Molecular findings and clinical data in a cohort of 150 patients with anophthalmia/microphthalmia.

Anophthalmia and microphthalmia (AM) are the most severe malformations of the eye, corresponding respectively to reduced size or absent ocular globe. Wide genetic heterogeneity has been reported and different genes have been demonstrated to be causative of syndromic and non-syndromic forms of AM. We screened seven AM genes [GDF6 (growth differentiation factor 6), FOXE3 (forkhead box E3), OTX2 (orthodenticle protein homolog 2), PAX6 (paired box 6), RAX (retina and anterior neural fold homeobox), SOX2 (SRY sex determining region Y-box 2), and VSX2 (visual system homeobox 2 gene)] in a cohort of 150 patients with isolated or syndromic AM. The causative genetic defect was identified in 21% of the patients (32/150). Point mutations were identified by direct sequencing of these genes in 25 patients (13 in SOX2, 4 in RAX, 3 in OTX2, 2 in FOXE3, 1 in VSX2, 1 in PAX6, and 1 in GDF6). In addition eight gene deletions (five SOX2, two OTX2 and one RAX) were identified using a semi-quantitative multiplex polymerase chain reaction (PCR) [quantitative multiplex PCR amplification of short fluorescent fragments (QMPSF)]. The causative genetic defect was identified in 21% of the patients. This result contributes to our knowledge of the molecular basis of AM, and will facilitate accurate genetic counselling.

Efficacy of ceftriaxone and doxycycline in the treatment of early syphilis.

INTRODUCTION: An increase of syphilis cases has been recorded in the past few decades, especially among HIV-infected patients. These patients often present with concomitant primary and secondary lesions or extensive presentations of syphilis. OBJECTIVE: Our goal was to compare alternative regimens to the recommended penicillin treatment. METHOD: We retrospectively studied 116 patient files (80% HIV1-infected) treated for a first episode of early syphilis. RESULTS: Patients mainly presented with symptoms of secondary syphilis. In 15.5% of the cases patients were asymptomatic and 17.2% of patients with secondary syphilis presented with neurologic or ophthalmic symptoms. Some less usual clinical presentations included diffuse polyadenopathy or isolated fever. The time to serological response was similar among those treated with benzathine-penicillin (n=52), ceftriaxone (n=49), or doxycycline (n=15). CONCLUSION: Ceftriaxone and doxycycline could be suitable alternatives to penicillin in the treatment of early syphilis in HIV-infected patients. These two treatments have a concomitant effectiveness even for asymptomatic forms of neurosyphilis.

[Request of epidural analgesia by women and obstetrical teams in four French areas. Part I: Request for analgesia]. / Demande d'analgésie périmédullaire par les patientes et les équipes obstétricales dans quatre régions françaises. Partie I : demande d'analgésie.

OBJECTIVE: Epidural analgesia (EA) is widely used in France to treat the labour pain. The aim of this study was to evaluate and analyse the reasons of EA requirements by parturients and obstetricians. STUDY DESIGN: An epidemiological survey was sent in all maternity units of four French areas. PATIENTS AND METHODS: Forty-eight of the 84 maternity units entered the study. In each centre, 25 patients fulfilled the questionnaire and the medical team fulfilled a questionnaire about the organisation of the ward. Demands of EA by parturients during pregnancy, labour and for the next delivery were recorded, and also the offer of EA by midwives and the possibility to move in an other town to obtain an EA. Student t-test, chi2 test and logistic regression model were used as requested. p<0.05 was considered as significant. RESULTS: One thousand one hundred forty-two women entered the study. During pregnancy 79% of women asked for an EA, they were 72% during labour. Factors influencing the request of an EA were parity, educational level, pain, preanaesthetic evaluation and the size and the kind of maternity unit (university, public or private hospital). During labour, the request of an EA was more frequent when anaesthesiologists were on call in the hospital (77.7 vs 66.7%, p<0.001). Midwives offered EA to 69% of women. For the next delivery 79.9% of women hoped an EA, the factors of this new request were parity, pain during this labour and EA during this labour; 42.8% would be ready to move in an other town to obtain an EA. The medical indications for EA occurred in 1.8% of patients. DISCUSSION: The request for EA mainly comes from women. Probably, in France, the request for EA will not diminish in the future. Analgesia networks could be considered.

[Request of epidural analgesia by women and obstetrical teams in four French areas. Part II: Management of epidural analgesia]. / Demande d'analgésie périmédullaire par les patientes et les équipes obstétricales dans quatre régions françaises. Partie II : réalisation des analgésies périmédullaires.

OBJECTIVE: Epidural analgesia (EA) is widely used in France to treat the labour pain. The aim of this study was to evaluate and analyse the rate of EA and the ratio between EA required by parturients and total EA realised (performance ratio). STUDY DESIGN: An epidemiological survey was sent in all maternity units of 4 French areas. Patients and methods. - 48 of the 84 maternity units were participating to the study. In each centre, 25 patients fulfilled the questionnaire and the medical team fulfilled a questionnaire about the organisation of the ward. Rate of EA and performance ratio were calculated. Student t test, chi2 test and logistic regression model were used as requested; p<0.05 was considered as significant. RESULTS: The mean rate of epidural analgesia (EA) rate, in the French areas studied, was 61.6%. It was significantly higher in university (79+/-13.7%) and private hospital (73.1+/-20.4%) than in general hospitals (54.6+/-19.6%, p<0.01), and also in those where anaesthesiologists are dedicated to the maternity unit (71.3+/-17.8 vs 54.6+/-22.1%, p<0.01) and in hospitals where anaesthesiologists were on duty in the hospital versus on call at home (69.8+/-21.4 vs 56.1+/-19%, p<0.02). Median duration of EA was 180 minutes, and 21.3% of them lasted more than five hours. Most of EA was performed between 8 AM and 6 PM. Patients' request was the major reason of EA insertion (OR=11.81), then the midwife request (OR=9.01). Other significant factors were the type of the hospital, the anaesthesiologist on duty and parity of women. The ratio between the number of EA requested by parturients and the total number of EA performed was significantly better in university hospitals (100.3+/-13%) and private hospitals (92.2+/-15.7%) than in general hospitals (79.4+/-17.3%, p<0.02). For the patients who had requested EA and did not have EA, the main reason was that labour was too fast (122/167) and then that there was a fail in anaesthesiological organization (59/167). The contraindications were rare (14/167). CONCLUSION: To correctly answer to the request of EA, it seems necessary that one or more anaesthesiologists were dedicated to the maternity units, and that they were on duty into the hospital. So it seems important to have large maternities with adequate number of anaesthesiologists.

At least five regions of imbalance on 6q in breast tumors, combining losses and gains.

The long arm of chromosome 6 is frequently rearranged in human cancer. In breast cancer, allelotyping studies have indicated the existence of three to four distinct regions of allelic imbalance. Chromosome transfer studies have shown the presence of several growth inhibiting or senescence promoting genes in the segment between 6q13 and 6q27. Moreover, results from comparative genomic hybridization (CGH) analyses have indicated that 6q was indeed a site of chromosomal losses, but that it was also involved in a substantial number of gains. In the present work, we allelotyped 178 pairs of breast tumor and normal tissue DNAs using 30 CA repeat markers from the Genethon collection. Seventy-six percent of the tumors in our panel displayed allelic imbalance (AI) of at least one locus, but patterns of AI could be complex. Whereas 11 tumors showed AI at all markers tested, 57 presented zebra profiles, and 28 showed AI at one site only. We characterized five distinct domains of AI defined, from centromere to telomere, by D6S300 (domain 1), D6S434 (domain 2), D6S261 (domain 3), D6S314-D6S409 (domain 4), and D6S441-D6S415 (domain 5). Some of the domains could be narrowed down to intervals of 1cM or less. We performed CGH analysis on a subset of 34 tumors presenting AI of variable extent at 6q. In 10/34 tumors, CGH did not reveal any anomaly on 6q. Most of these presented AI on short intervals, thus being below the detection threshold by CGH. Of the remaining 24 tumors presenting anomalies by CGH, 11 presented gains involving all or portions of 6q and 15 losses (2 presented combined losses and gains). By CGH, the 6q21-22 region was most commonly involved in gains, whereas 6q13-14 and 6q25-27 were frequently lost. Thus, allelic imbalances on 6q can either represent a gain or a loss depending on the region involved. Genes Chromosomes Cancer 27:76-84, 2000.

Quantification of ERBB2 protein expression in breast cancer: three levels of expression defined by their clinico-pathological correlations.

Activation of the ERBB2 oncogene seems to be an early event in breast cancer progression and prevalent in in situ carcinomas. However, its prognosis value, albeit recognized for node-positive patients, remains controversial for those without apparent nodal involvement. One possible reason for this problem is likely to be the difficulty of defining threshold levels for ERBB2 protein overexpression. ERBB2 protein expression was therefore analyzed in primary invasive breast tumors. Quantification of the gene product by a commercial ELISA test was compared to results obtained by immunohistochemistry and western blotting, as well as to gene amplification status determined by Southern blotting. Correlations between results obtained by the different techniques were highly significant (P value < 10(-6)). Nevertheless, ELISA permitted us to determine three levels of protein expression corresponding to distinct tumor subsets. 1) Tumors with p185/ERBB2 expression levels exceeding 10 U/microgram exhibited in most cases amplification of the gene (83% of cases), DNA aneuploidy (81%) and absence of estrogen receptor (ER) (44%). Such high levels of protein expression were exclusively observed in invasive ductal carcinomas and were prevalent in those showing a significant in situ component. 2) "Intermediate" levels of expression (3-10 U/micrograms) were rarely observed in tumors exhibiting gene amplification (9%), but were preferentially found in cancers of more favorable prognosis (only 49% were aneuploid and 9% estrogen receptor negative). 3) Levels of p185/ERBB2 below 3 U/micrograms were detected in benign mastopathies and, thus, carcinomas presenting such levels were scored ERBB2 negative. Interestingly, invasive lobular carcinomas were rarely ERBB2 positive, and if so, only at intermediate levels. Moreover, our data show a complex interrelationship between p185/ERBB2 expression and ER levels. Indeed, tumors with more than 10 U/micrograms of p185 were prevalently ER, whereas those with p185 ranging from 3 to 10 U presented elevated levels of ER.

Enzyme immunoassay for human chorionic gonadotropin using monoclonal antibodies elicited with a synthetic peptide.

We recently described the generation of mouse monoclonal antibodies directed against hCG using a synthetic peptide immunogen: the carboxy-terminal peptide (CTP) of beta-hCG. The CTP, residues 109-145, is a portion unique to hCG and is not present in other glycoprotein hormones. These monoclonal antibodies react with beta-hCG-CTP and whole hCG but do not react with hLH. In the present study, a two-site sandwich ELISA specific for hCG has been developed using these CTP-induced monoclonal antibodies. Two monoclonal antibodies were paired: beta-hCG-CTP-a6 was immobilized to give a hCG-specific solid phase, and a biotinylated monoclonal antibody against beta-hCG was used as the indicator antibody. This second antibody was introduced during the hCG-capture step because of its agonist effect on hCG-capture by beta-hCG-CTP-a6 solid phase. This ELISA system can detect 2.2 IU/l hCG in a serum-free medium and 10.4 IU/l in a medium containing 20% human serum. We did not observe any cross-reactivity with hLH up to concentrations of 5000 IU/l. The hCG-ELISA correlated well (r = 0.99) with a control RIA in an assay of 78 human sera from healthy males, pregnant or non-pregnant females and from patients with choriocarcinoma, hydatidiform mole, or teratocarcinoma.

Human T-lymphocyte colonies: generation of colonies in different lymphocyte subpopulations.

The generation of human T-lymphocyte colonies from different lymphocyte subpopulations in the presence of PHA alone, or PHA plus media conditioned by PHA-stimulated lymphocytes (PHA-LCM) has been investigated. The separation technique consisted of phagocytic cell depletion by carbonyl iron treatment and fractionation of non-phagocytic cells (NP cells) into B cells and T + null cells by affinity chromatography on an anti-F(ab')2 column. The T cells were separated from the null cells by E-rosette sedimentation. Under these conditions, we showed that: no T-lymphocyte colonies were obtained from the null-cell subset in the presence of PHA of PHA + PHA-LCM; T-lymphocyte-colony formation potential was retained in the T-cell subset. Some variability was observed in the production of T colonies using peripheral blood lymphocytes (PBL) from different donors. Low producers and high producers of T-lymphocyte colonies were encountered. The low production of T-lymphocyte colonies observed in some donors was due to a suppressive effect mediated by the phagocytic cells, probably monocytes. The anti-F(ab')2 immunoadsorbent retained a cell population necessary for T-lymphocyte colony growth.

Human T lymphocyte colonies. I. Surface markers and cytotoxic potential of colony cells.

Colonies were obtained from peripheral blood lymphocytes (PBL) grown in soft agar in the presence of PHAM or PHAp mitogens. One out of 130 PBL was able to generate a colony. Colony cells were mass harvested and assayed for surface markers and cytotoxic potential. Most of the colony cells (83%) form spontaneous rosettes with sheep-red blood cells (RBC) and bear the human T lymphocyte antigens (HTLA) (92%). A significant amount of colony cells able to bind autologous RBC was detected (24%). The capacity of PBL and colony cells to bind Ox-RBC sensitized with rabbit anti-Ox-RBC IgM (EAM complexes) was measured: only 15% of colony cells compared to 49% of the PBL formed EAM rosettes. The capacity of cells to bind the Fc portion of antigen-complexed IgG was investigated by two rosette assays: using Chicken or Ox-RBC sensitized with a rabbit anti-Chicken-RBC or Ox-RBC IgG (Chicken EAG or Ox-EAG complexes). The percentage of colony cells forming Chicken EAG rosettes was low (3.6%) compared to PBL (12%). This percentage was significantly increased with PHAp, and not PHAM stimulation (11%). Using Ox-EAG complexes, we confirmed the low percentage of EAG rosettes in colony cells under PHAM stimulation (4.7%) compared to PBL (21%). A significant cytotoxic capacity (spontaneous or antibody dependent) was found in colony cells after PHAM stimulation. This method of culture is able to generate clones of T cells and conserve T cell subsets and cytotoxic potential usually found in a T purified population. In further studies, it will be interesting to investigate if each clone possesses specific markers and cytotoxic potential and is able to maintain this differentiation step in long term culture.