Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Respiration activity monitoring system (RAMOS), an efficient tool to study the influence of the oxygen transfer rate on the synthesis of lipopeptide by Bacillus subtilis ATCC6633.

The effect of oxygen transfer rate (OTR) on the synthesis of mycosubtilin, a non ribosomal lipopeptide antifungal biosurfactant, was investigated in the respiration activity monitoring system (RAMOS) for two Bacillus subtilis strains. These cultures were performed under definite oxygen-limited conditions without the adding of any anti-foam in the culture medium. By using four different filling volumes (FV) in the shaken bioreactors, different levels (20, 14, 9 and 7 mmol O(2)l(-1)h(-1)) of oxygen-limited growth could be obtained. A 25-fold increase of the specific productivity of mycosubtilin was observed for B. subtilis ATCC6633 in the case of the most severe oxygen limitation. But nearly no effect could be found with strain BBG100 carrying the constitutive P(repU) promoter instead of the natural P(myc) promoter. Transcript analysis of the fenF gene belonging to the myc operon indicated that the P(myc) promoter regulation could be slightly oxygen sensitive. Additionally, different patterns of the synthetised mycosubtilin homologues were obtained for different level of oxygen-limited growths. At the present state of investigation, oxygen regulation was thus shown to act at different levels suggesting the existence of a complex regulatory system of NRPS lipopeptide synthesis in the natural B. subtilis ATCC6633 strain.

No loss of genomic imprinting of IGF-II and H19 in placentas of diabetic pregnancies with fetal macrosomia.

OBJECTIVES: Fetal macrosomia is a common complication of maternal diabetes mellitus and is associated with substantial morbidity, but the precise cellular and molecular mechanisms that induce fetal macrosomia are not well understood. The imprinted genes IGF-II and H19 are crucial for placental development and fetal growth. The term placentas from diabetic pregnancies express more insulin-like growth factor II (IGF-II) than those from normal pregnancies. Deregulation of their imprinting status is observed in the macrosomia-associated syndrome, the Beckwith-Wiedemann syndrome. The aim of this study was to determine whether loss of imprinting hence biallelic expression was also a hallmark of macrosomia in diabetic pregnancies. DESIGN AND METHODS: IGF-II and H19 maternal and paternal expressions were studied in placentas from two groups of type 1 diabetic mothers: one with macrosomic babies and the other with babies of normal weight. Maternal or paternal allele specific expressions were defined by using DNA polymorphic markers of the IGF-II and H19 genes. RFLP analysis was performed on PCR products from genomic DNA of the father, the mother and the child, and on RT-PCR products from placental mRNA. RESULTS: RFLP analysis showed that the IGF-II gene remains paternally expressed and the H19 gene remains maternally expressed in all placentas examined, independently of the birth weight status. CONCLUSIONS: These results suggest that, in contrast with Beckwith-Wiedemann syndrome-associated macrosomia, loss of imprinting for IGF-II or H19 is not a common feature of diabetic pregnancies associated with macrosomia.

Polymorphisms of the receptor of advanced glycation endproducts (RAGE) and the development of nephropathy in type 1 diabetic patients.

OBJECTIVES: We investigated the association of the RAGE (Receptor for Advanced Glycation End products) exon3 gene polymorphisms with stages of nephropathy in type 1 diabetes. METHODS: The RAGE exon 3 genotype was assessed by Denaturing Gradient Gel Electrophoresis (DGGE) procedure in 487 type 1 diabetic patients with proliferative retinopathy subdivided into four groups according to their level of renal involvement and in 351 control subjects (GENEDIAB study). RESULTS: We reported here three main low frequency dimorphisms, previously submitted to data banks, Gly82Ser, Val89 CTC/CTG, and Arg77Cys. The genotype distribution of these polymorphisms was not statistically different in type 1 diabetic patients compared to healthy controls (p=0.37). Among the three described polymorphisms, only the RAGE Gly82Ser genotype frequency was significantly increased in the group with advanced nephropathy (11%) defined by a chronic renal failure compared to the three others groups: no nephropathy, 5%; incipient (microalbuminuria) 5%; established (macroalbuminuria), 2%) (P=0.04). The 82 Ser allele was identified as an independent risk marker for the stage of advanced nephropathy: adjusted odds ratio 3.17(95% CI 1,32-7,85, p=0.008). CONCLUSION: These data suggest that the 82 Ser allele of the RAGE gene is a risk allele for developing advanced nephropathy. This suggests that some RAGE gene polymorphisms may be associated with progression to diabetic advanced nephropathy in Caucasian type 1 diabetic patients.

[Micro-array based technologies to study the proteome: technological progress and applications]. / Nouvelle approche globale en protéomique: les biopuces à protéines. Techniques actuelles et applications.

Since these twenty last years, there is an increasing interest for large-scale analysis of biological function. In the field of transcriptome, the emergence of microarray-based technologies and the design of DNA biochips allow high-throughput studies of RNA expression in cell and tissue at a given moment. In the field of proteome, methods of reference are still the 2D electrophoresis followed by analysis with mass spectrometry. Technological progress makes it possible to apply microarray methods to proteomics study : they are protein biochips or protein arrays. Expression analysis of proteins in a cell or a tissue in simultaneous and highly parallel way give further information for large-scale studies of signaling pathway. Numerous applications of protein microarray-based assays are described in basic biological research and in medical research to identify diagnostic biomarkers of inflammatory and cancerous pathologies and to find out news drugs and new therapeutic targets. This review summarizes concrete applications of microarray-based technology in the field of proteome, describes fundamental technical stages in protein array development and highlights critical points which will be useful to improve this emerging proteomic method.

CTLA-4 49 A/G dimorphism and type 1 diabetes susceptibility: a French case-control study and segregation analysis. Evidence of a maternal effect.

Several studies have demonstrated an association of cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) (IDDM 12) alanine 17 with type 1 diabetes, but we wished to study the parental effect of CTLA-4 49 A/G dimorphism in diabetic families. The CTLA-4 exon 1 polymorphism (49 A/G), HLA-DRB1 and insulin gene (INS) variable number tandem repeats (VNTR) were analysed in 134 type 1 diabetic patients vs. 273 control subjects. The segregation analysis for transmission was carried out in 70 informative diabetic families using the transmission distortion test (TDT). All genotyping was performed by PCR-RFLP. CTLA-4 49 G allele frequency was not increased in diabetic patients compared to controls (41 vs. 38%, not significant). The distribution of GG, AG and AA CTLA-4 genotypes was similar in the two groups (13, 57 and 30% vs. 11, 54 and 35%, respectively) and was independent of HLA-DRB1 or INS VNTR polymorphism. The CTLA-4 49 G allele showed weak distorted transmission to the diabetic offspring, whereas random transmission was observed in unaffected offspring. This distortion is attributable to a maternal effect (71% compared to the 50% expected ratio; tdt = 4.8; P < 0.03). The combined transmission of maternal CTLA-4 G with HLA-DRB1*03 (90%; tdt = 6.4; P < 0.01) and VNTR class I (80%; tdt = 5.4; P < 0.02) enhanced the susceptibility effect of each marker separately. We noted a slight CTLA-4 49 G and HLA-DRB1*04 distortion of transmission shared in paternal and maternal diabetic meiosis. In non-diabetic offspring, the CTLA-4 49 A allele confers a protective effect in the presence of maternal HLA-DRB1*03 and paternal HLA-DRB1*04 alleles. Despite the absence of a positive association of the CTLA-4 49 G allele with type 1 diabetes, our segregation analysis supports the hypothesis of a modulation by CTLA-4 49 G/A dimorphism of the susceptibility conferred by maternal HLA-DRB1*03 inheritance. This potential parental effect needs to be confirmed in a larger data set.

Progression of actinic keratosis to squamous cell carcinoma of the skin correlates with deletion of the 9p21 region encoding the p16(INK4a) tumor suppressor.

Actinic keratoses (AKs) are pre-neoplastic lesions that can develop into squamous cell carcinomas (SCCs) of the skin. Often AK and SCC have commonly altered p53. A status of another tumor suppressor, the p16(INK4a), was reported for SCC but not for AK. A comparative study of SCC and AK human samples by loss of heterozygosity (LOH) analysis determined that the p16(INK4a/ARF) locus is less frequently altered in AKs than in SCCs. These LOH data highly correlated with immunohistochemical findings demonstrating the presence of p16(INK4a) in the AK skin samples but its absence in SCC lesions. Our results imply that progression of AK into SCC may involve inactivation of p16(INK4a).

HLA alleles in relation to specific immunity to liver stage antigen-1 from plasmodium falciparum in Gabon.

Cellular responses to synthetic peptides from the Liver Stage Antigen-1 (LSA-1) from Plasmodium falciparum were determined in 229 Gabonese children. HLA class I and II typing (by PCR-SSP and -RFLP, respectively) revealed that HLA-A*19, -B*17 (and -B*70), -DRB1*05, -DQA1*0102, -DQB1*0602 and -DPB1*0402 were the most frequent types or alleles at each locus. The DQB1*0201 and DQB1*0301 alleles were present at a higher frequency among IL-6 and IFN-gamma responders to the LSA-Rep and LSA-CTL peptides, respectively, and a higher proportion of these responders carried A*19 or B*53. The DRB1*06 type was positively related to the IL-10 production in response to the LSA-CTL peptide, and responders presented mainly A*2. The specificity A*10 was negatively associated with the cellular response to the LSA-J peptide. These results suggest a degree of genetic regulation of specific immune responses by HLA-A, operating at the pre-erythrocytic stage of development of P. falciparum in this Central African population.

Human RAGE GLY82SER dimorphism and HLA class II DRB1-DQA1-DQB1 haplotypes in type 1 diabetes.

Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs increase in diabetes and modulate cellular functions through binding to a specific cell surface receptor (RAGE). The RAGE gene maps to chromosome 6p in the HLA class III area and is telomeric to the class II region at 250 kb from DRA. A recent report described the characterization of a major RAGE gene variant as a biallelic single base polymorphism (G/A 557) in the exon 3 sequence leading to a change of a glycine to a serine at position 82. Using DGGE and PCR-RFLP, we have investigated the distribution of this dimorphism in conjunction with HLA class II genes in large populations of type 1 diabetic patients and healthy subjects. Although no association of this RAGE gene polymorphism with disease susceptibility was found, we report a strong linkage disequilibrium between the variant carrying the serine amino acid at position 82 and two HLA-DR2 and HLA-DR4 specificities. In particular, we describe two major extensive HLA class II haplotypes associated with this serine variant and identified as DRB1*0401-DQA1*0301-DQB1*0301 in the diabetic group and DRB1*1501-DQA1*0102-DQB1*0602 in control individuals. These data were partially confirmed by family transmission analysis.

HLA class II polymorphism in a Gabonese Banzabi population.

The HLA class II typing of 167 unrelated Gabonese individuals from the Banzabi ethnic group was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The most frequent alleles at each locus were DRB1*1501-3 (0.31), DQA1*0102 (0.50), DQB1*0602 (0.42) and DPB1*0402 (0.29). The estimation of the haplotype frequencies as well as the observation of the segregation of several haplotypes using additional HLA typing of relatives, revealed that the three-locus haplotype DRB1*1501-3-DQA1*0102-DQB1*0602 was found at the highest frequency (0.31) among these individuals. This haplotype is not typically African and has already been described in Caucasians, but its presence at high frequency is exclusive to populations originating from Central Africa, and can thus be designated as a particular genetic marker of these populations.

Evidence for the involvement of both retinoic acid receptor- and retinoic X receptor-dependent signaling pathways in the induction of tissue transglutaminase and apoptosis in the human myeloma cell line RPMI 8226.

In this study, we show that both all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA) are potent inducers of tissue transglutaminase (TGase II), an enzyme involved in apoptosis, at the level of both enzyme activity and mRNA in the human myeloma cell line RPMI 8226. RPMI 8226 cells were shown to express mRNAs for all the retinoid receptors subtypes, ie, RARalpha, RARbeta, RARgamma, RXRalpha, RXRbeta, and RXRgamma. To identify which of these receptors are involved in regulating TGase II expression, several receptor-selective synthetic retinoids were used. Neither CD367, a very potent retinoid that selectively binds and activates receptors of the RAR family, nor CD2425, an RXR-selective agonist, induced TGase II when used alone. However, combination of CD367 and CD2425 resulted in nearly full induction of the enzyme. Moreover, when used in combination with atRA, CD367 partially inhibited the atRA-dependent induction of TGase II, whereas CD2425 enhanced it. The effects of Am 580, CD417, and CD437, three synthetic retinoids selective for the RARs subtypes RARalpha, RARbeta, and RARgamma, respectively, were also investigated. None of these compounds was able to induce TGase II when used alone; however, the combination of each of them with CD2425 resulted in strong induction of the enzyme activity, reaching 30% to 50% of the values obtained in the presence of retinoic acid and suggesting functional redundancy between the RAR subtypes. Finally, treatment with atRA or the combination of CD367 and CD2425, but not with CD367 or CD2425 alone, was also shown to trigger apoptosis in RPMI 8226 cells, with prominent accumulation of TGase II immunoreactivity in apoptotic cells. Taken together these data suggest that the induction of TGase II expression and apoptosis in the RPMI 8226 myeloma cell line required ligand-dependent activation of both the RAR and RXR receptors.

Association of rheumatoid arthritis with an amino acid allelic variation of the T cell receptor.

OBJECTIVE: To investigate allelic variations of T cell receptor residues for a contribution to rheumatoid arthritis (RA) susceptibility. METHODS: We conducted an RA case-control study involving 1,579 northwest Europeans: 766 patients with erosive and rheumatoid factor-positive disease and 813 control subjects. Productive changes of segments TCRAV6S1, TCRAV7S1, TCRAV8S1, TCRAV10S2, and TCRBV6S1, TCRBV6S7 were investigated by single-strand conformation polymorphisms. The TCRAV8S1 association was confirmed by restriction fragment length polymorphism. RESULTS: In the systematic study (77 patients and 119 controls), an increase in 1 TCRAV8S1 genotype was found in the RA patients (P = 0.0004). This finding was replicated in 2 further populations, one from France (212 patients and 254 controls) and the other from Britain (477 patients and 440 controls), with a similar odds ratio (OR), which allowed pooling of the data and confirmation of the association (OR 1.3 [95% confidence interval 1.1-1.7], P = 0.008). CONCLUSION: These findings show evidence that TCRA is an RA susceptibility locus.

[The HLA system: a clinical help?]. / Le système HLA, une aide à la clinique?

UNLABELLED: Chronic diseases are polymorph and influenced by many environmental and genetic factors. The HLA system is implicated in the modulation the onset and the evolution of chronic diseases. These observations are important to be considered for the prediction, prognosis and treatment adaptation. Evaluation tests are mainly statistical and are based on epidemiological studies. Thus, results must be considered with caution. Two aspects are to be considered: DIAGNOSIS: Associations between HLA alleles and chronic diseases are well known but concern very few diseases like narcolepsy or rheumatoid arthritis. Insulin dependent diabetes mellitus is particular because the prediction is limited to familial studies. PROGNOSIS: This point is less documented in clinical applications but is of interest particularly in inflammatory bowel diseases or systemic affections. This observation can be considered as a compartmental response which is a kind of adaptation to stress.

[Genetics of insulin-dependent diabetes mellitus. Value in biological practice]. / La génétique du diabète insulinodépendant. Intérêt dans la pratique biologique.

Insulin-dependent diabetes mellitus is a polygenic disease with an environmental component. Technological advances and large collection families allowed genetic factors understanding. On clinical practice, two questions could be raised. First, will the genetic markers be of interest in disease prediction either in families studies or in the general population? Second, will the genetic approach explain the physiopathological process of the disease? Initially, the gene candidate approach led to the identification of two important loci. Linkage with the HLA locus showed the importance of the autoimmune part. Linkage of insulin-dependent diabetes mellitus with insulin locus gave a mechanistic answer for disease susceptibility. These two loci can be used as prediction markers, but only in family studies. Since 1993, a whole genome approach was performed and led to the identification of other susceptibility loci. These initial results are in progress and should have important implications for public health strategies.

Gestational diabetes mellitus and HLA class II (-DQ, -DR) association: The Digest Study.

Gestational diabetes mellitus (GDM) and impaired glucose tolerance during pregnancy (IGT) are associated with an increased risk of perinatal morbidity and then further development of diabetes among 30-50% of affected women. This is a real public health problem that deserves investigation of phenotypic and genotypic predisposing markers. However, the involvement of genetic background in GDM and IGT remains unclear. In particular, association with HLA class II polymorphism has been poorly studied and has produced conflicting results. In attempt to clarify these discrepancies, we investigated HLA class II polymorphism in 95 GDM and 95 IGT women from the north of France using DNA amplification followed by restriction enzyme digestion (PCR-RFLP). Ninety-five pregnant women with normal glucose tolerance (NGT) were chosen as a control reference group. The distribution of HLA class II polymorphism was not found to be significantly different between GDM, IGT and NGT samples. In particular, we did not find any significant variation of DRB1*03 and DRB1*04 allele frequencies between these three groups. These data provide further evidence that insulin-dependent diabetes mellitus (IDDM) HLA class II susceptibility alleles cannot serve as genetic markers for susceptibility to glucose intolerance during pregnancy. However, GDM and IGT were not equivalent to the NGT control group and presented particular HLA patterns. In particular, we observed an increase of the DRB1*0701-DQA1*0201-DQB1*02 haplotype in GDM women (P = 0.02; Pc not significant) and an increase of DRB1*0101-DQA1*0101-DQB1*0501 and DRB1*1302-DQA1*0102-DQB1*0604 haplotypes in the IGT group (P = 0.02 and 8 x 10(-3), respectively; Pc not significant). In contrast, we found a decrease in the DRB1*1101 allele in IGT samples (P = 0.03; Pc not significant) and a decrease of DRB1*1103-*1104 alleles in the GDM group (P = 9 x 10(-3); Pc not significant). Although these findings are only descriptive, it points out the genetic heterogeneity of glucose intolerance during pregnancy.

Are HLA-DR or TAP genes genetic markers of severity in ulcerative colitis?

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n = 91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n = 200) were included. HLA-DRB1*03 was less frequent in UC patients than in healthy controls (8% vs 28%, PC < 0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively, (PC < 0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.

Association of HLA class II genes with susceptibility to Crohn's disease.

BACKGROUND: Published studies on the association between HLA class II genes and inflammatory bowel disease are contradictory perhaps because of the limited size and ethnic heterogeneity of the populations studied. AIM: To compare the frequencies of HLA class II genes in a large number of French patients with Crohn's disease and in an ethnically matched control group. METHODS: 344 patients (196 F, 148 M, mean age 23.6 years) with Crohn's disease were molecularly genotyped for the HLA-DQB1 and DRB1 alleles. The results were compared with those for an ethnically matched control population of 488 white adults. RESULTS: There were two significant variations of alleles at the DQB1 locus: an increase in DQB1*0501 allele frequency (chi 2 = 10.6, corrected p value (pc) = 0.01, odds ratio (OR) = 1.61) and a decrease in DQB1*0602/0603 allele frequencies (chi 2 = 8.43, pc = 0.037, OR = 0.66). DRB1 analysis showed associations with three allelic variations: an increase in the frequencies of DRB1*01 (chi 2 = 12.86, pc = 0.003, OR = 1.75) and DRB1*07 alleles (chi 2 = 11.18, pc = 0.008, OR = 1.58) and a very significant decrease in that of the DRB1*03 allele (chi 2 = 19.7, pc = 9.10(-5), OR = 0.46). CONCLUSION: The alleles DRB1*01 and DRB1*07 are associated with susceptibility to Crohn's disease. The strong negative association between the DRB1*03 allele and Crohn's disease suggests that the HLA-DRB1*03 allele mediates 'resistance' to Crohn's disease.