C-reactive protein, bone loss, fracture, and mortality in elderly women: a longitudinal study in the OPRA cohort.

SUMMARY: This longitudinal study investigates the association between C-reactive protein (CRP), osteoporosis, fractures, and mortality in 1044 elderly women. CRP was not an indicator for low bone mineral density (BMD), bone loss, or fracture in elderly women; however, women with elevated CRP levels over a prolonged period lost more bone over the 10-year follow-up, although fracture risk was not increased. INTRODUCTION: Inflammation may contribute to the pathophysiology underlying impaired bone metabolism. This study investigates the association between CRP, BMD, bone loss, fracture risk, and mortality in women aged 75 and above. METHODS: This longitudinal study is based on 1044 women, all age 75 at inclusion, reassessed at ages 80 and 85, with a mean follow-up time of 11.6 years (maximum 16.9 years). RESULTS: Women in the lowest CRP quartile (mean 0.63 mg/L) had lower BMD compared to those in the highest CRP quartile (mean 5.74 mg/L) at total hip (TH) (0.809 vs. 0.871 g/cm2, p<0.001) and femoral neck (FN) (0.737 vs. 0.778 g/cm2, p=0.007). A single measurement of CRP was not associated with bone loss; however, women with persistently elevated CRP, i.e., ≥3 mg/L at ages 75 and 80 had significantly higher bone loss compared to women with CRP<3 mg/L (TH -0.125 vs. -0.085 g/cm2, p=0.018 and FN -0.127 vs. -0.078 g/cm2, p=0.005) during 10 years of follow-up. Women in the highest CRP quartile had a lower risk of osteoporotic fractures (hazard ratios (HR) 0.76 (95% confidence intervals (CI) 0.52-0.98)) compared to those in the lowest, even after adjusting for weight and BMD. Mortality risk was only increased among women with the highest CRP levels. CONCLUSION: CRP was not an indicator for low BMD, bone loss, or fracture in elderly women in this study. Persistently elevated CRP however seemed to be detrimental to bone health and may be associated with a higher rate of bone loss. Only the highest CRP levels were associated with mortality.

Genetic analysis of non-insulin dependent diabetes mellitus in the GK rat.

Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.

Genetic variation in the PTH pathway and bone phenotypes in elderly women: evaluation of PTH, PTHLH, PTHR1 and PTHR2 genes.

INTRODUCTION: Parathyroid hormone (PTH) is a key regulator of calcium metabolism. Parathyroid hormone-like hormone (PTHrP) contributes to skeletal development through regulation of chondrocyte proliferation and differentiation during early bone growth. Both PTH and PTHrP act through the same receptor (PTHR1). A second receptor, PTHR2, has been identified although its function is comparatively unknown. PTH hyper-secretion induces bone resorption, whereas intermittent injection of PTH increases bone mass. To explore the effects of genetic variation in the PTH pathway, we have analysed variations in PTH, PTHLH, PTHR1 and PTHR2 in relation to bone mass and fracture incidence in elderly women. MATERIALS AND METHODS: This study includes 1044 elderly women, all 75 years old, from the Malmö Osteoporosis Prospective Risk Assessment study (OPRA). Single nucleotide polymorphisms (SNPs) from 4 genes and derived haplotypes in the PTH signaling pathway were analysed in 745-1005 women; 6 SNPs in the PTH gene and 3 SNPs each in the PTHLH, PTHR1 and PTHR2 genes were investigated in relation to BMD (assessed at baseline), fracture (434 prevalent fractures of all types over lifetime, self-reported and 174 incident fractures up to 7 years, X-ray verified) and serum PTH. RESULTS AND CONCLUSION: Individually, SNPs in the 4 loci did not show any significant association with BMD. Neither were PTHLH, PTHR1 and PTHR2 polymorphisms associated with fracture. Three of 5 common haplotypes, accounting for >98% of alleles at the PTH locus, were identified as independent predictors of fracture. Haplotype 9 (19%) was suggestive of an association with fractures of any type sustained during lifetime (p=0.018), with carriers of one or more copies of the haplotype having the lowest incidence (p=0.006). Haplotypes 1 (13%) and 5 (37%) and 9 were suggestive of an association with fractures sustained between 50 and 75 years (p=0.02, p=0.013 and p=0.034). Carriers of haplotypes 1 and 5 were more likely to suffer a fracture (haplotype 1, p=0.045; haplotype 5, p=0.008). We conclude, that while further genotyping across the gene is recommended, in this cohort of elderly Swedish women, polymorphisms in PTH may contribute to the risk of fracture through mechanisms that are independent of BMD.

Differences in the ratio of RNA encoding two isoforms of the insulin receptor between control and NIDDM patients. The RNA variant without Exon 11 predominates in both groups.

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 +/- 1%) compared with control subjects (31 +/- 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.

Glucose potentiation of arginine-induced insulin secretion is impaired in subjects with a glucokinase Glu256Lys mutation.

Insulin and glucagon release and insulin sensitivity were investigated in patients with glucokinase deficiency. Five subjects with a missense mutation (Glu256Lys) were studied. They were compared with six healthy subjects with low insulin response but normal glucose tolerance. Insulin and glucagon levels were measured at blood glucose 7.1 +/- 0.1 mmol/l and at 10.9 +/- 0.2 mmol/l with or without arginine (5 g i.v.). Insulin sensitivity was assessed as the ratio between infused glucose and the insulin level (M:I) during hyperglycemic clamps. Glu256Lys subjects were nonobese and had fasting blood glucose 6.7 +/- 0.1 mmol/l (P < 0.001 vs. control group). Insulin release was reduced in response to 11 mmol/l glucose (61% of control group, P < 0.05) as well as to arginine in the presence of 11 mmol/l glucose (54% of control group, P < 0.01). Also, the slope of potentiation, i.e., the enhancement of arginine-induced release as a function of prevailing glucose concentration, was reduced (delta insulin/delta glucose, 47% of control group, P < 0.05). As for glucagon release, the response to arginine was not inhibited normally by glucose, resulting in threefold higher levels at 11 mmol/l glucose versus control subjects. Insulin sensitivity, assessed as M:I, was significantly (P < 0.05) reduced (55% of control group). Glucokinase deficiency thus affects not only insulin responses to glucose per se but also glucose potentiation of responses to non-nutrient secretagogues. Abnormalities in glucagon release and insulin sensitivity coexist with attenuated insulin responses in glucokinase-deficient subjects.

Increased mRNA levels of Mn-SOD and catalase in embryos of diabetic rats from a malformation-resistant strain.

Previous studies have suggested that reactive oxygen species (ROS) are mediators in the teratogenic process of diabetic pregnancy. In an animal model for diabetic pregnancy, offspring of the H rat strain show minor dysmorphogenesis when the mother is diabetic, whereas the offspring of diabetic rats of a sister strain, U, display major morphologic malformations. Earlier studies have shown that embryonic catalase activity is higher in the H than in the U strain, and maternal diabetes increases this difference in activity. The aim of this study was to characterize the influence of genetic predisposition on diabetic embryopathy by comparing the mRNA levels of ROS-metabolizing enzymes in the two strains. We determined the mRNA levels of catalase, glutathione peroxidase, gamma-glutamylcystein-synthetase, glutathione reductase, and superoxide dismutase (CuZn-SOD and Mn-SOD) in day 11 embryos of normal and diabetic H and U rats using semiquantitative reverse transcription-polymerase chain reaction. The mRNA levels of catalase and Mn-SOD were increased in H embryos as a response to maternal diabetes, and no differences were found for the other genes. Sequence analysis of the catalase promoter indicated that the difference in mRNA levels may result from different regulation of transcription. Sequence analysis of the catalase cDNA revealed no differences between the two strains in the translated region, suggesting that the previously observed difference in the electrophoretic mobility in zymograms is due to posttranslational modifications. An impaired expression of scavenging enzymes in response to ROS excess can thus be an integral part of a genetic predisposition to embryonic dysmorphogenesis.

Impact of diabetic inheritance on glucose tolerance and insulin secretion in spontaneously diabetic GK-Wistar rats.

The impact of genetic factors and maternal diabetes on glucose tolerance and pancreatic beta-cell function was studied in first generation (F1) offspring generated in crosses between the spontaneously diabetic Goto-Kakizaki (GK)-Wistar rat and normoglycemic control Wistar rats (W). The (GK x W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK x fW) or male Wistar (mW) and female GK (fGK) (mW x fGK) rats. Already at 8 days of age, blood glucose levels were elevated in GK (7.6 +/- 0.5 vs. 4.8 +/- 0.3 mM in W; P < 0.001) and in F1 rats (6.0 +/- 0.3 in mGK x fW and 6.6 +/- 0.4 mM in mW x fGK; both P < 0.01 vs. W). In 2-month-old male rats, glucose (2 g/kg, intraperitoneally) markedly increased blood glucose levels after 60 min in GK rats (18.1 +/- 0.6 vs. 5.5 +/- 0.3 mM in W; P < 0.001) and moderately increased levels in F1 rats (9.9 +/- 0.9 in mGK x fW and 11.6 +/- 1.0 mM in mW x fGK, both P < 0.01 vs. W). Similar patterns were obtained in female rats. Repeated backcrossing of F1 with W rats successively improved glucose tolerance. In perfused pancreases of male rats, the 20-min insulin response to 16.7 mM glucose was -7.44 +/- 5.18 pmol in GK rats, 71.57 +/- 12.25 pmol in W rats, 9.00 +/- 0.89 pmol in mGK x fW rats, and 18.20 +/- 3.97 pmol in mW x fGK rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiological and genetic characterization of the major diabetes locus in GK rats.

Genetic studies of the type 2 diabetes-like GK rat have revealed several susceptibility loci for the compound diabetes phenotype. Congenic strains were established for Niddm1, the major quantitative trait locus (QTL) for postprandial glucose levels, by transfer of GK alleles onto the genome of the normoglycemic F344 rat. Despite the polygenic nature of diabetes in GK, the locus-specific diabetes phenotype was retained in the congenic strain Niddmla, containing a GK-derived genomic fragment of 52 cM from the Niddm1 locus. Furthermore, Niddm1 was divided into two non-overlapping loci, physically separated in the two congenic strains Niddmlb and Niddm1i with distinct metabolic phenotypes. Both strains displayed postprandial hyperglycemia and reduced insulin action in isolated adipose cells. Furthermore, Niddm1i already exhibits a pronounced in vivo insulin secretion defect at 65 days, while Niddm1b develops a relative insulin secretory defect at 95 days. This suggests that Niddm1i impairs mechanisms common to insulin secretion in pancreatic B-cells and insulin action in adipocytes. Niddm1b rats show signs of increasing insulin resistance with age associated with obesity, hyperinsulinemia, and dyslipidemia. Moreover, the data indicated nonallelic interaction (epistasis) between Niddm1b and Niddm1i on the postprandial glucose levels. These data emphasize the pathophysiological complexity of diabetes, even within an apparently single QTL, and demonstrate the potential of the GK model in transforming the multifactorial diabetes phenotype into single traits, suitable for positional cloning.

Phenotyping of individual pancreatic islets locates genetic defects in stimulus secretion coupling to Niddm1i within the major diabetes locus in GK rats.

The major diabetes quantitative trait locus (Niddm1), which segregates in crosses between GK rats affected with spontaneous type 2-like diabetes and normoglycemic F344 rats, encodes at least two different diabetes susceptibility genes. Congenic strains for the two subloci (Niddm1f and Niddm1i) have been generated by transfer of GK alleles onto the genome of F344 rats. Whereas the Niddm1f phenotype implicated insulin resistance, the Niddm1i phenotype displayed diabetes related to insulin deficiency. Individual islets from 16-week-old congenic rats were characterized for insulin release and oxygen tension (pO(2)). In the presence of 3 mmol/l glucose, insulin release from Niddm1f and Niddm1i islets was approximately 5 pmol. g(-1). s(-1) and pO(2) was 120 mmHg. Similar recordings were obtained from GK and F344 islets. When glucose was raised to 11 mmol/l, insulin release increased significantly in Niddm1f and F344 islets but was essentially unchanged in islets from GK and Niddm1i. The high glucose concentration lowered pO(2) to the same extent in islets from all strains. Addition of 1 mmol/l tolbutamide to the perifusion medium further increased pulsatile insulin release threefold in all islets. The pulse frequency was approximately 0.4 min(-1). alpha-Ketoisocaproate (11 mmol/l) alone increased pulsatile insulin release eightfold in islets from Niddm1f, Niddm1i, and control F344 rats but had no effect on insulin release from GK islets. These secretory patterns in response to alpha-ketoisocaproate were paralleled by reduction of pO(2) in Niddm1f, Niddm1i, and control F344 islets and no change of pO(2) in GK islets. The results demonstrate that Niddm1i carries alleles of gene(s) that reduce glucose-induced insulin release and that are amenable to molecular identification by genetic fine mapping.

Non-contiguous segments of the polyoma genome required in cis for DNA replication.

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 basepairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A . T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions. The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.

Insulin receptor ribonucleic acid levels and alternative splicing in human liver, muscle, and adipose tissue: tissue specificity and relation to insulin action.

The absolute levels and alternative splicing of insulin receptor RNA molecules were determined in samples from liver, muscle, and adipose tissue from 17 nondiabetic individuals. Both the absolute levels and alternative splicing varied in a tissue-specific manner. In all tissues, a majority of the insulin receptor RNA molecules contained exon 11. Liver tissue had a lower percentage of RNA molecules without exon 11 (Ex 11-) than muscle and adipose tissue, but the absolute number of Ex 11- RNA copies was higher due to higher overall levels of insulin receptor RNA. Insulin receptor RNA levels in adipose tissue showed significant correlation with obesity, expressed as body mass index (kilograms per m2) as well as with in vivo insulin action, as measured by the insulin tolerance test. In this study, obesity and insulin action were not correlated with insulin receptor RNA expression in liver or muscle. Within individuals, no relation was detected between the number of insulin receptor RNA copies in a tissue and the number or percent Ex 11- RNA in the same tissue. Also, the absolute levels or Ex 11- percentages in one tissue could not predict corresponding measurements in the other two investigated tissues from the same individual.

21-hydroxylase deficiency: disease-causing mutations categorized by densitometry of 21-hydroxylase-specific deoxyribonucleic acid fragments.

The types of disease-causing mutations were studied in 43 unrelated patients with 21-hydroxylase deficiency. Densitometry of Southern blots after cleavage with the restriction enzymes TaqI, PvuII, and BglII was used to measure the ratio of the copy-number of the 21-hydroxylase gene (CYP21) to the copy-number of its pseudogene (CYP21P). DNA from 16 unrelated patients showed equal hybridization intensities of the 2 genes, indicating that point mutations caused the enzyme deficiency. One of the 2 haplotypes in 7 patients showed evidence of a large gene conversion between the CYP21 and the CYP21P gene without loss of the total number of 21-hydroxylase genes. Deletion of at least 1 21-hydroxylase gene was found in 11 patients. DNA from 8 of these patients had relative hybridization intensities compatible with a deletion of the active 21-hydroxylase gene, CYP21. Two patients with the salt-wasting form of the disease showed homozygous loss of DNA fragments that are specific for the 5' end of the active 21-hydroxylase gene. Nine patients showed relative 21-hydroxylase hybridization intensities compatible with duplication of the gene in 1 or both haplotypes. In conclusion, point mutations, gene conversions, or CYP21 gene deletions are the typical mutations in patients with the simple virilizing and salt-wasting forms of the disease, while duplications of the locus are overrepresented in patients with nonclassical 21-hydroxylase deficiency.

Synergistic effect of partially inactivating mutations in steroid 21-hydroxylase deficiency.

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. Mild forms of the disease cause late-onset symptoms of hyperandrogenism and are thought to be largely underdiagnosed. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. We previously reported a rare allele in two siblings with late-onset disease. This allele contained three sequence alterations, a C to T transition 4 bases upstream of translation initiation, a CCG to CTG change at codon 105 (P105L), and a CCC to TCC transition at codon 453 (P453S). The latter mutation has been found in other ethnic groups, whereas P105L seems to be unique to this family. We have now analyzed the functional consequences of the -4, P105L, and P453S sequence alterations by in vitro translation and after expression of mutant enzyme in cultured cells. As expected, the base substitution at position-4 had no measurable effect on gene expression. The P105L mutation reduced enzyme activity to 62% for 17-hydroxyprogesterone and 64% for progesterone, and the P453S mutation reduced activity to 68% and 46%, respectively. When present in combination, the two mutations caused a reduction of enzyme activity to 10% for 17-hydroxyprogesterone and 7% for progesterone. These results indicate that P105L and P453S can be expected to result in a very subtle disease manifestation when not found in combination, motivating their inclusion when genotyping to ascertain undiagnosed patients with the mildest forms of 21-hydroxylase deficiency.

Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation.

We have characterized the disease-causing mutations in the steroid 21-hydroxylase genes of 127 patients with different clinical forms of congenital adrenal hyperplasia, representing 186 unrelated chromosomes. The gene was completely absent on 29.8% of the chromosomes, and this together with the I2 splice (27.7%), I173N (20.8%), V282L (5.4%), and R357W (3.8%) mutations constitute 87.5% of all affected chromosomes. In total, 15 different sequence aberrations combine to form 19 different disease-causing alleles. The results confirm that genotyping is an efficient means of diagnosing steroid 21-hydroxylase deficiency, although special consideration is needed to resolve genotypes when full families are not available. Clinical presentations of the different combinations of mutations indicate that genotyping is reliable for prediction of clinical outcome in patients with 21-hydroxylase deficiency. It is especially helpful in determining whether in utero treatment of affected females is indicated and in classifying the severity of 21-hydroxylase deficiency in children diagnosed through neonatal screening, before symptoms have appeared.

High diagnostic accuracy for idiopathic Addison's disease with a sensitive radiobinding assay for autoantibodies against recombinant human 21-hydroxylase.

Autoantibodies against 21-hydroxylase (P450c21) are common in idiopathic autoimmune Addison's disease. In the present work, we have developed a sensitive radiobinding assay using in vitro translated recombinant human 35S-P450c21. Levels of P450c21 antibodies (P450c21-Ab) were expressed as a relative index (P450c21 index) using a P450c21-Ab positive Addisonian serum and two antibody-negative healthy sera as positive and negative standards in healthy individuals. The upper level of normal was the mean + 3 SD. Positivity for P450c21-Ab was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated 35S-P450c21. In 38 Addisonian patients, P450c21-Ab were found in 24/28 (86%) idiopathic, 0/5 post-tuberculosis, 0/3 adrenoleukodystrophy, and 0/2 post-adrenalectomy sera. Among healthy individuals, 1/70 (1.4%) were positive. The P450c21 index, as an estimate of P450c21-Ab levels, correlated inversely with the duration of idiopathic Addison's disease (r = -0.527; P = 0.007): 16/16 (100%) positive in patients with less than 20 yr and 8/12 (67%) positive in patients with more than 20 yr disease duration. The availability of this simple and sensitive radiobinding assay to evaluate levels of P450c21-Ab will permit large clinical studies as well as screening subjects at risk. In addition, the general population can now be screened to evaluate the predictive value of P450c21-Ab for Addison's disease.

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

Changes in cytoplasmic ATP concentration parallels changes in ATP-regulated K+-channel activity in insulin-secreting cells.

Changes in cytoplasmic ATP concentration were monitored in intact insulin-producing cells and correlated to changes in the activity of ATP-sensitive K+-channels (KATP channels). Luciferase was introduced into HIT M2.2 cells and whole pancreatic islets by transient expression of firefly (Photinus pyralis) luciferase cDNA. In transfected cells, extracellular addition of luciferin increased the luminescence signal to a maximum within 50-120 s. Addition of 1 microM of the mitochondrial uncoupler FCCP decreased the luminescence, an effect partly reversed upon withdrawal of the compound. High concentrations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP-sensitive K+-channel. Transfection per se did not deteriorate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+-concentration, [Ca2+li, in both transfected and non-transfected cells. By measuring the cytoplasmic ATP concentration and KATP channel activity under similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of KATP channel activity under physiological conditions.

Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats.

Genetic analysis of experimental autoimmune encephalomyelitis (EAE) can provide clues to the etiology of multiple sclerosis (MS). Identifying the susceptibility genes of DA rats may be particularly rewarding since they are prone to develop a remarkably MS-like chronic and demyelinating disease. As a first step in this direction, we investigated the role of DA genes within and outside the major histocompatibility complex (MHC) for susceptibility to severe protracted and relapsing EAE (SPR-EAE). This form of EAE developed in DA rats but not in LEW. ACI and BN rats after immunization with syngeneic spinal cord and complete Freund's adjuvant. Studies of crosses between DA and BN rats revealed that non-MHC genes determine susceptibility to SPR-EAE. A role for MHC-genes was also established using MHC-congenic rat strains, in which the DA MHC haplotype (av1) associated with relapsing EAE. Again, non-MHC genes were decisive since a high incidence of SPR-EAE only occurred in rats with DA non-MHC genes. Analysis of cytokine mRNA expression and infiltrating cells in the spinal cords of congenic strains revealed that the av1 haplotype associated with a high CD4/CD8 ratio and expression of mRNA for interferon-gamma (IFN-gamma), but not for transforming growth factor-beta (TGF-beta) or interleukin-10 (IL-10). In contrast, the other MHC haplotypes (h, l, u) associated with low CD4/CD8 ratios and mRNA expression for TGF-beta and IL-10, but not for IFN-gamma. DA non-MHC genes determined the intensity of inflammation since the number of cells expressing MHC class II, CD4 and interleukin-2 receptor (IL-2R) was higher in DA rats than in LEW.1AV1 and PVG.1AV1 rats which also carry the av1 haplotype. We conclude that the MHC haplotype of DA rats favors a prolonged proinflammatory autoimmune response associated with relapses, while the DA background intensifies inflammation correlating with a high incidence of relapsing disease.

Polygenic control of autoimmune peripheral nerve inflammation in rat.

Experimental autoimmune neuritis (EAN) is the principal animal model for Guillain-Barré syndrome (GBS), an inflammatory disease of the peripheral nervous system. Little is known on the genetic regulation of these diseases. We provide the first genetic linkage analysis of EAN. Susceptibility to EAN in a rat F2 population segregated with high levels of anti-PNM IgG, as well as IgG2b and IgG2c isotype levels, which support that disease genes regulate preferential Th1/Th2 differentiation. Linkage analysis demonstrated co-localization of EAN loci with reported susceptibility loci for experimental arthritis and/or encephalomyelitis and a new region on chromosome 17. Further dissection of these loci may disclose disease pathways in GBS.

Effect of growth hormone treatment on insulin action in adipocytes from children with Prader-Willi syndrome.

OBJECTIVE: To study the effect of growth hormone (GH) treatment (2-4 months) on insulin action in adipocytes isolated from children with Prader-Willi syndrome (PWS), in whom GH deficiency appears to be a primary defect. We investigated the complex effects of GH on carbohydrate metabolism, as part of a current clinical trial of GH treatment in children with PWS. METHODS: Biopsies of subcutaneous abdominal adipose tissue were obtained from 12 children with PWS before and after 2-4 months of GH treatment. Lipogenesis was determined by the incorporation of radiolabelled glucose into lipids in isolated adipocytes, and glycerol release to the incubation medium was used as an index of lipolysis. GLUT4 RNA was measured by solution hybridization. RESULTS: With low glucose concentrations, at which glucose transport is rate-limiting, maximal insulin-induced lipogenesis was increased by 120% after GH treatment (P < 0.05), but the sensitivity to insulin (half-maximum effective hormone concentration) was unchanged. This was not accompanied by a significant change in the RNA expression of GLUT4. Neither responsiveness (maximum effect) nor sensitivity of insulin-induced inhibition of lipolysis was affected by GH treatment. CONCLUSIONS: GH treatment of children with PWS results in an upregulation of insulin-induced lipogenesis in isolated adipocytes, with no effect on insulin-induced inhibition of lipolysis. The data suggest that the site of the effect of GH on lipogenesis is distal to the insulin hormone-receptor interaction, but does not involve altered GLUT4 expression.