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Fetal and neonatal nicotine exposure causes beta-cell apoptosis and loss of beta-cell mass, but the underlying mechanisms are unknown. The goal of this study was to determine whether maternally derived nicotine can act via the pancreatic nicotinic acetylcholine receptor (nAChR) during fetal and neonatal development to induce oxidative stress in the pancreas. Female Wistar rats were given saline or nicotine (1 mg/kg/day) via subcutaneous injection for 2 weeks prior to mating until weaning (postnatal day 21). In male offspring, nAChR subunit mRNA expression was characterized in the developing pancreas and various oxidative stress markers were measured at weaning following saline and nicotine exposure. The nAChR subunits alpha2-alpha4, alpha6, alpha7, and beta2-beta4 were present in the pancreas during development. Fetal and neonatal exposure to nicotine significantly increased pancreatic GPx-1 and MnSOD protein expression, as well as islet ROS production. Furthermore, protein carbonyl formation was higher in nicotine-exposed offspring relative to controls, particularly within the mitochondrial fraction. There was also a nonsignificant trend toward higher serum 8-isoPG levels. These data suggest that beta-cell apoptosis in the fetal and neonatal pancreas may be the result of a direct effect of nicotine via its receptor and that this effect may be mediated through increased oxidative stress.

Nicotine replacement therapy (NRT) is currently recommended as a safe smoking cessation aid for pregnant women. However, fetal and neonatal nicotine exposure in rats causes mitochondrial-mediated beta cell apoptosis at weaning, and adult-onset dysglycemia, which we hypothesize is related to progressive mitochondrial dysfunction in the pancreas. Therefore in this study we examined the effect of fetal and neonatal exposure to nicotine on pancreatic mitochondrial structure and function during postnatal development. Female Wistar rats were given saline (vehicle control) or nicotine bitartrate (1 mg/kg/d) via subcutaneous injection for 2 weeks prior to mating until weaning. At 3-4, 15 and 26 weeks of age, oral glucose tolerance tests were performed, and pancreas tissue was collected for electron microscopy, enzyme activity assays and islet isolation. Following nicotine exposure mitochondrial structural abnormalities were observed beginning at 3 weeks and worsened with advancing age. Importantly the appearance of these structural defects in nicotine-exposed animals preceded the onset of glucose intolerance. Nicotine exposure also resulted in significantly reduced pancreatic respiratory chain enzyme activity, degranulation of beta cells, elevated islet oxidative stress and impaired glucose-stimulated insulin secretion compared to saline controls at 26 weeks of age. Taken together, these data suggest that maternal nicotine use during pregnancy results in postnatal mitochondrial dysfunction that may explain, in part, the dysglycemia observed in the offspring from this animal model. These results clearly indicate that further investigation into the safety of NRT use during pregnancy is warranted.

Background: Several estimates of the risk of progression to diabetes in people with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) have been reported., Objective: To provide an estimate of the risk of progression to diabetes and regression to normoglycemia in these populations., Design: Systematic overview and meta-analysis of prospective cohort studies published from 1979 until 2004., Setting: Global cohort studies., Patients: People with IFG or IGT detected by a screening oral glucose tolerance test., Measurements: Fasting and post-load plasma glucose levels., Results: The absolute annual incidence of diabetes in individuals with various categories of IFG or IGT varied from 5 to 10%. Compared to normoglycemic people the meta-analyzed relative risk and 95% confidence interval for diabetes was: 6.35 (4.87-7.82) in people with IGT; 5.52 (3.13-7.91) in people with isolated IGT; 4.66 (2.47-6.85) in people with IFG; 7.54 (4.63-10.45) in people with isolated IFG; and 12.13 (4.27-20.00) in people with both IFG and IGT. People with IGT were 0.33 times as likely to be normoglycemic after 1 year compared to people with normal glucose tolerance (95% CI 0.23-0.43)., Limitations: Studies that used differing criteria for IFG and IGT were included, and participants were classified on the basis of only one test., Conclusion: IFG and IGT are associated with similar, high relative risk for incident diabetes. The combined abnormality of IFG plus IGT is associated with the highest relative risk.

Fetal and lactational exposure to nicotine at concentrations comparable with those in women who smoke causes impaired glucose tolerance in male offspring in postnatal life. It remains unknown whether there are critical windows of susceptibility to nicotine exposure. Female nulliparous Wistar rats were given saline vehicle or nicotine bitartrate (1 mg/kg per day) prior to pregnancy, which was then: A) discontinued during pregnancy and lactation; B) continued until parturition; C) continued until weaning; and D) discontinued during pregnancy and restarted from lactation until weaning. At 26 weeks of age, offspring in each group were challenged with an oral glucose load. Beta-cell mass, apoptosis, and proliferation were measured at birth, and at 4 and 26 weeks of age. The animals in group C (exposed to nicotine throughout pregnancy and lactation) had reduced beta-cell mass from birth through 26 weeks of age and impaired glucose homeostasis at 26 weeks of age. beta-Cell mass was also reduced at birth and at 4 weeks of age in animals exposed to nicotine during pregnancy alone (group B). However, enhanced proliferation following weaning led to recovery of this defect to 98% of control levels by week 26. The response to the glucose load in groups A, B, and D did not differ from controls. Continued exposure to nicotine from conception through lactation results in permanent beta-cell loss and subsequent impaired glucose tolerance. This model of type 2 diabetes requires that nicotine exposure occurs both in utero and during lactation.

A wide variety of in utero insults are associated with an increased incidence of metabolic disorders in the offspring and in subsequent generations. We have shown that fetal and neonatal exposure to nicotine results in endocrine and metabolic changes in the offspring that are consistent with those observed in type 2 diabetes. This study examines whether fetal and neonatal exposure to nicotine has transgenerational effects in the F2 offspring. Female Wistar rats were given either saline or nicotine (1 mg/kg/d) during pregnancy and lactation to create saline- and nicotine-exposed female F1 progeny. These F1 females were then bred to produce F2 offspring. We examined glucose homeostasis, serum lipids and fat pad weights, mitochondrial enzyme activity in skeletal muscle and blood pressure in these F2 offspring between 13 and 15 weeks of age. Offspring of nicotine- versus saline-exposed mothers had elevated fasting serum insulin concentrations and an enhanced total insulin response to the glucose challenge. This apparent insulin resistance was unrelated to changes in skeletal muscle mitochondrial volume or activity. The offspring of nicotine-exposed mothers also had elevated blood pressure. These data demonstrate that adverse effects of fetal and neonatal exposure to nicotine can influence aspects of metabolic risk in subsequent generations.

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for its variability in disease outcome. Apolipoprotein E (APOE) is involved in neuronal remodelling and several studies have attempted to examine the effect of APOE on MS disease severity, but its function in modifying the course of MS is controversial. It has been suggested recently that PVRL2, not APOE, is the locus on chromosome 19 which influences clinical outcome of MS. A cohort of sporadic MS cases, taken from opposite extremes of the putative distribution of long-term outcome using the most stringent clinical criteria to date, was used to determine the role of APOE and PVRL2 on MS disease severity. The MS cases selected represent the prognostic best 5% (benign MS) and worst 5% (malignant MS) of cases in terms of clinical outcome assessed by the EDSS. Genotyping the two sets of MS patients (112 benign and 51 malignant) and a replication cohort from Sardinia provided no evidence to suggest that APOE or PVRL2 have any outcome modifying activity. We conclude that APOE and PVRL2 have little or no effect on the clinical outcome of MS.

Objective: The usefulness of measuring the GH-dependent acid-labile subunit (ALS) in the management of GH deficiency (GHD) and acromegaly remains in question and is investigated in this study, comparing several different immunoassays for ALS., Method: We compared the diagnostic accuracy of a commercially available polyclonal Ab-based ELISA with SDS pre-treatment (SDS-ELISA) with a monoclonal Ab-based immunofluorometric assay, using two unfolding methods (urea (UREA) and Glycine-HCl (Gly)). The corresponding molecular weight (MW) of ALS and IGFBP-3 immunoreactivity was determined. The clinical usefulness of each assay was examined in adult GH disorders., Results: ALS was lower in GHD and higher in acromegaly using all assays. In GHD, UREA had higher sensitivity and specificity than SDS-ELISA (59 and 69% versus 41 and 51% respectively). In acromegaly, sensitivity and specificity was 94 and 87% for UREA, 81 and 36% for Gly, and 44 and 44% for SDS-ELISA. After UREA, immunoreactivity for ALS and IGFBP-3 eluted at their predicted free MW using size-exclusion chromatography, whereas ALS immunoreactivity in SDS (300-600 kDa) and Gly (250-500 kDa) was at a high apparent MW consistent with aggregation., Conclusion: The diagnostic accuracy of ALS varies with assay choice and pre-treatment modality. UREA, which results in migration of ALS at the expected MW on a sizing column, has the highest specificity and sensitivity. Thus, if measured in an assay in which ALS is unfolded without aggregation, ALS is a clinically highly useful parameter for the assessment of GH.

Background: The classifications of impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) represent glucose levels above normal, but below the decision threshold for diabetes. We sought to determine what the reproducibility of these classifications was when repeat tests were performed by conducting a systematic review of the literature., Methods: All primary studies published in English of any study design were included. Studies were excluded if they did not follow the World Health Organization or American Diabetes Association diagnostic criteria, used whole blood as the specimen type, a glucose meter for analysis, or performed repeat testing greater than 8 weeks apart., Results: Five papers had reproducibility data for IGT or IFG, two of which where from the same population but sampled differently. The kappa coefficients, indicating agreement between repeat tests that exceeded chance, indicated poor to fair agreement for IGT (0.04, 0.22, 0.38, 0.42) and moderate agreement for IFG (0.44 and 0.56). Similarly, the observed reproducibility was slightly lower for IGT (33%, 44%, 47%, 48%) compared to IFG (51%, 64%). In two studies for which data were available for both IGT and IFG, the average reproducibility was lower (49%) for the prediabetes group compared to the diabetes group (73%) or the normal group (93%)., Conclusions: Poor reproducibility of IGT and IFG classification suggests caution should be exercised when interpreting a single test result.

Microchimerism, the persistence of foreign cells thought to derive from previous pregnancies, has been associated with autoimmune diseases. A maternal parent-of-origin effect in MS remains unexplained. We tested for microchimerism in monozygotic and dizygotic twin-pairs with MS. Microchimerism was associated with MS in affected females from monozygotic concordant pairs when compared to both affected (p=0.020) and unaffected (p=0.025) females in monozygotic discordant pairs. Microchimerism was increased in affected females of dizygotic discordant pairs (p=0.059). The rate of microchimerism was significantly higher in affected twins than in unaffected co-twins (p=0.0059). These observations show an association in twins between the presence of microchimerism and having MS.

Aims/hypothesis: Epidemiological studies report an increased risk of obesity and type 2 diabetes in children born to women who smoked during pregnancy. This study examines the effect of fetal and neonatal exposure to nicotine, the major addictive component of cigarettes, on postnatal growth, adiposity and glucose homeostasis., Methods: Female Wistar rats were given either saline (vehicle) or nicotine (1 mg kg(-1) day(-1)) during pregnancy and lactation. Serum and pancreas tissue were collected from the infant rats at birth. Postnatal growth was assessed weekly until the rats reached 26 weeks of age and glucose homeostasis was examined by OGTTs performed at 7 and 26 weeks of age., Results: Exposure to nicotine resulted in increased postnatal growth and adiposity. Nicotine exposure also resulted in dysglycaemia at 7 and 26 weeks of age. Serum insulin concentrations were decreased in the pups exposed to nicotine at birth. This was associated with increased beta cell apoptosis (pups of saline-treated mothers 8.8+/-1.21% apoptotic beta cells; pups of nicotine-treated mothers 27.8+/-3.1% apoptotic beta cells)., Conclusions/interpretation: Fetal and neonatal exposure to nicotine results in metabolic changes in the offspring that are consistent with obesity and type 2 diabetes. We propose that these metabolic changes may be a consequence of the initial insult to the beta cell during fetal life and that this animal model has many characteristics of diabetes in humans.

BACKGROUND: Asthma is a sometimes severe respiratory illness with an increased prevalence, especially in low-income, minority, and inner-city populations, over the past 2 decades. Prior reports have suggested a link between depression and asthma deaths. However, no studies have examined the relationship between objective measures of asthma severity and clinician-rated depressive symptom severity. METHOD: In this pilot study, 46 children receiving treatment at an inner-city asthma clinic were assessed with the Children's Depression Rating Scale, Revised (CDRS-R). The current percentage of forced expiratory volume in 1 second (FEV(1)%) predicted and the inhaled steroid dose were recorded, as were oral steroid use, emergency room visits, and hospitalizations in the preceding year. RESULTS: Depressive symptoms were common in this sample, with 30% (N = 14) of the participants having CDRS-R scores consistent with likely, very likely, or almost certain major depressive disorder. When mean CDRS-R scores were compared between the sample divided by these asthma severity measures, only hospitalizations in the past year was associated with higher depressive symptom scores (p =.03). CONCLUSION: These findings suggest that in the patient sample studied, depressive symptoms appear to be common. However, depressive symptom severity is related only to hospitalization, not other measures of asthma severity. Larger studies are needed to confirm these findings and determine if other variables such as family history of depression or subjective assessment of asthma severity explain the high prevalence of depressive symptoms in these patients.

Panels of monoclonal antibodies (mAbs) were raised against recombinant human leptin and the recombinant human soluble leptin receptor. Using these mAbs, we established a ligand-mediated immunofunctional assay (LIFA) to quantify concentrations of the soluble leptin receptor, which has been shown to be a major binding protein for leptin in human serum. In performing the assay, a monoclonal antibody (mAb 2H6) against the soluble leptin receptor, which binds an epitope outside the leptin-binding site and equally recognizes both, free and leptin-occupied soluble leptin receptor, is used to capture the soluble leptin receptor on a microtiter plate. Recombinant human leptin is added to saturate all binding sites, and a biotinylated anti-leptin mAb (4D3) detects the amount of leptin (endogenous and exogenous) bound to the soluble leptin receptor. The same procedure, but without adding exogenous leptin, allows for measurement of the circulating endogenous leptin/soluble leptin receptor complexes. The LIFA assay has a linear working range of 0.5-200 microg/liter, intra- and interassay coefficients of variation ranged from 3.2-6.3% and from 5.2-7.9%, respectively. The assay has a linearity of 102.2 +/- 5.2% (mean +/- SD) and a recovery of 100.7 +/- 6.9%. Size-exclusion chromatography revealed that the assay measures a protein with a main peak eluted at 340 kDa. The soluble leptin receptor concentration (63.3 +/- 22.8 microg/liter (mean +/- SD), range 17.9-129.2 microg/liter, n = 43) in normal subjects (body mass index = 22.3 +/- 2.3 kg/m(2)) was not different from the concentration (54.4 +/- 19.8 microg/liter, range 23.7-104.8 microg/liter, n = 34, P > 0.05) found in obese subjects (body mass index = 40.9 +/- 15.7 kg/m(2)). However, the percentage of the total soluble leptin receptor complexed with endogenous leptin was significantly higher in obese subjects, compared with normal subjects (74.9% +/- 23.5% vs. 33.1% +/- 19.5%, P < 0.001). Higher serum leptin levels in obese subjects (38.4 +/- 23.7 microg/liter vs. 7.8 +/- 5.5 microg/liter in normal subjects, P < 0.001) together with comparable soluble leptin receptor levels result in a lower proportion of leptin bound to the soluble leptin receptor in obese subjects (19.3% +/- 19.4%, range 4.9-97.2%) than in normal subjects (39.0% +/- 22.5%, range 15.3-96.5%, P < 0.001). The development of this LIFA for the rapid and accurate quantification of total soluble leptin receptor and circulating leptin/soluble leptin receptor complexes provides a valuable tool for the further understanding of the role of leptin and its soluble receptor in health and disease.

Growth hormone (GH) action induces a variety of biochemical factors of which insulin-like growth factor I (IGF-I) is conventionally measured for the diagnosis and monitoring of GH-related disorders such as GH deficiency or acromegaly. IGF-I circulates predominantly as a ternary complex with IGF-binding protein 3 (IGFBP-3) and the acid labile subunit (ALS). In the treatment of active acromegaly with the GH receptor antagonist pegvisomant, ALS showed a closer correlation with the change in ring size, measured as a clinical indicator of disease activity, than did IGF-I or IGFBP-3. ALS thus seems to be a clinically useful marker of disease activity in acromegaly.

Quantification of the acid-labile subunit (ALS) has to date been restricted to immunoassays utilizing polyclonal antibodies. By immunization with N-terminal and C-terminal specific ALS oligopeptides, we generated monoclonal antibodies (mAbs) that target ALS-specific sequences outside the nonspecific leucine-rich repeats in the ALS molecule. For mAb selection, a special screening method was developed. Monoclonal antibody 5C9, which targets the N-terminus of ALS, is immobilized and the anti-ALS mAb 7H3, directed against the C-terminus, is biotinylated and used as tracer Ab. Due to the extreme pH-lability of ALS, changes in immunorecognition of ALS were investigated after acidification for protein unfolding in different pH ranges and in a time-dependent manner. It was determined that acidification of the serum samples to pH 2.7 for 30 min, followed by neutralization and dilution to 1:100 was the optimal acid-neutralization method. For standardization purposes, a serum pool derived from healthy volunteers was assigned the value 1 U/ml ALS. The sandwich assay has a working range with a linear dose-response curve in a log/log system between 0.005 and 10 U/ml. ALS levels in seven acromegalic patients ranged from 2.0 to 4.2 U/ml, and in 12 untreated growth hormone deficient patients from 0.036 to 0.986 U/ml (mean=0.45 U/ml). After 12 months of growth hormone therapy, ALS levels increased significantly to 1.18+/-0.45 U/ml (mean+/-SD; p<0.0006). The increase ranged from 0.48 to 1.4 U/ml. The change in ALS with growth hormone (GH) therapy correlated closer with the change in IGF-I (r=0.798, p=0.0057; Spearman rank correlation) than with the change in insulin-like growth factor binding protein (IGFBP3; r=0.549, p=0.057). This specific sandwich assay for the measurement of ALS provides a potentially valuable indicator of growth hormone secretory status. With this mAb-based immunofluorometric assay, the nonspecific detection of other proteins containing leucine-rich repeat sequences can be excluded.

Loss of appetite and weight are frequently observed at altitudes above 5000 m. However, the pathophysiology behind changes in body composition at extreme altitude is still not fully understood. Proper acclimatization to altitude and high caloric intake minimizes, but can not completely prevent significant weight loss under the influence of hypobaric hypoxia. The discovery of leptin in 1994 has initiated a new research area investigating molecular networks that connect peripheral organs with the central nervous system to sense and regulate energy intake as well as energy expenditure. Since then, a whole microcosm of new hormones, neurotransmitters and receptors has been discovered and studied with respect to body weight control. Those agents include neuropeptide Y (NPY), agouti-related protein (AGRP), melanocortin receptors (MC-R), cocaine-amphetamine regulated transcript (CART), pro-opiomelanocortin (POMC), orexin A and B (hypocretins), melanin-concentrating hormone (MCH) and ghrelin (endogenous ligand of the growth hormone secretagogue receptor). This overview will introduce the current concepts of the molecular control of energy homeostasis and attempt to reexamine the effects of altitude on appetite and body composition in light of these concepts. An overview of studies on changes of appetite and body composition at high altitude will be followed by the presentation of recent data on changes of endocrine parameters at hypobaric hypoxia that could be involved in the pathophysiology of weight loss.

The concentration of circulating insulin-like growth factor I (IGF-I) is a potential marker for growth hormone (GH) deficiency in adults. Indeed, researchers have shown that IGF-I levels are of greater diagnostic value than other possible markers, such as IGF-binding protein 3 (IGFBP-3) and the acid-labile subunit (ALS). Accurate age-matched normative data are essential to give patient data diagnostic meaning. Such data are assay specific and must exclude those individuals with certain confounding medical conditions. Post-diagnosis, monitoring of IGF-I and IGFBP-3 levels can be used to assess the efficacy and safety of GH replacement therapy. Furthermore, IGF-I levels, and possibly ALS levels, can be used to aid the diagnosis and monitoring of acromegaly. For example, acromegaly can be excluded in patients with normal IGF-I levels if liver failure and malnutrition/malabsorption are ruled out., (Copyright 2001 S. Karger AG, Basel)

The purpose of this study was to explore the potential of applying arthroscopic techniques to autogenous bone grafting of long bone fracture delayed union. There were 9 patients in this initial series, including 4 patients (average age, 37 years) with humeral lesions and 5 patients (average age, 25 years) with tibial fractures. There were 6 men and 3 women. Techniques customarily employed in arthroscopy were used to visualize, expose, and deliver the onlay cancellous bone grafts. Bony union occurred in all but 1 patient in an average of 4 months. This patient had a fibrous union and sustained a reinjury that led to successful repeat open bone graft surgery. The arthroscopic approach for bone grafting of certain long bone delayed union appears to be a safe and effective procedure. The procedure is best suited for patients with mechanically stabilized fragments, and it lends itself to those with overlying skin or soft tissue compromise. There are some relative contraindications: grossly unstable fragments, severe malunion, and/or infection.

Severe occupational traumatic injuries represent a challenge to workers and physicians. Efforts to prevent occupational injuries, such as education, protective equipment, adherence to safety procedures, and personal responsibility, are of critical importance. The physician's role as educator, consultant, and on-site manager of these injuries forms the basis for effective management of severe occupational traumatic injuries. Rapid assessment and treatment by coworkers trained in first aid can be vital in preventing more serious sequelae.

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.

There is added stability when plate fixation of the fibula supplements the external fixator used in treating tibial fractures associated with a segmental defect. An anatomic specimen model of a Grade III open fracture of the tibia with an associated fibular fracture was used in this study. Fibular internal fixation using an AO standard plate was performed on one of each of five matched pairs of legs. These were then subjected to identical loads in compression and torsion. The numeric values obtained during loading in all modes were converted to ratios between each of the tested pair to directly compare the effect of stabilization of the fibula. The final results showed that in each case, the preparation with fibular plating had improved resistance to deformation in axial loading. On average, the plated specimens were 2.2 times stiffer. In torsion, plating the fibula did not appear to affect the resistance of the construct to the applied load. Supplemental plate fixation of the fibula in fractures of the tibia with segmental defects may improve subsequent morbidity. The increased rigidity of the system may be sufficient to allow partial to full weight bearing immediately after surgery. This may have significant value in the management of the polytraumatized patient.

The use of interventional angiography has been limited in the past to the angiography suite. Significant technical difficulties have been associated with transfer of the unstable patient from the operating room for this procedure. However, current radiographic sophistication now exists to allow rapid intraoperative digital subtraction angiography of uncompromised quality to be performed. An animal model using these techniques is presented.