HLA DQA1 genes generate multiple transcripts by alternative splicing and polyadenylation of the 3' untranslated region.

Regulation of the human leucocyte antigen (HLA) class II genes expression is an important field in immunology, because these molecules play a crucial role in the function of the immune system. HLA DQ genes expression is a complex phenomenon regulated at both transcriptional and post-transcriptional levels. In this study, we have investigated the post-transcriptional mechanisms accounting for alleles-dependent length polymorphism of DQA1 mRNA. We have first sequenced the genomic DNA encoding the 3' untranslated region (UTR) of DQA1 *0101, *0102, *0103, *0201, *0301, *0401, and *0501 alleles. We have identified two competing splicing sites: a unique splicing donor site AG/GTA located 20 nucleotides downstream from the stop codon associated to two spliced acceptor sequences, approximately 165 and approximately 370 nucleotides downstream. In addition, three polyadenylation signals have been identified, respectively, at approximately 475, approximately 795, and approximately 855 nucleotides downstream from the stop codon. Subsequently, we have analyzed mRNAs derived from DQA1 alleles in homozygous B lymphoblastoid cell lines by reverse transcriptase-polymerase chain reaction. We show that allele-dependent length polymorphism of DQA1 mRNA-3' UTR results from a combination of differential splicing and alternative polyadenylations. Four mRNA isoforms (two spliced variant cleaved at two distinct polyadenylation sites) were detected in DQA1 *0101, *0102, and *0103 homozygous cell lines, and six mRNA species (three spliced variant cleaved at two polyadenylation-sequence signal) were generated by the other four alleles. Possible advantages for cells to generate multiple transcripts previously undetected are discussed.

Comparison of TAP2 frequencies in type 1 diabetes patients and healthy controls from three ethnic groups indicates an African origin for the TAP2 G allele.

In order to determine the ethnic origin of the transporter associated with antigen processing 2 (TAP2) G allele, initially discovered by us in a group of type 1 diabetes (insulin-dependent diabetes mellitus) patients living on Reunion Island, HLA TAP2 typing was performed using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) method in type 1 diabetes patients and unrelated healthy controls of three different ethnic groups (Caucasians, Indians and black Africans from Senegal and Mauritius). The comparison of TAP2 allele frequencies in controls showed significant racial (ethnic) differences. The TAP2*0101 and TAP2 C alleles were increased, respectively, in the Caucasian (50% in Caucasians vs. 40% in other groups) and Senegalese (27% in Senegalese vs. 10% in other groups) populations. In comparison with Caucasians, the TAP2*0201 variant was significantly increased in the Indian population and decreased in the Senegalese black population. In addition, the TAP2 G allele was observed in the two African populations studied but not in the Caucasian or Indian population. This observation is consistent with the view that this allele is restricted to populations of African origin. In addition, we have determined the large extended haplotype DQA1-DQB1-DRB1 associated with TAP2 G. We found that this allele is preferentially associated with the large conserved haplotype HLA DQA1*0501-DQB1*0201-DRB1*0301.

[Is HbA1c appropriate for the screening of diabetes in general pratice?]. / L'HbA1c peut-elle être utilisée par le praticien pour le dépistage du diabète?

The measurement of glycated haemoglobin (HbA(1c)) is a practical and more sensitive tool than fasting plasma glucose (FPG) in screening type 2 diabetes in current practice. Its use has been limited so far by the variability of the analytical methods. The standardization process is going on, and many laboratories are currently using valid methods. Our study is consistent with the results of other groups who recommended this measurement to identify undiagnosed diabetic patients, that are about 25% to 30% in the French population. The demonstration was provided through a survey including a screening step by both HbA(1c) and G0, and a second exam with a 2 hr OGTT in a sample of positive screenees according to at least one criterion (HbA(1c) >=6% or G0 >=1.26 g/L), as well as in a sample of negative screenees. We showed that nine confirmed diabetic subjects out of ten had HbA(1c) >=6% at the screening step, while only a half had G0 >=1.26 g/L. Conversely, 22% of the positive screenees according to HbA(1c) were not confirmed as diabetic by the OGTT, including however more than half with abnormal glucose values. A chart for practical use is proposed to define patients at risk, the process of screening, and the patient follow-up according to the results of the tests.

In vivo analysis of HLA-DQ gene expression in heterozygous cell lines.

Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism associated with these genes and their promoters is a putative source of differential allele expression. Both transcriptional and post-transcriptional regulation could account for the density of the molecules expressed at the cell surface and then for the specificity of the immune response. Different methods have been developed to evaluate the functional consequences of this polymorphism, but at present no universal method allows measurement of either the steady-state level or the half-life time of mRNA species of both DQA1 and DQB1 polymorphic genes in heterozygous cell lines. Here, we propose a potent method, based on relative quantification of reverse transcriptase-polymerase chain reaction products, which analyzes the differential expression of all DQA1 or DQB1 allele combinations. This method is used to analyze the differential expression of HLA-DQB1*020110402 alleles in the human heterozygous lymphoblastoid B-cell line. Nucleotidic sequences of the proximal upstream regulatory region of these alleles exhibit significant differences. We show that the DQB1*0402 promoter is able to mediate a transcription strength twice as efficiently as *0201. In addition, the *0402 mRNA steady-state level is also governed by a remarkable post-transcriptional regulation. Indeed, an important part (20%) of the *0402 primary transcript is derived by alternative splicing in a short mRNA translated into a nonfunctional protein. Despite their variable sequence and length, no difference in the half-life of DQB1*0201 and both DQB*0402 mRNAs was observed in B-lymphoblastoid cells. The implications of these findings are discussed.

Influence of metabolic and genetic factors on tumour necrosis factor-alpha and lymphotoxin-alpha production in insulin-dependent diabetes mellitus.

The potential role of tumour necrosis factors (TNFs) in autoimmunity and insulin-dependent diabetes mellitus (IDDM) led us to determine in vitro TNF-alpha and lymphotoxin-alpha (LT-alpha, TNF-beta) production in IDDM patients according to TNF polymorphism. LT-alpha production of peripheral blood mononuclear cells (PBMC) was lower in diabetic subjects (m = 0.30 +/- 0.2 ng.10(-6) cells) than controls (m = 0.68 +/- 0.3 ng.10(-6) cells, p < 0.05), and early age-at-onset was correlated with low LT-alpha production (rs = 0.8, p = 0.0006). TNF-alpha production was the same in patients and controls, but patients with HbA1c > or = 8% had a higher TNF-alpha production (m = 3.05 +/- 1.2 ng.10(-6) cells) than those with HbA1c < 8% (m = 1.31 +/- 0.33 ng.10(-6) cells, p < 0.05). A study of the microsatellite TNFa region close to the LTA gene showed that the presence of the TNFa1 allele in HLA-(DR3) subjects was associated with increased risk of IDDM. TNFa1-positive subjects (both patients and controls) also had lower LT-alpha production than other subjects. These results indicate that low LT-alpha production is an additional risk factor for IDDM and that poor glycaemic control in patients is associated with enhanced PBMC TNF-alpha production which causes an imbalance between TNF-alpha and LT-alpha production in IDDM patient.

Analysis of HLA haplotypes in families with type 1 diabetes mellitus in La Réunion island.

To analyse HLA and insulin-dependent diabetes mellitus (IDDM) association in the ethnically mixed population of La Réunion island, we carried out a family study on 70 diabetic subjects. HLA-DQA1, -DQB1 and -DRB1 typing was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), completed by PCR-sequence-specific oligonucleotide (SSO) and PCR-sequence-specific priming (SSP). Haplotype-relative risks (HRR) were determined with the non-transmitted parental haplotypes as controls, and relative risks (RR) were calculated with a classical case-control study. The most significant risks were found for the cis and trans combinations between DQA1*03 or *0501 (Arg52+) and DQB1*02 or *0302 (Asp57-) alleles, suggesting a direct role for the HLA-DQ heterodimer in IDDM susceptibility. Interestingly, due to the mixed origin of the population, the trans-encoded DQ molecules in the (DR3)-DQA1*0501-DQB1*02/(DR4)-DQA1*03-DQB1*0302 subjects were also found cis-encoded in patients with the (DR7 or 9)-DQA1*03-DQB1*02 haplotype and in a patient with the rare (DR11)-DQA1*0501-DQB1*0302 haplotype. A relative predispositional effect (RPE) analysis gave significant haplotype-IDDM+ associations in the following order: (DR3)-DQA1*0501-DQB1*02 > (DR4)-DQA1*03-DQB1*0302 > (DR9)-DQA1*03- DQB*02 > (DR7)-DQA1*03-DQB1*02 > (DR2)-DQA1*01-DQB1*0502. No protective effect remained significant once the susceptible haplotypes were removed. A stratification study showed a stronger influence of the DQ genes than DRB1 alleles within the DR7 haplotypes. On the other hand, IDDM subjects with only one susceptible haplotype had inherited this haplotype more often from their father than from their mother. This paternal effect could be related to the greater risk of IDDM in offspring of diabetic fathers than the risk in offspring of diabetic mothers.

[Dysregulation of in vitro TNF-beta production in insulin-dependent diabetes mellitus]. / Dysrégulation de la production in vitro de TNF-beta au cours du diabète insulinodépendant.

The possible role of tumor necrosis factors (TNF) in autoimmunity led us to explore the relationship between TNF production, polymorphism of the TNF-beta gene and type one diabetes. In the diabetic group we found a low production of TNF-beta. This abnormality appeared not to be exclusively related to the TNF-beta gene and could be also due to polymorphic regulatory sequences in the major histocompatibility complex region.

Renal excretion of antidiuretic hormone in healthy subjects and patients with renal failure.

Urinary clearance of antidiuretic hormone (ADH) has been measured under basal conditions and during intravenous administration of arginine vasopressin in ten healthy subjects, and only under basal conditions in 18 patients with chronic renal failure and seven patients with acute renal failure at the polyuric phase of the disease. In healthy subjects studied under conditions of mild water diuresis plasma concentration, urinary excretion rate, urinary clearance and fractional clearance of ADH were 3.3 +/- 0.36 pg/ml, 25.2 +/- 5.5 pg/min, 7.5 +/- 1.2 ml/min and 6.4 +/- 1.0% (means +/- SEM) respectively. When plasma ADH was raised to levels between 7 and 26 pg/ml during intravenous administration of the hormone, urinary excretion rate and urinary clearance of ADH increased. Tubular reabsorption of ADH did not reach a plateau but progressively increased in the range of plasma ADH studied. In patients with chronic renal failure, plasma concentration, urinary excretion rate, urinary clearance and fractional clearance of ADH were 2.8 +/- 0.19 pg/ml, 9.4 +/- 2.0 pg/min, 3.4 +/- 0.6 ml/min and 10.0 +/- 2.9% (means +/- SEM) respectively. Urinary excretion rate and urinary clearance were significantly lower than in healthy subjects. In patients with acute renal failure, plasma concentration, urinary excretion rate, urinary clearance and fractional clearance of ADH were 4.6 +/- 0.47 pg/ml, 52.8 +/- 15.8 pg/min, 9.5 +/- 2.7 ml/min and 24.9 +/- 4.4% (means +/- SEM) respectively. Urinary excretion rate and fractional clearance were higher than in healthy subjects and patients with chronic renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)

High plasma antidiuretic hormone in patients with cardiac failure: influence of age.

Plasma antidiuretic hormone (ADH) was greater in patients with cardiac failure than in healthy subjects. Plasma ADH increased significantly with age in both groups. Covariance analysis showed that the difference between patients and controls occurred whatever the age. Plasma sodium and plasma osmolality were lower in cardiac patients than in healthy subjects. This showed that increase in plasma ADH observed in patients was inappropriate since it coexisted with inhibitory levels of plasma osmolality. Plasma creatinine was greater in cardiac patients than in healthy subjects and for each group in elderly than in young people. But there was no significant correlation between plasma ADH and creatinine. This suggested that increase in plasma ADH observed in cardiac failure was due more to an augmented release than to a diminished catabolism. No clear influence of etiology, severity, length of the disease and treatment with diuretics could be demonstrated.

[Serum amylase determination : automated continuous flow method (author's transl)]. / Mesure de l'amylase sérique : méthode automatisée en flux continu.

A simple automated assay was described for the determination of serum amylase using a chromogenic substrate. The reaction was stopped by filtration across a cellulose nitrate membrane. The analytic parameters of this method were studied. Repeatability, reproducibility and precision were convenient. The calibration curve was linear up to 850 U/I. An excellent correlation was obtained after comparison with the manual method (Phadebas amylase test). The effects of haemolysis and very high ascorbic concentrations of bilirubin, glucose, uric acid and ascorbic acid were negligible on the value measured. The serum turbidity was alone to induce a variation by excess.

Effects of furosemide-induced plasma volume reduction on plasma antidiuretic hormone in normal and hypertensive subjects.

Plasma antidiuretic hormone (ADH) was measured before and after furosemide administration in hypertensive patients (essential benign hypertensions with low plasma renin activity) and in normal subjects. Furosemide-induced reduction of plasma volume was about 10% after 2 hours. In normal subjects, plasma ADH rose progressively till the end of the study (1.5 pg/ml per hour corresponding to about 3 pg/ml per liter of plasma water lost) whereas it remained unchanged in hypertensive patients. There was an early increase of plasma renin activity (PRA) in normal subjects followed by a progressive fall. PRA response was blunted in hypertensive patients. These results show that volume-dependent ADH secretion is inhibited in patients with essential benign hypertension.

[Serum isoamylases determination. Analytical and critical study of a new method using an inhibitor protein]. / Evaluation des isoamylases sériques. Etude analytique et critique d'une méthode nouvelle utilisant une protéine inhibitrice.

A new method for the serum isoamylase assay is studied. Its principle is the use of an inhibitor protein from wheat, mainly active on the salivary type isoamylases. Amylase remaining in the presence of the inhibitor (R) and total amylase (T) are measured and the R/T ratios calculated. The kinetic of the inhibition is studied with various samples of pancreatic juice and saliva, of which the isoenzymatic pattern has been evidenced by electrofocusing. The results show that 80% of the salivary type isoamylases (S) and 20% of those from pancreatic type (P) are inhibited in the operational conditions described. The inhibition is stopped, but not modified, by addition of amylase activity substrate. The analytic study of the kit showed convenient results. The chemical inhibition methodology is compared with a modified Davies' electrophoretic technic on cellulose acetate membrane. The results are shown on a diagram: R/T ratio on the ordinate and total amylasemia on the abscissa. In the middle of the diagram appears an area which corresponds to normal values obtained with 69 healthy subjects from 20 to 30 years old.

[A study of the specificity of amylases. Measurement of pancreatic isoamylases (author's transl)]. / Spécificité des amylases. Détermination des isoamylases pancréatiques.

Serum lipase, pancreatic isoamylases and serum inhibitory activity on proteases were measured in 29 patients, 17 of whom had pancreatic disorders. The new test used to measure pancreatic isoamylases is rapid and useful, as it is relatively specific of pancreatic lesions. In acute abdominal syndromes, it undoubtedly enhances the value of biochemical investigations by confirming or excluding pancreatic lesions or involvement.

[The effect of inhibition of angiotensin II synthesis on the response of plasma antidiuretic hormone (ADH) to the osmotic and volume-dependent stimuli in uremic patients (author's transl)]. / Effet de l'inhibition de la synthèse de l'angiotensine II sur la sécrétion de l'hormone antidiurétique chez l'urémique en réponse aux stimuli osmotique et volumique.

The effect of inhibition of angiotensin II synthesis by captopril on the response of plasma ADH to the osmotic and volume-dependent stimuli has been studied in 15 uremic patients. Captopril administration had no effect either on basal plasma ADH or on plasma ADH response to the osmotic stimulus. Time-course of plasma ADH following hypertonic saline administration and sensitivity of the response (increase of plasma ADH related to increase of plasma sodium) were not modified. On the contrary, the response of plasma ADH to the volume-dependent stimulus induced by hemofiltration was markedly blunted by captopril administration. The sensitivity estimated from the slope of the regression line relating plasma ADH to the cumulated lost volume was clearly diminished. These results suggest that angiotensin II mediates ADH response only to the volume-dependent stimulus.

[Comparative study of digoxin determination by E.L.I.S.A. and radioimmunoassay techniques (author's transl)]. / Etude comparative du dosage de la Digoxine ar méthodes Immunoenzymologique (E.L.I.S.A.) et radio-immunologique.

This study is based on a double blind interlaboratory comparison between radioimmunoassay and E.L.I.S.A. digoxin determination. The correlation between digoxin values found with these two methods is good (r = 0.96 for therapeutic and toxic ranges 1,0 ng/ml - 5,5 ng/ml). The results indicate good repeatability, reproducibility and precision. The determination by E.L.I.S.A. can be performed with sera or plasma. The presence of haemolysis makes no appreciable difference in results. No variation is observed when different kits are used from an identical lot. However digoxin gives an important cross reactivity with digitoxin in Enzymeimmunoassay. Therefore it is necessary to know exactly the digitalis glycoside before the determination in order to avoid significant error.

[Biological changes observed after infusion of modified gelatin in women who had cesarean sections]. / Modifications biologiques observées après perfusion de gélatine modifiée chez la femme césarisée.

We suggest to study some biologic parameters after perfusion by 1,000 ml modified gelatin to young women who have a caesarian operation. The level of gelatin is determined by previously proposed method. Gelatin is also revealed at electrophoresis. Concurrently, protein contents in sera and hematocrit are estimated. These investigations are also made at the same time for a reference population who has a ceasarian operation without perfusion of gelatin. The highest level of gelatin is reached at t(0) + 90 mn. Gelatin is not present in sera at t(0) + 6 hr. At electrophoresis the beta-globulins raise concurrently with the level of gelatin. The alpha-globulins decrease but that variation is small and later. Protein level decreases also in conjunction with hematocrit. Hematocrit value is about 30 p. cent. It seems to the authors that this value allows a better oxygen transmission to tissues. For reference population these biologic parameters are not modified.