The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy.

BACKGROUND: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. METHODS: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. RESULTS: Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. CONCLUSION: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

A new simple method to perform pressure-volume curves obtained under quasi-static conditions during mechanical ventilation.

OBJECTIVE: To describe a fast, simple method to acquire pressure-volume curves of the respiratory system and to compare this with a classic method in terms of reliability of the data and speed. DESIGN: Acquisition of pressure-volume curves by low flow inflation technique (P-Vlf) versus the occlusion technique (P-Vst) using the standard equipment of a Cesar ventilator. SETTING: General ICU - Aix en Provence Hospital. PATIENTS: Ten sedated, curarized patients undergoing mechanical ventilation. INTERVENTIONS: P-Vlf curves were acquired by setting the ventilator parameters at f = 5 c./min, duty time Ti/Ttot = 80 %, VT = 1100 ml, pause time = 0. The pressure and volume data were collected directly on the ventilator screen. P-Vst curves were acquired using an airway occlusion technique. The pressures obtained for the same inflation volumes and times necessary for performance of the two techniques were compared. RESULTS: The time needed to acquire a P-Vlf curve was 3 min versus 38 min for P-Vst curve. Concordance analysis between the two methods showed a 95 % confidence interval of (-0.5 cm H2O, + 1.8 cm H2O) for pressure. CONCLUSIONS: P-Vlf curves are close to P-Vst curves, are much less time-consuming, easy to acquire with Cesar ventilator equipment, and may be used in clinical routine to assess the elastic properties of the respiratory system.

[CFTR gene analyis in 207 patients with cystic fibrosis in southwest France: high frequency of N1303K and 1811+1.6bA>G mutations]. / Etude du gène CFTR chez 207 patients du Sud-Ouest de la France atteints de mucoviscidose: fréquence élevée des mutations N1303K et 1811 + 1,6 kbA > G.

UNLABELLED: The large molecular heterogeneity in cystic fibrosis (CF) represents the main difficulty for the genotype characterization. Moreover, numerous studies have reported considerable variations in frequencies of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in different populations. MATERIAL AND METHODS: We analyzed the genotype of 207 CF children living in southwest France. RESULTS: Among 50 identified mutations, we report for some of them a widely modified incidence compared with those observed in other regions of France. These differences were more significant in the subset of the CF chromosomes originating in southwest France. Thus, the 1811 + 1.6 kbA > G mutation, rarely observed in the other French regions (< 0.5%), proved to be, with a frequency of 8.8%, the most frequent mutation after the F508 deletion (57%). The frequencies of N1303K, 1811 + 1.6 kbA > G and R334W mutations were also clearly increased: 7.9 and 2.6%, respectively. CONCLUSION: We show that the southwest of France is characterized by a specific mutational spectrum. We consider that these regional data on the spectrum of CF mutations are crucial to develop more accurate and less expensive molecular screening strategies for cystic fibrosis in France.

[Pyoderma gangrenosum and biclonal gammapathy probably due to multiple myeloma (author's transl)]. / Pyoderma gangrenosum et gammapathie biclonale d'origine myélomateuse probable.

The authors report the observation of pyoderma gangrenosum (P. G.) leading to the discovery of an underlying biclonal gammapathy, which despite the absence of bony lesions, almost certainly represents a malignant myeloma. The authors have reviewed the different known associations with pyoderma gangrenosum, with special reference to benign and malignant gammapathies. Rare cases of biclonal gammapathies with pyoderma gangrenosum have been published but none of a malignant nature. This therefore appears to be the first reported cases. The authors are aware of the reported tendency of pyoderma gangrenosum lesions to appear in areas of previous trauma, as may be the case in this patient. Finally systemic corticosteroid therapy produced a rapid remission of the skin lesions, but with the reactivation of known treated pulmonary tuberculosis.

Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Unexpectedly, the signal corresponding to the maternally inherited F508del allele appeared much more intense than the paternally derived wild allele. Molecular analysis including polymorphic marker studies, microsatellites and single-nucleotide polymorphisms subsequently showed that the boy was a carrier of a de novo mosaic maternal isodisomy of a chromosome 7 segment while there was a biparental inheritance of the rest of the chromosome. This is the first report of a mosaic partial UPD7. The matUPD7 segment at 7q21-qter extends for 72.7 Mb. The karyotype (550 bands) of our patient was normal, and fluorescence in situ hybridization with probes mapping around the CFTR gene allowed us to rule out a partial duplication. The detection of this chromosomal rearrangement confirms the hypothesis that the 7q31-qter segment is a candidate for the localization of human imprinted genes involved in the control of IUGR and PNGR. It also emphasizes the importance of searching for UPD7 in severe, isolated and unexplained IUGR and PNGR.

Cross-reactivity among evolutionarily distant major histocompatibility complex class I molecules (HLA-B27 and H-2Kk) revealed by xenoreactive T lymphocytes.

A set of mouse HLA-B27-reactive cytotoxic T lymphocyte clones were found to recognize the HLA-B27 molecule in an H-2-unrestricted manner, i.e. independently of any mouse major histocompatibility complex (MHC) molecule. The reactivity patterns of these clones on HLA-B27 variants (positive only on HLA-B*2702 and HLA-B*2701) allowed the identification of residues N77 and A81 of the HLA-B27 molecule as important for their reactivity. The location of these residues in the peptide-binding groove (specificity pocket F) suggested that the reactivity of the clones is dependent on HLA-B27-bound peptide(s). However, several other class I molecules sharing these residues (N77 and A81) were not recognized, indicating that other residues might also be involved. One of the clones was found to display an interesting cross-reactivity with allogeneic H-2Kk molecules, sharing N77 and A81 with HLA-B*2702. Sequence comparison suggested the involvement of residue H9, located in specificity pocket B of the peptide-binding groove, and revealed some similarity of pockets B in HLA-B27 and H-2Kk. The structural basis of such T cell-mediated MHC cross-reactions across species barriers is discussed.

Strong alloantigenicity of the alpha-helices residues of the MHC class I molecule.

To evaluate the role of single residues of a MHC class I molecule in the induction of a primary allogeneic response, we have tested the ability of various point mutants (of the alpha-helices or beta-sheet of the alpha1 and alpha2 domains) of the Kd molecule to induce a primary cytotoxic T cell response in mice carrying the wild-type molecule. For that, we have used an in vivo model in which cells expressing mutant molecules were injected into the hind footpads of mice carrying wild-type Kd, and the recipient graft-draining popliteal lymph nodes were tested for the presence of alloreactive CTL. Under these experimental conditions, only 7 of the 25 mutant Kd molecules induced a primary allogeneic response. All of these mutations (positions 62, 65, 69, 72, 155, 163, 166) concern residues of the alpha-helices, demonstrating that very small variances from self in a class I molecule, located outside the peptide-binding groove, can be antigenic. To determine the peptide requirements for the generation of a primary allogeneic response, we have analyzed the repertoire of peptides selected by individual mutant molecules shown to be able or unable to induce a CTL response. No correlation was observed between the peptidic make-up presented by a given mutant and its capacity to induce a primary allogeneic response. On the whole, our data point to the alloantigenicity of potentially TCR-contacting surface residues of the MHC class I molecules.

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse model.

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2-restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27-induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse specificities contribute to HLA-B27-xenorecognition.

Alloreactive monoclonal antibodies select Kd molecules with different peptide profiles.

As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies. First, we have studied the capacity of five different Kd-reactive mAbs to recognize a panel of 25 site-directed mutants of the H-2Kd molecule. Both the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indicating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab binding. Mutation of two alpha-helix-situated residues, 58 and 166, completely abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb. The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs. We have addressed this issue by comparison of the repertoire of peptides eluted from Kd molecules precipitated by different Kd-reactive mAbs. The results reveal that the two-dimensional profile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different. The use of 21 single amino acid variants of a Kd-restricted 10-mer peptide allowed us to identify the residue of the bound peptide contributing to the epitope recognized by this mAb. Thus, we have shown that at least in some instances, changes induced in the MHC molecules by the binding of distinct peptides can be recognized as alterations in serologic determinants expressed on the class I molecules.

[Migrating and recurrent superficial phlebitis and Takayasu's disease (apropos of a case)]. / Phlébite superficielle migratrice et récidivante et maladie de Takayashu (A propos d'une observation)

This case of recurrent migratory superficial phlebitis is reported because of the highly unusual nature of the lesion observed. The picture was dominated by periphlebitis with inflammatory granuloma, giant cells and elastophagia. When the condition had been present for sixteen months, an aortic arch syndrome developed in an inflammatory context. In this light, various auto-immunological etiologies were considered, among them Takayashu's arteritis of which, in the author's opinion, this would be the first case to be combined with venopathy.

Anti-MHC immunity detected prior to intentional alloimmunization. IV. Natural monoclonal H-2-specific antibodies.

Naturally occurring H-2-specific antibodies can be detected rather frequently in sera of non-alloimmunized mice by sufficiently sensitive techniques (Cerny-Provaznik et al., 1985a; Cerny-Provaznik & Ivanyi, 1985). In this report, we summarize our experiences with the preparation of monoclonal anit-H-2 antibodies obtained from hybridization experiments from non-alloimmunized mice. From a total of 30 spleen cell hybridization experiments, we could isolate only four anti-H-2 monoclonal antibodies (mAB). Two of the mAB are described in this report. Monoclonal antibody By-2 is anti-Kf and mAB By-3 is anti-Db, Ds. We investigated which conditions favour the isolation of monoclonal H-2-specific antibodies from non-alloimmunized mice. The presence of naturally occurring serum antibodies, the age of the spleen donor mouse or non-specific B cell stimulation were not critical for the isolation of natural anti-H-2 mAB. We hypothesise that the 'natural' H-2-specific antibodies represent compartments of the B cell repertoire which were triggered by modified or aberrant self-MHC expression.

HLA antigens in 16 families with xeroderma pigmentosum.

Xeroderma pigmentosum is an autosomal recessive disease. HLA-A and -B typing was performed on peripheral blood lymphocytes and platelets. Sixteen Tunisian families were typed with 37 patients and 108 relatives. Genetic transmission of the disease and of the HLA system seemed to be independent in this study. Comparison of HLA gene frequencies between (unrelated) parents of patients and a control population showed no difference, proving that there is no clear association in populations between deleterious XP genes and a particular HLA gene. However, an excess of identical HLA among pairs of diseased siblings would suggest that the disease is polymorphic and a form of the XP could be linked to HLA.

HL-A phenotyping in an Indonesian population.

The frequencies of 30 HL-A antigens were studied in an Indonesian population of 95 individuals from the city of Jakarta. The antigens HL-A9, or more precisely W24, and HL-A11 (first series) and W15 (second series) occurred with high frequencies, whereas HL-A8, W14 and W22 were completely absent. These results are consistent with previous reports of HL-A typing in South East Asian populations.