The Val985Met insulin-receptor variant in the Danish Caucasian population: lack of associations with non-insulin-dependent diabetes mellitus or insulin resistance.
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Blood Glucose↗
Biomedical subjects
T Hansen
Publications and source records attributed to T Hansen.
Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin.
The role of the endothelial cytoskeleton for the structural integrity of the pulmonary gas exchange area was probed with the use of Clostridium botulinum C2 toxin. This agent causes selective loss of nonmuscle F-actin. In buffer-perfused rabbit lungs, vascular pressures were kept within physiological ranges. In different groups, low-dose [0.3 (C2,I)/0.6 (C2,II) ng/ml] and high-dose [10 (C2,I)/20 (C2,II) ng/ml] toxin were applicated into the buffer fluid; experiments were terminated after a total weight gain of either 1 or 7.5 g. Electron microscopy revealed extensive attenuations, undulations, and protrusions of the endothelial layer, suggestive of "remodeling" and "flowing" of the cell membrane in low C2 toxin-treated lungs accompanied by few disruptions of the endothelial layer and edema formation. In addition, endothelial cells displayed vesiculation and bleb formation. Lungs that were exposed to high-toxin doses displayed marked attenuations of the endothelial layer in addition to large endothelial cell disruptions, which did not include interendothelial junctions. Interestingly, type II epithelial cells displayed fusion of lamellar bodies. Collectively, these data suggest that the actin microfilament system is instrumental in supporting endothelial cell membrane configuration and integrity and maintains the intimal barrier function of the lung microvasculature.
Animals↗
Studies of the impact of a liver glucokinase promoter variant on glucose tolerance and insulin sensitivity index.
Because a frequently occurring nucleotide substitution at position -258 in the liver glucokinase promoter has been reported to be associated with impaired promoter activity, we have examined in Danish Caucasians whether this variant is associated with alterations in glucose tolerance and/or the insulin sensitivity index (Si). Among 246 Danish Caucasian patients with noninsulin-dependent diabetes mellitus, the allelic frequency of the -258 promoter variant was 15.2% (95% confidence interval: 12.0-18.4%) vs. 16.5% (13.2-19.8%) among 242 matched control subjects. In the control group, the glucokinase variant was not related to serum insulin or plasma glucose levels before or during an oral glucose tolerance test. Neither was the gene variant among 380 young, healthy subjects associated with altered Si or altered insulin secretion after an i.v. glucose load. We conclude that in Danish Caucasians, the -258 glucokinase promoter variant has no impact on glucose tolerance, whole-body Si, or insulin secretion.
Adult↗
Identification of a common amino acid polymorphism in the p85alpha regulatory subunit of phosphatidylinositol 3-kinase: effects on glucose disappearance constant, glucose effectiveness, and the insulin sensitivity index.
Phosphatidylinositol 3-kinase (PI3-K) may regulate the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool in addition to being an insulin signal for translocation of glucose transporters to the plasma membrane. The objectives of the present study were to examine for genetic variability in the human regulatory p85alpha subunit of PI3-K, to look for an association between gene variants and NIDDM in a case-control study, and to relate identified variability to potential changes in whole-body insulin sensitivity and glucose turnover in a phenotype study. Single-strand conformational polymorphism and heteroduplex analysis of the coding region of the regulatory p85alpha subunit in cDNA isolated from human muscle tissue from 70 insulin-resistant NIDDM patients and 12 control subjects revealed three silent polymorphisms and a missense mutation at nucleotide position 1020 (G-->A), changing a Met to Ile at codon 326. Using allele-specific oligohybridization, we found a similar allelic frequency of the codon 326Met-->Ile variant in 404 NIDDM patients (0.15 [95% CI 0.13-0.17]) and 224 matched glucose tolerant control subjects (0.16 [0.13-0.19]). In a random sample of 380 unrelated healthy young Caucasians aged 18-32 years, in whom we have performed a tolbutamide modified intravenous glucose tolerance test, we identified 263 wildtype subjects, 109 heterozygous subjects, and 8 subjects homozygous for the codon 326 variant (allelic frequency = 0.16 [0.13-0.19]). No difference in glucose disappearance constant (KG), insulin sensitivity index (SI), and glucose effectiveness (SG) was observed between wildtype and heterozygous subjects. However, compared with the combined values for wildtype and heterozygous carriers, KG was reduced by 40% (P = 0.004) and SG by 23% (P = 0.03) in homozygous carriers of the p85alpha variant. Moreover, in homozygous carriers, a 32% reduction was found in SI (P = 0.08). In conclusion, a codon 326Met-->Ile variant in the gene encoding the PI3-K p85alpha regulatory subunit is found in 31% of a random sample of young healthy Caucasians. About 2% of the subjects in this population carry the gene variant in its homozygous form, and these carriers are characterized by significant reductions in whole-body glucose effectiveness and intravenous glucose disappearance constant. In itself, the gene variant does not confer an increased risk of diabetes.
Adolescent↗
Amino acid polymorphisms in the ATP-regulatable inward rectifier Kir6.2 and their relationships to glucose- and tolbutamide-induced insulin secretion, the insulin sensitivity index, and NIDDM.
Kir6.2 is an inwardly rectifying potassium channel that is expressed in pancreatic beta-cells and cardiac and skeletal muscle. Expressed together with the high-affinity sulphonylurea receptor, it reconstitutes a sulphonylurea- and also ATP-sensitive potassium channel resembling the native beta-cell channel. The objective of this study was to search for mutations in the Kir6.2 gene that might be associated with NIDDM or related to altered insulin secretion, insulin action, or glucose metabolism in healthy subjects. Using polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) on genomic DNA from 69 Danish NIDDM patients and 66 matched control subjects, we report the finding of three missense polymorphisms in otherwise conserved codons and three silent polymorphisms in the gene encoding Kir6.2: codon 23 (GAG/AAG), Glu-->Lys; codon 190 (GCT/GCC), Ala-->Ala; codon 267 (CTC/CTG), Leu-->Leu; codon 270 (CTG/GTG), Leu-->Val; codon 337 (ATC/GTC), Ile-->Val; codon 381 (AAG/AAA), Lys-->Lys. The codon 23 and codon 337 amino acid polymorphisms were always coupled. The allelic frequencies of the polymorphisms were similar in NIDDM patients and control subjects. The amino acid polymorphisms were not associated with altered insulin secretion after intravenous glucose or tolbutamide injections or with altered glucose effectiveness in a phenotype study of 346 young healthy subjects. However, carriers of the maximal load of amino acid variants, the compound homozygous codon 23/337 and heterozygous codon 270, had on average a 62% higher insulin sensitivity index (P = 0.006), compared with noncarriers. We conclude that a combination of common Kir6.2 amino acid variants may contribute to the genetic background behind the large variation of the insulin sensitivity index in the general population.
Adenosine Triphosphate↗
Novel MODY3 mutations in the hepatocyte nuclear factor-1alpha gene: evidence for a hyperexcitability of pancreatic beta-cells to intravenous secretagogues in a glucose-tolerant carrier of a P447L mutation.
One form of maturity-onset diabetes of the young (MODY3) results from mutations in the hepatocyte nuclear factor (HNF)-1alpha gene, located on chromosome 12q24.2. The primary objective of the present study was to search for genetic variation in the HNF-1alpha gene in nine nonrelated Danish Caucasian subjects with MODY. Direct sequencing of the coding region and intron-exon boundaries of the HNF-1alpha gene revealed 2 novel and 1 previously reported missense mutations and 2 novel frameshift mutations in five of nine MODY subjects. These five mutations were found in neither 84 NIDDM patients nor 84 control subjects. One glucose-tolerant lean male with a P447L missense mutation, which in his relatives caused MODY, underwent an oral glucose tolerance test (OGTT), a tolbutamide modified frequently sampled intravenous glucose tolerance test, and a glucagon test to examine for a possible early beta-cell abnormality. He had a low insulin secretion rate during an OGTT, but a twofold increase in pancreatic beta-cell response after intravenous glucose and a 2.5- to 4-fold increase in beta-cell response after either intravenous tolbutamide or intravenous glucagon loads. In conclusion, 1) mutations in the HNF-1alpha gene are common in Danish Caucasian MODY patients, and 2) early stages in the pathogenesis of MODY3 caused by the P447L mutation may be characterized by a hyperexcitability of beta-cells to intravenous secretagogues.
Adolescent↗
A prevalent amino acid polymorphism at codon 98 in the hepatocyte nuclear factor-1alpha gene is associated with reduced serum C-peptide and insulin responses to an oral glucose challenge.
Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause the type 3 form of maturity-onset diabetes of the young (MODY3), which is characterized by a severe impairment of insulin secretion. In addition to disease-associated mutations, three common amino acid polymorphisms have been identified in the HNF-1alpha gene: Ile/Leu27, Ala/Val 98, and Ser/Asn487. We have addressed the question of whether these variants of the HNF-1alpha gene are associated with altered glucose-induced C-peptide and insulin responses or late-onset NIDDM. Among 245 NIDDM patients, the allelic frequency of the Val 98 variant was 3.7% (95% CI 2.0-5.4%) vs. 4.4% (2.6-6.2%) among 240 glucose tolerant control subjects (NS). Studies of genotype-phenotype interactions in 240 middle-aged control subjects showed, however, that heterozygous subjects (i.e., genotype Ala/Val 98) had an 18% decrease in 30-min serum C-peptide level (P = 0.004) as well as a 23% decrease in 30-min serum insulin level (P = 0.03) during an oral glucose tolerance test. One Val 98 homozygote subject had a more severe reduction in stimulated insulin and C-peptide levels. The impact of the homozygous carrier status was similar in a study of 377 healthy young subjects. In contrast, the Ile/Leu27 and Ser/Asn487 polymorphisms were not associated with altered C-peptide and insulin release or NIDDM. In conclusion, 8% of white subjects of Danish ancestry are heterozygous for the Ala/Val 98 polymorphism in the HNF-1alpha gene, which in middle-aged subjects is associated with a approximately 20% reduction in serum C-peptide and insulin responses 30 min after an oral glucose challenge. Val 98 homozygotes may exhibit a more severe defect in the early glucose-induced insulin response.
Administration, Oral↗
Prevalence of a polymorphism of the phosphatidylinositol 3-kinase p85 alpha regulatory subunit (codon 326 Met-->Ile) in Japanese NIDDM patients.
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Base Sequence↗
Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3)
The disease non-insulin-dependent (type 2) diabetes mellitus (NIDDM) is characterized by abnormally high blood glucose resulting from a relative deficiency of insulin. It affects about 2% of the world's population and treatment of diabetes and its complications are an increasing health-care burden. Genetic factors are important in the aetiology of NIDDM, and linkage studies are starting to localize some of the genes that influence the development of this disorder. Maturity-onset diabetes of the young (MODY), a single-gene disorder responsible for 2-5% of NIDDM, is characterized by autosomal dominant inheritance and an age of onset of 25 years or younger. MODY genes have been localized to chromosomes 7, 12 and 20 (refs 5, 7, 8) and clinical studies indicate that mutations in these genes are associated with abnormal patterns of glucose-stimulated insulin secretion. The gene on chromosome 7 (MODY2) encodes the glycolytic enzyme glucokinases which plays a key role in generating the metabolic signal for insulin secretion and in integrating hepatic glucose uptake. Here we show that subjects with the MODY3-form of NIDDM have mutations in the gene encoding hepatocyte nuclear factor-1alpha (HNF-1alpha, which is encoded by the gene TCF1). HNF-1alpha is a transcription factor that helps in the tissue-specific regulation of the expression of several liver genes and also functions as a weak transactivator of the rat insulin-I gene.
Animals↗
Donor nutritional status--a determinant of liver preservation injury.
In liver transplantation, the quality of the liver is determined by a number of factors including donor nutritional status. Livers from fasted donors appear to tolerate long-term preservation better than livers from fed donors. In this study we repeated earlier results and obtained 31% (4/13) survival after 40-hr preservation of livers from fed donor Brown Norway rats and 67% (8/12) survivors with donor livers from 4-day-fasted rats (P = 0.154). The explanation for this improvement is not known but may be due to inactivation of Kupffer cells due to nutritional depletion of the liver. Kupffer cell activation has been one explanation advanced to explain how cold storage injuries livers during reperfusion (transplantation). In this study, we have measured how donor fasting affects Kupffer cell function (phagocytosis of colloidal carbon) after preservation of the rat liver. In addition, we measured how enhancing liver glycogen by feeding glucose to the rat donors affected outcome and liver functions tested by isolated perfusion after 24- and 40-hr cold storage of the liver. Preservation did not cause inactivation or activation of Kupffer cell phagocytosis of colloidal carbon. In livers with 0-hr preservation, colloidal carbon uptake was 3.1 +/- 0.2 mg/g/hr, after 40-hr preservation uptake was 3.8 mg/g/hr (P < 0.05 vs. 0 hr) (fed) and 2.7 +/- 0.3 mg/g/hr (fasted, P, 0.05 vs. 0-hr and 40-hr-fed). Thus, the improved survival obtained with livers from fasted donors does not appear related to inactivation of Kupffer cell phagocytosis. Although livers from fasted donors showed improved survival, there was extensive hepatocellular injury as indicated by large LDH release from the livers after 40-hr cold storage as tested by isolated perfusion. LDH released into the perfusate increased from 35.8 +/- 10.1 U/L (fed, 40-hr CS) to 301 +/- 65 U/L (fasted, 40-hr CS) after 1-hr reperfusion. AST release showed a similar pattern and bile production was suppressed more in livers from fasted donors than fed donors. Feeding rats glucose elevated liver glycogen and significantly reduced hepatocellular injury as measured by LDH release and AST release in the isolated perfused liver after 40-hr cold storage. Feeding rats glucose (40% in drinking water for 4 days) also improved survival: fed+glucose = 85% survival versus 31% survival with no glucose and fasted+glucose = 92% survival versus 67% survival with no glucose. These results show that both extensive donor fasting and glucose feeding enhanced outcome in orthotopic liver transplantation. This dilemma (both fasting and feeding improved survival) are discussed in terms of how the interactions between Kupffer cells and hepatocytes affect liver viability. Donor fasting is probably impractical clinically as a method to improve the donor liver, but elevating liver glycogen by glucose supplementation is possible and may lead to improved preservation and outcome in liver transplantation.
Animals↗
Lens autofluorescence is increased in newly diagnosed patients with NIDDM.
Lens and cornea autofluorescence has been shown to be increased in patients with insulin-dependent diabetes mellitus and to be positively correlated to glycaemic control and duration of diabetes. We have studied lens and cornea autofluorescence at the clinical onset of non-insulin-dependent diabetes mellitus (NIDDM), in comparison with age-matched subjects with normal glucose tolerance. Fourteen subjects with NIDDM diagnosed less than 6 months prior to the examination were characterised by ocular fluorometry, glycosylated hemoglobin A1c, plasma lipid status, arterial blood pressure, and an oral glucose tolerance test (OGTT). Eleven age- and gender-matched healthy subjects without a family history of diabetes and with a normal glucose tolerance underwent the same examinations. In 11 of the 14 diabetic patients lens autofluorescence was increased to levels higher than the age-related mean + 2SD of healthy subjects. For the entire study population, control and diabetic subjects, lens fluorescence was positively correlated with HbA1c (p < 0.0001, r = 0.73), fasting plasma glucose (p = 0.002, r = 0.60) and the plasma glucose level 2 h after an OGTT (p = 0.004, r = 0.55). Cornea autofluorescence was also significantly increased in the group of newly diagnosed NIDDM patients, but only 9 patients had values above the mean + 2SD of the healthy subjects. NIDDM could be detected by ocular fluorometry with a sensitivity of 79% and a specificity of 100%. We conclude that lens and cornea autofluorescence is abnormally increased in the majority of patients with newly diagnosed NIDDM. The sensitivity and specificity of the method indicate that lens fluorometry may potentially be useful for screening for undiagnosed NIDDM in the general population. Additionally, we propose that the method may be a clinically useful indicator of cumulative glycaemia and risk of development of secondary complications in patients with diabetes.
Adult↗
Differential role of actin in lung endothelial and epithelial barrier properties in perfused rabbit lungs.
Lung fluid balance is critically dependent on capillary endothelial and alveolar epithelial barrier properties, and cytoskeletal components have been implicated in these barrier functions. In an earlier study, we perfused Clostridium botulinum C2 toxin, which effects selective loss of non-muscle F-actin, through isolated rabbit lungs: a severalfold increase in the capillary filtration coefficient (Kfc) was noted, together with attenuations and disruptions of endothelial cells upon electron microscopic examination. In this model we have investigated the influence of the C2 toxin on alveolar epithelial barrier properties. Epithelial permeability was assessed by continuous monitoring of the transepithelial passage of technetium-labelled diethylenetriamine penta-acetic acid (99mTc-DTPA), offered to the alveolar surface by aerosol technique. Intravascular administration of hydrogen peroxide, used as control agent, was shown to provoke a four- to fivefold increase in the clearance rate of 99mTc-DTPA under conditions of severe fluid leakage into the lung interstitial and alveolar space. Intravascular administration of C2 toxin caused a dose- and time-dependent increase in Kfc values (8-15 fold), but the Tc-DTPA clearance rate was entirely unaffected. Moreover, transbronchial application of C2 toxin again reproduced the manifold increase in Kfc data (about six fold), but the rate of transepithelial passage of the hydrophilic Tc-DTPA complex remained unchanged. We conclude that the barrier properties of the lung microvascular endothelial and epithelial layer are differentially regulated. It is suggested that the actin microfilament system plays a decisive role in the structural and functional integrity of the endothelial but not the epithelial barrier.
Actins↗
Insulin sensitivity and body weight changes in young white carriers of the codon 64 amino acid polymorphism of the beta 3-adrenergic receptor gene.
Recently, a missense mutation replacing tryptophan with arginine at codon 64 of the beta 3-adrenergic receptor gene was shown to be associated with insulin resistance in nondiabetic subjects and to an earlier onset of NIDDM in Pima Indians. We studied whether the codon 64 amino acid polymorphism of the beta 3-adrenergic receptor gene in a cohort of young healthy Danes was associated with high birth weight, accelerated weight gain during childhood and adolescence, present obesity, or impaired insulin sensitivity. The protocol included 380 unrelated white subjects in whom insulin sensitivity and secretion were measured during a combined intravenous glucose and tolbutamide tolerance test. A number of biochemical and anthropometric characteristics were determined for each subject. The subjects were genotyped for the codon 64 polymorphism by applying polymerase chain reaction restriction fragment-length polymorphism screening with the use of endonuclease BstN1. The allelic frequency of the mutated allele was 7% (95% CI: 5-10%), and it was similar in obese and nonobese subjects. The beta 3-adrenergic receptor gene variant was not related to birth weight or weight gain during childhood or adolescence. In its heterozygous form, the gene variant was not associated with an altered insulin sensitivity index (SI) or other features of the insulin resistance syndrome (BMI, blood pressure, fasting serum lipid levels, or fasting serum fibrinolytic variables). Three homozygous carriers of the polymorphism were identified, and each had a significantly higher BMI (27.4 +/- 1.3 vs. 23.5 +/- 3.7 kg/m2 [mean +/- SD]; P = 0.032), lower SI [4.9 +/- 2.9 vs. 15.4 +/- 9.0 10(-5) x (min x pmol/l)-1; P = 0.013], and higher fasting serum C-peptide (730 +/- 155 vs. 471 +/- 158 pmol/l; P = 0.016) than the wild-type carriers. The homozygous carriers also had significantly higher levels of fasting serum triglyceride (P = 0.042) and serum LDL cholesterol (P = 0.013). When adjustments were made for age, sex, BMI, and VO2max in a multiple regression analysis, a significantly negative association was found between homozygosity for the codon 64 variant and the SI (P = 0.009). We conclude that in young healthy Danes, the homozygous form but not the heterozygous form of the codon 64 amino acid polymorphism of the beta 3-adrenergic receptor may be associated with obesity and, independent of BMI, with a low SI. Since only three homozygous carriers were identified among 380 subjects, the results must be interpreted with caution, and studies of larger population samples are needed.
Adult↗
Insulin resistance: interactions between obesity and a common variant of insulin receptor substrate-1.
We previously discovered two aminoacid polymorphisms in codons 513 and 972 of the protein insulin receptor substrate-1 (IRS-1), which is important in cellular insulin action. We have investigated whether these polymorphisms are associated with changes in insulin sensitivity in a random sample of young healthy adults. Insulin sensitivity and secretion were measured during a combined intravenous glucose and tolbutamide tolerance test in 380 unrelated white subjects aged 18-32. IRS-1 polymorphisms were examined by single-strand conformation polymorphism and verified by restriction-enzyme digestion. No homozygous carrier of the codon-513 variant was identified, but one non-obese man had the codon-972 mutation on both alleles. He had low fasting-serum insulin and C-peptide concentrations and low insulin sensitivity and glucose effectiveness. During a 24 h dexamethasone test, he developed transient diabetes. In their heterozygous forms the codon-513 and codon-972 variants of IRS-1 were found in 3% and 9% of the subjects. Non-obese carriers of either polymorphism had similar insulin sensitivity and pancreatic beta-cell function to non-obese wild-type subjects (no known variants of IRS-1). Analysis of variance showed, however, a significant interaction between obesity (body-mass index > or = 25 kg/m2) and the heterozygous form of the codon-972 variant (p < 0.003); obese polymorphism carriers had lower insulin sensitivity than obese non-carriers (mean 6.0 [SD 3.3] vs 12.3 [9.5] x 10(-5) L min-1 pmol-1). The obese carriers of the codon-972 variant were also characterised by a clustering of metabolic cardiovascular risk factors, with raised fasting concentrations of plasma glucose, serum triglyceride, and plasma tissue-plasminogen-activator and its fast-acting inhibitor. With adjustment for known modulators of insulin sensitivity, multivariate analyses showed that the combination of obesity and the codon-972 variant was associated with a 50% reduction in insulin sensitivity (p = 0.0008). Our results suggest that the codon-972 IRS-1 gene variant may interact with obesity in the pathogenesis of common insulin-resistant disorders.
Adolescent↗
Photoperiod regulation of plasma growth hormone levels during parr-smolt transformation of Atlantic salmon: implications for hypoosmoregulatory ability and growth.
The influence of photoperiod on plasma growth hormone (GH) levels during parr-smolt transformation was studied in Atlantic salmon, using a specific salmon GH radioimmunoassay; in addition, the impact of endogenous changes in GH levels on hypoosmoregulatory ability and growth was assessed using seawater challenge tests and measurements of growth rates. Groups of fish were kept on simulated natural photoperiod or continuous light, and subgroups were subjected to reciprocal transfers between these two light regimes at different times. For Atlantic salmon on simulated natural photoperiod, GH levels increased during the parr-smolt transformation, from 0.6 ng ml-1 in January-March to 6.6 ng ml-1 in June, while the corresponding change in fish on continuous light was from 0.4 to 1.0 ng ml-1. The study demonstrates that photoperiod is a major zeitgeber for the increased GH levels during the parr-smolt transformation of Atlantic salmon. The data further support the view that exposure to continuous light into fall and winter causes a "free running" of an endogenous rhythm governing smolting and a subsequent phase-delay of the parr-smolt transformation-related increase in plasma GH levels. A strong positive nonlinear correlation was found between specific growth rate and GH levels, with growth rate increasing rapidly with increasing GH levels up to 2-3 ng ml-1 at which point near-maximal growth rate is reached, revealing that relatively small increases in GH levels may be of great importance for an increased specific growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Hydrogen peroxide-induced increase in lung endothelial and epithelial permeability--effect of adenylate cyclase stimulation and phosphodiesterase inhibition.
Neutrophil-derived hydrogen peroxide (H2O2) is believed to play an important role in inflammatory lung injury. We investigated the influence of pharmacological agents that increase intracellular c-AMP levels on endothelial and epithelial leakage in response to intravascular H2O2 challenge in buffer-perfused rabbit lungs. Endothelial permeability was assessed by determination of the capillary filtration coefficient (Kfc) and lung weight gain. Measurement of the clearance rate of inhaled aerosolized technetium-99m-labeled diethylenetriamine pentaacetic acid ([99mTc]DTPA) from the lungs into the perfusion fluid was used as an index of alveolar epithelial permeability. Experiments were performed in the presence of acetylsalicylic acid to suppress H2O2-induced lung prostanoid generation and concomitant vasoconstriction. Under these conditions, H2O2 admixture to the perfusate (250 microM) caused a greater than eight-fold increase in Kfc values, resulting in > 30 g lung weight gain within 30 min in the absence of any significant vasopressor response. Pretreatment with the adenylate cyclase activators prostaglandin E1 (0.1 microM) and forskolin (0.1 microM), the dual phosphodiesterase type III/IV inhibitor zardaverine (10 microM) as well as combinations of these drugs all caused a nearly complete suppression of this early Kfc increase; and severe edema formation (> 30 g) was retarded to approximately 50-55 min. In addition to the microvascular leakage response, H2O2 caused a four- to five-fold increase in the [99mTc]DTPA clearance rate, starting within 15 min and culminating after approximately 35 min. Adenylate cyclase activation reduced this epithelial leakage response by approximately 30%, whereas zardaverine exerted no significant effect. We conclude that both microvascular endothelial and alveolar epithelial barrier function are severely compromised by intravascular H2O2 challenge in intact lungs. Pharmacological approaches to increase c-AMP levels, including both adenylate cyclase activation and phosphodiesterase inhibition, partially block the endothelial response and, to a lesser extent, the epithelial response.
Adenylyl Cyclases↗
HLA-DQ2-restricted T-cell recognition of gluten-derived peptides in celiac disease. Influence of amino acid substitutions in the membrane distal domain of DQ beta 1*0201.
CD is precipitated in susceptible individuals by ingestion of wheat gluten. The disease is strongly associated to the HLA-DQ(alpha 1*0501, beta 1*0201) (DQ2) heterodimer, where both the DQ alpha and DQ beta chains are required for susceptibility. We have recently shown that gluten-specific CD4+ T cells from the small intestinal mucosa of CD patients are predominantly restricted by the CD-associated HLA-DQ(alpha 1*0501, beta 1*0201) heterodimer. Here we report studies on the influence of aa substitutions in the DQ beta 1*0201 chain on DQ2-restricted T-cell recognition of gluten antigens. A B-LCL expressing the DQ(alpha 1*501, beta 1*0301) heterodimer was transfected with the DQB1*0201 gene, or with DQB1*0201 genes altered by site-directed mutagenesis. Surface expression of the wild-type or mutated DQ(alpha 1*0501, beta 1*0201) heterodimers was observed in the transfectants. Seven DQ2-restricted, gluten-specific TCCs were then investigated with respect to their ability to recognize antigen presented by the transfectants. All TCCs were sensitive to one or more of the aa substitutions induced but showed different response patterns. The results demonstrate that single aa substitutions of the DQ beta 1*0201 chain at positions in the peptide-binding cleft of DQ(alpha 1*0501, beta 1*0201) may affect binding of gluten-derived peptides and/or interfere with T-cell recognition. Because all seven TCCs studied were differently affected, they probably differ with respect to glutenpeptide and/or DQ(alpha 1*0501, beta 1*0201) restriction fine specificity.
Amino Acid Sequence↗
The cytotoxic HLA-DQ3 reactive human hybridoma antibody 4166 that may distinguish DQ7 + 8 from DQ9.
The human cytotoxic hybridoma antibody 4166 (IgM kappa) was generated by fusing an in vitro EBV-transformed B-LCL from a multiparous woman with the mouse-human heteromyeloma fusion partner CB-F7. In microcytotoxicity and IIF tests with B-LCLs as target cells, the mAb 4166 was specific for DQ3 (= DQ7 + 8 + 9). However, when used for DQ typing of class-II-positive PBMCs, 4166 could be rendered functionally specific for DQ7 + 8 and did not react with DQ9+ PBMCs. Binding of mAb 4166 to DQ8-positive cells was efficiently blocked by several allotype-specific mAbs recognizing DQ8. Other HLA class-II-specific mAbs were unable to inhibit. With the use of mAb 4166, it is possible to discriminate DQ7 + 8 from DQ9 in serologic DQ typing.
Amino Acid Sequence↗