy shown with IGF-I treatment in these studies and the fidelity of the rat model to findings in diabetic human sympathetic ganglia provide promise for the development of new clinical therapeutic strategies.

Leptin elimination in hyperleptinaemic peritoneal dialysis patients.

BACKGROUND: Elevated plasma concentrations of leptin, a hormone thought to regulate body composition by influencing food intake/metabolic rate, are prevalent in renal failure patients. The mechanism for these increases is not known, but evidence suggests that simple accumulation due to decreased elimination is insufficient explanation. METHODS: We studied the incidence of hyperleptinaemia in 28 end-stage renal disease patients treated with continuous ambulatory peritoneal dialysis (CAPD), compared with body-mass-index-and sex-matched controls. Results were separated by gender because women have higher leptin concentrations than men. Excretion of leptin and other substances in dialysis fluid was also studied. RESULTS: Hyperleptinaemia was prevalent in women CAPD subjects, but not in men. Plasma leptin concentrations correlated strongly with the daily excretion of leptin in dialysis fluid. Clearance of leptin in dialysis fluid was greater in men than women CAPD subjects. Single regression analysis found that fasting insulin, glucose content of dialysis fluid, plasma albumin, C-reactive protein, erythropoietin dose, urinary creatinine clearance and plasma beta2-microglobulin were not determinants of plasma leptin concentrations. Stepwise forward multiple regression, examining the dependence of plasma leptin on body mass index, renal creatinine clearance, plasma albumin, daily dialysis fluid glucose load, daily leptin in dialysis fluid, erythropoietin dose and plasma C-reactive protein found only erythropoietin dose as a consistent negative predictor of plasma leptin concentrations. CONCLUSIONS: The results suggest that hyperleptinaemia of CAPD was due to predisposing loss of renal elimination capacity combined with increased production due to obesity (more prevalent in women subjects of this study) and potentially female gender.

Tpm1, a locus controlling IL-12 responsiveness, acts by a cell-autonomous mechanism.

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 x BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.

Hyperleptinaemia of end-stage renal disease is corrected by renal transplantation.

BACKGROUND: Previous studies have reported that patients with end-stage renal disease (ESRD) have elevated plasma leptin concentrations, but the cause and significance of the elevations are unknown. We studied leptin concentrations in 29 adults undergoing renal transplantation, to determine if restoration of renal function reduced leptin concentrations in ESRD. METHODS: Leptin concentrations were measured by radioimmunoassay in plasma specimens collected within 1 week before transplant, 6 days post-transplant, and 60 days post-transplant. RESULTS: Mean plasma leptin concentrations were higher in both male and female ESRD patients compared with a control population of similar age and body mass index (BMI), but most of the disparity was due to a minority of patients with grossly elevated concentrations; the majority of ESRD patients had normal or near-normal leptin concentrations after accounting for their adiposity with BMI. Six days after successful renal transplantation, average plasma leptin concentrations decreased to control levels. The grossly elevated pretransplant concentrations in a minority of patients were greatly reduced in relation to BMI, and the reduction persisted to 60 days post-transplant. The decrease in creatinine with transplant did not correlate with the decrease in leptin. CONCLUSIONS: These results demonstrate that restoration of renal function in ESRD patients reduces hyperleptinaemia, which provides further evidence of a cause/effect relationship between impaired renal function and abnormal leptin metabolism.

Combining laboratory data sets from multiple institutions using the logical observation identifier names and codes (LOINC).

A standard set of names and codes for laboratory test results is critical for any endeavor requiring automated data pooling, including multi-institutional research and cross-facility patient care. This need has led to the development of the logical observation identifier names and codes (LOINC) database and its test-naming convention. This study is an expansion of a pilot study using LOINC to exchange laboratory data between Columbia University Medical Center in New York and Barnes Hospital at Washington University in St. Louis, where we described complexities and ambiguities that arose in the LOINC coding process (D.M. Baorto, J.J. Cimino, C.A. Parvin, M.G. Kahn, Proc. Am. Med. Inf. Assoc. 1997). For the present study, we required the same two medical centers to again extract raw laboratory data from their local information system for a defined patient population, translate tests into LOINC and provide aggregate data which could then be used to compare laboratory utilization. Here we examine a larger number of tests from each site which have been recoded using an updated version of the LOINC database. We conclude that the coding of local tests into LOINC can often be complex, especially the 'Kind of Property' field and apparently trivial differences in choices made by individual institutions can result in nonmatches in electronically pooled data. In the present study, 75% of failures to match the same tests between different institutions using LOINC codes were due to differences in local coding choices. LOINC has the potential to eliminate the need for detailed human inspection during the pooling of laboratory data from diverse sites and perhaps even a built-in capability to adjust matching stringency by selecting subsets of LOINC fields required to match. However, a quality standard coding procedure is required and examples highlighted in this paper may require special attention while mapping to LOINC.

Vacuolar neuritic dystrophy in aged mouse superior cervical sympathetic ganglia is strain-specific.

We have developed a model of autonomic nervous system aging using the mouse superior cervical sympathetic ganglion (SCG) which is characterized by the reproducible development of distinctive, markedly-enlarged neuritic swellings (vacuolar neuritic dystrophy, VND). These structures contained an admixture of lucent vacuoles and subcellular organelles, and involved both presynaptic and postsynaptic ganglionic elements. Quantitation of the frequency of VND was accomplished at the light microscopic level and validated by ultrastructural examination. VND lesions were 30-100-fold more frequent in the aged mouse paravertebral SCG than in the prevertebral celiac/superior mesenteric (C/SMG) sympathetic ganglia. Although VND was identified in all ages of mice examined, the number of lesions increased significantly with age. The frequency of VND was a function of the strain of mouse examined with a 40-fold difference in VND frequency between C57BL6 mice, the least involved strain, and the DBA/2J strain, which was most affected and began to develop significant numbers of lesions at an early age. As in our human studies of aging in the sympathetic nervous system, there was a prominent gender effect with males developing twofold greater numbers of VND lesions than females. Mice maintained on a significant calorie restricted diet for 30 months developed 70% fewer lesions than ad libitum-fed, age and sex matched controls. The aging mouse SCG, therefore, represents a robust animal model with reproducible, quantifiable and unambiguous neuropathology. Insights into pathogenetic mechanisms gained in the subsequent analysis of this relatively simple peripheral sympathetic nervous system model may contribute to the understanding of some of the most complex and significant problems involving higher brain function.

Patterns and potential value of cardiac troponin I elevations after pediatric cardiac operations.

BACKGROUND: Perioperative myocardial injury is a major determinant of postoperative cardiac dysfunction for congenital heart disease, but its assessment during this period is difficult. The objective of this study was to determine the suitability of using postoperative serum concentrations of cardiac troponin I (cTnI) for this purpose. METHODS: Cardiac troponin I levels were measured serially in the serum of patients undergoing uncomplicated repairs of atrial septal defect (n = 23), ventricular septal defect (n = 16) or tetralogy of Fallot (n = 16). The concentrations were correlated with intraoperative parameters (cardiopulmonary bypass time, aortic cross-clamp time, and cardiac bypass temperature), and postoperative parameters (magnitude of inotropic support, duration of intubation, and postoperative intensive care and hospital stay). RESULTS: Postoperative absolute cTnI levels were lesion specific, with a pattern of increase and decrease similar for each lesion. For the total cohort, significant correlations between postoperative cTnI levels at all times (r = 0.43 to 0.83, p < 0.05) until 72 hours were noted for all parameters, except for cardiac bypass temperature. When evaluated as individual procedure groups, no significant relationships were noted in the atrial septal defect group, whereas postoperative cTnI levels were more strongly correlated with all intraoperative and postoperative parameters in the ventricular septal defect group than in the tetralogy of Fallot group. CONCLUSIONS: This study suggests that cTnI values immediately after operation reflect the extent of myocardial damage from both incisional injury and intraoperative factors. Cardiac tropinin I levels in the first hours after operation for congenital heart disease are a potentially useful prognostic indicator for difficulty of recovery.

Effect of analytical run length on quality-control (QC) performance and the QC planning process.

The performance measure traditionally used in the quality-control (QC) planning process is the probability of rejecting an analytical run when an out-of-control error condition exists. A shortcoming of this performance measure is that it doesn't allow comparison of QC strategies that define analytical runs differently. Accommodating different analytical run definitions is straightforward if QC performance is measured in terms of the average number of patient samples to error detection, or the average number of patient samples containing an analytical error that exceeds total allowable error. By using these performance measures to investigate the impact of different analytical run definitions on QC performance demonstrates that during routine QC monitoring, the length of the interval between QC tests can have a major influence on the expected number of unacceptable results produced during the existence of an out-of-control error condition.

Dystrophic axonal swellings develop as a function of age and diabetes in human dorsal root ganglia.

Neuroaxonal dystrophy, characterized by swollen axon terminals and, to a lesser degree, enlarged initial segments of axons or perikaryal projections, develops in human dorsal root sensory ganglia as a function of aging and diabetes. Lesions are typically located within the satellite cell capsule and are intimately applied to sensory neuronal perikarya, which are compressed and distorted but are otherwise normal. Swollen axons contain large numbers of neurofilaments that are immunoreactive with antisera to highly phosphorylated neurofilament epitopes but fail to stain with antisera directed against hypophosphorylated neurofilament epitopes. Other dystrophic swellings contain collections of tubulovesicular profiles admixed with neurotransmitter granules. Neuroaxonal dystrophy involves subpopulations of intraganglionic axons and apparent terminals, notably those containing CGRP, while apparently sparing others, including noradrenergic sympathetic axons. Diabetic subjects develop lesions prematurely and in greater numbers than in aged subjects. Individual dystrophic axons in diabetics and aged human subjects are identical in their light microscopic, immunohistochemical and ultrastructural appearance, suggesting the possibility of shared pathogenetic mechanisms.

Quality-control (QC) performance measures and the QC planning process.

Numerous outcome measures can be used to characterize and compare the performance of alternative quality-control (QC) strategies. The performance measure traditionally used in the QC planning process is the probability of rejecting an analytical run when a critical out-of-control error condition exists. Another performance measure that naturally fits within the total allowable error paradigm is the probability that a reported test result contains an analytical error that exceeds the total allowable error specification. In general, the out-of-control error conditions associated with the greatest chance of reporting an unacceptable test result are unrelated to the traditionally defined "critical" error conditions. If the probability of reporting an unacceptable test result is used as the primary performance measure, worst-case QC performance can be determined irrespective of the magnitude of any out-of-control error condition that may exist, thus eliminating the need for the concept of a "critical" out-of-control error.