Maintenance of methylation profile in imprinting control regions in human induced pluripotent stem cells.

BACKGROUND: Parental imprinting is an epigenetic mechanism that leads to monoallelic expression of a subset of genes depending on their parental origin. Imprinting disorders (IDs), caused by disturbances of imprinted genes, are a set of rare congenital diseases that mainly affect growth, metabolism and development. To date, there is no accurate model to study the physiopathology of IDs or test therapeutic strategies. Human induced pluripotent stem cells (iPSCs) are a promising cellular approach to model human diseases and complex genetic disorders. However, aberrant hypermethylation of imprinting control regions (ICRs) may appear during the reprogramming process and subsequent culture of iPSCs. Therefore, we tested various conditions of reprogramming and culture of iPSCs and performed an extensive analysis of methylation marks at the ICRs to develop a cellular model that can be used to study IDs. RESULTS: We assessed the methylation levels at seven imprinted loci in iPSCs before differentiation, at various passages of cell culture, and during chondrogenic differentiation. Abnormal methylation levels were found, with hypermethylation at 11p15 H19/IGF2:IG-DMR and 14q32 MEG3/DLK1:IG-DMR, independently of the reprogramming method and cells of origin. Hypermethylation at these two loci led to the loss of parental imprinting (LOI), with biallelic expression of the imprinted genes IGF2 and DLK1, respectively. The epiPS™ culture medium combined with culturing of the cells under hypoxic conditions prevented hypermethylation at H19/IGF2:IG-DMR (ICR1) and MEG3/DLK1:IG-DMR, as well as at other imprinted loci, while preserving the proliferation and pluripotency qualities of these iPSCs. CONCLUSIONS: An extensive and quantitative analysis of methylation levels of ICRs in iPSCs showed hypermethylation of certain ICRs in human iPSCs, especially paternally methylated ICRs, and subsequent LOI of certain imprinted genes. The epiPS™ culture medium and culturing of the cells under hypoxic conditions prevented hypermethylation of ICRs in iPSCs. We demonstrated that the reprogramming and culture in epiPS™ medium allow the generation of control iPSCs lines with a balanced methylation and ID patient iPSCs lines with unbalanced methylation. Human iPSCs are therefore a promising cellular model to study the physiopathology of IDs and test therapies in tissues of interest.

Role for cementoplasty in intra-articular distal radius fractures: Cadaver study and application to arthroscopy.

BACKGROUND: Die-punch intra-articular fractures of the distal radius raise surgical challenges. The residual articular step-off must be less than 1mm to prevent the development of radio-carpal osteoarthritis. The objectives of this cadaver study were to evaluate whether cementoplasty was effective in reducing die-punch fractures and to determine whether this technique was feasible as an arthroscopic procedure. HYPOTHESIS: Cementoplasty performed as an arthroscopic procedure is effective in treating die-punch fractures. MATERIAL AND METHODS: Eleven cadaver forearms collected at a laboratory were studied. In each, a depressed fracture of the lunate fossa of the radial articular surface was created using a Tinius Olsen H25K-S compression test machine. A Kyphon XPander® balloon (Medtronic) was used to lift the depressed area, and calcium-phosphate cement was then injected to stabilise the reduction. Cementoplasty under arthroscopic guidance was performed on an additional forearm. RESULTS: Computed tomography of the wrists after fracture induction showed a mean depression of 4.66mm (range, 4.01-5.25mm). Arthroscopic cementoplasty proved feasible with the arthroscope inserted through the 3-4 radio-carpal portal. Positioning the balloon under the depressed area ensured satisfactory reduction and allowed the injection of cement. DISCUSSION: Cementoplasty may be useful for the treatment of die-punch fractures. Additional indications may be other types of distal radius fractures with articular surface depression. LEVEL OF EVIDENCE: IV, cadaver study.

Descriptive study of the parkinsonian population in the north of France: Epidemiological analysis and healthcare consumption.

BACKGROUND: The "neurodegenerative diseases plan" under elaboration for the Hauts-de-France region requires better knowledge of the patient population and care pathways. In France, the prevalence of Parkinson's disease (PD) has been estimated from cohorts to be about 1-3 per 1000 inhabitants, but exhaustive data are scarce for the general population. The purpose of this study was to evaluate the prevalence of PD in the Hauts-de-France region and to assess PD-related healthcare consumption. METHOD: A descriptive study was conducted to identify the parkinsonian population in the Hauts-de-France region (including the administrative districts of Pas-de-Calais and Picardie) for the year 2014. Parkinsonian patients were identified from health insurance fund reimbursement data using the following criteria: (i) reimbursement for a PD-specific medication; (ii) attribution of long-duration disease status coded as PD; (iii) hospital stay with PD diagnosis in the standard discharge report contained in the French medico-economic database on hospital activity (PMSI). RESULTS: The raw prevalence of PD in the region was 5.03 per 1000 inhabitants aged 20 years and older. The standardized prevalence by health territory ranged from 4.0 to 9.0 per 1000 inhabitants aged 20 years and older. During the 1-year study period, 33.5% of patients had a neurology consultation, 57.1% attended a physiotherapy session, and 7.7% received speech therapy. Most of patients (79.6%) were treated with levodopa, sometimes in combination with a catechol-O-methyl transferase inhibitor (14.4%). Dopaminergic agonists were prescribed in 33.5% of cases. A neuroleptic was prescribed for 6.9% of the population (clozapine for 25.9%). CONCLUSION: The prevalence of PD is high in the Hauts-de-France region with a heterogeneous distribution by health territory. Neurology consultations were attended by a minority of patients in 2014. This work provides perspectives for necessary improvement in specialized care for this disease, both in terms of follow-up consultations and home care.

Prevalencia de la desnutrición crónica en niños menores de 5 años atendidos en el Centro de Salud Tacopaya, primer semestre gestión 2014 / Prevalence of chronic under nutrition of children under 5 years served in the Center of Health management Tacopaya, first half 2014

Objetivo: determinar la prevalencia de la desnutrición crónica en menores de 5 años, atendidos en el Centro de Salud de Tacopaya de la Provincia Arque de Cochabamba, en el primer semestre de la gestión 2014. Material y métodos: se realizó un estudio descriptivo, cuantitativo y transversal, incluyendo 654 menores de 5 años que asistieron al Centro de Salud Tacopaya, durante el primer semestre 2014, reportados en el SNIS. Los datos sobre el grupo de talla, peso, edad, sexo y grupo etáreo fueron recogidos y analizados de acuerdo con los estándares de la OMS; para determinar la desnutrición crónica, se utilizó el indicador antropométrico talla para la edad. Nuestros datos tabulados en Excel, fueron estratificados por sexo, grupo etáreo y comparados con otros periodos de otros años. Resultados: 361 (55,20%) niños y 293 (40,8%) niñas fueron atendidos para su control talla, peso en el Centro de Salud Tacopaya, 106 (16,2%) presentaron desnutrición crónica. El número más alto de desnutrición crónica fue en el grupo etáreo de 1 año a menor de 2 años y de sexo masculino, y el registro más bajo de desnutrición crónica fue para menores de 1 año. En el primer semestre del 2012, la prevalencia de desnutrición crónica fue de un 20,2%, en 2013, 19,1% y en 2014, 15,9%. Conclusiones: La desnutrición crónica en niños menores de 5 años de edad, que asistieron al Centro de Salud Tacopaya, para pacientes ambulatorios alcanzó el 16,2% en 2014, mostrando mayor prevalencia en los niños que en las niñas. Los niños de 1 año hasta menos de 2 años fueron los más afectados. La desnutrición sigue siendo un problema muy grave que sigue afectando a la población boliviana. Esta información puede ser útil para la planificación de intervenciones nutricionales dirigidas a reducir desnutrición crónica.

Objective: determine the prevalence of chronic malnutrition in children < 5 years, treated at the outpatient Health Center Tacopaya of the Cochabamba Arque province in the first half of 2014. Material and methods: A descriptive, quantitative and cross-sectional study was performed, including 654 children, reported in the SNIS. Data on height, weight, age, sex and age group were collected and analyzed, in agreement to WHO standards, to determine the height-for-age chronic malnutrition anthropometric indicator. Our data, tabulated in Excel, were stratified by sex, age group and compared to other year periods. Results: among the 361 (55,20%) boy and 293 (40,8 %) girls treated for height control weight at the Tacopaya Health Center, 106 (16,2%) showed chronic malnutrition. The highest number of chronic malnutrition was in the age group of 1 year to less than 2 years and in the male group. The lowest record of chronic malnutrition was for children less than 1 year. Conclusions: chronic malnutrition in children less than 5-year-old, attending the Tacopaya outpatient Health Center, reached 16,2% in 2014, showing higher prevalence in boys than in girls. Children up to 1 year to less than 2 years were the most affected. Malnutrition remains a serious problem still affecting the Bolivian population. This information may be useful for planning nutritional interventions aiming to reduced chronic malnutrition.

Social, medical and self-perceived factors influencing postpartum screening of diabetes after gestational diabetes.

To assess factors related with postpartum testing for diabetes and the mothers' knowledge about their infant's future risk for diabetes and obesity, we asked 87 women with gestational diabetes who delivered during a 28-month period, to perform a postpartum glucose screening. They were then asked to answer a telephone questionnaire regarding risk perception of diabetes and infant's risk, and were invited to undergo further glucose testing if they missed the first test. Postpartum screening was assessed according to medical, sociodemographic and interview characteristics. A total of 42.5% of women escaped initial postpartum screening and 43.0% escaped secondary testing. Performance of postpartum testing was related with sub-Saharan origin, BMI < 25 kg/m(2) and with high risk perception of developing permanent diabetes. Few women were informed about their infant's future risk.

The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L.

An understanding of the genetic determinism of frost tolerance is a prerequisite for the development of frost tolerant cultivars for cold northern areas. In legumes, it is not known to which extent vernalization requirement or photoperiod responsiveness are necessary for the development of frost tolerance. In pea (Pisum sativum L.) however, the flowering locus Hr is suspected to influence winter frost tolerance by delaying floral initiation until after the main winter freezing periods have passed. The objective of this study was to dissect the genetic determinism of frost tolerance in pea by QTL analysis and to assess the genetic linkage between winter frost tolerance and the Hr locus. A population of 164 recombinant inbred lines (RILs), derived from the cross Champagne x Terese was evaluated both in the greenhouse and in field conditions to characterize the photoperiod response from which the allele at the Hr locus was inferred. In addition, the population was also assessed for winter frost tolerance in 11 field conditions. Six QTL were detected, among which three were consistent among the different experimental conditions, confirming an oligogenic determinism of frost tolerance in pea. The Hr locus was found to be the peak marker for the highest explanatory QTL of this study. This result supports the hypothesis of the prominent part played by the photoperiod responsiveness in the determinism of frost tolerance for this species. The consistency of three QTL makes these positions interesting targets for marker-assisted selection.

Topology of the fibrinolytic system within the mural thrombus of human abdominal aortic aneurysms.

Development and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. 20 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(2)-antiplasmin complexes (PAPs) and D-dimers release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and D-dimer plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and D-dimers. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and D-dimers found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.

Extracellular matrix remodeling in the vascular wall.

The extracellular matrix provides a structural framework essential for the functional properties of vessel walls. The three dimensional organization of the extracellular matrix molecules--elastin, collagens, proteoglycans and structural glycoproteins--synthesized during fetal development--is optimal for these functions. Early in life, the vessel wall is subjected to injury: lipid deposition, hypoxia, enzyme secretion and reactive oxygen species production during inflammatory processes, and the extracellular matrix molecules are hydrolyzed by proteases--matrix metalloproteinases, leukocyte elastase, etc. In uninjured arteries and veins, some proteases are constitutively expressed, but through the control of their activation and/or their inhibition by inhibitors, these proteases have a very low activity. During the occurrence of vascular pathologies--atherosclerosis, hypertension, varicosis, restenosis, etc.--the balance between proteases and their inhibitors is temporally destroyed through the induction of matrix metalloproteinase gene expression or the secretion of enzymes by inflammatory cells. Smooth muscle cells, the most numerous cells in vascular walls, have a high ability to respond to injury through their ability to synthesize extracellular matrix molecules and protease inhibitors. However, the three dimensional organization of the newly synthesized extracellular matrix is never functionally optimal. In some other pathologies--aneurysm--the injury overcomes the responsive capacity of smooth muscle cells and the quantity of extracellular matrix decreases. In conclusion, care should be taken to maintain the vascular extracellular matrix reserve and any therapeutic manipulation of the protease/inhibitor balance must be perfectly controlled, because an accumulation of abnormal extracellular matrix may have unforeseen adverse effects.

Inhibition of human papillomavirus-16 long control region activity by interferon-gamma overcome by p300 overexpression.

Although interferons (IFNs) are currently used in the treatment of various human papillomavirus (HPV)-associated lesions, their mechanisms of action are still unclear. In this study, we clearly demonstrated that IFN-gamma was a strong inhibitor of HPV-16 long control region (LCR) activity in two human cervical carcinoma cell lines. The effect of IFN-gamma was dose dependent. We investigated whether the effect of IFN-gamma on HPV-16 LCR could involve the inhibition of the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. In support of this model, we demonstrated by transfection experiments that a 12S E1A mutant (RG2), which interacts poorly with p300 and CBP in comparison to wild-type E1A, was less able to repress human papillomavirus (HPV) 16 long control region (LCR) than wild-type E1A. More important, overexpression of p300 was able to increase the HPV-16 LCR activity and to overcome inhibition by IFN-gamma. Finally, we demonstrated that p300 could cooperate with c-jun to activate HPV-16 LCR. According to our results, IFN-gamma might inhibit HPV-16 LCR transcription by activating the signal transducer and activator of transcription 1alpha, which in turn might compete for p300/CBP binding with specific transcription factors involved in LCR activation.

Efficacy of brief coping skills interventions that match different personality profiles of female substance abusers.

Female substance abusers recruited from the community were randomly assigned to receive 1 of 3 brief interventions that differentially targeted their personality and reasons for drug use. The 90-min interventions were: (a) a motivation-matched intervention involving personality-specific motivational and coping skills training, (b) a motivational control intervention involving a motivational film and a supportive discussion with a therapist, and (c) a motivation-mismatched intervention targeting a theoretically different personality profile. Assessment 6 months later (N = 198) indicated that only the matched intervention proved to be more effective than the motivational control intervention in reducing frequency and severity of problematic alcohol and drug use and preventing use of multiple medical services. These findings indicate promise for a client-treatment matching strategy that focuses on personality-specific motives for substance abuse.

Molecular plasticity of vascular wall during N(G)-nitro-L-arginine methyl ester-induced hypertension: modulation of proinflammatory signals.

It has previously been reported that hypertension induced by the chronic blockade of NO production is characterized by a proinflammatory phenotype of the arterial wall associated with a periarterial accumulation of inflammatory cells. In the present study, the cellular and molecular mechanisms involved in the luminal and perivascular accumulation of inflammatory cells were evaluated in the aortas of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats. Because the medial layer remains intact, putative markers of the resistance of the vascular wall to cell migration and to oxidative stress were also explored. For this purpose, monocyte adhesion, cytokine expression, superoxide anion production, and nuclear factor-kappa B (NF-kappa B) activation were assessed in the aortas of L-NAME-treated rats. Expressions of tissue inhibitor of metalloproteinases-1 (TIMP-1) and heme oxygenase-1 (HO-1) in the aortic wall were also studied as possible markers of such resistance. Chronic blockade of NO production increased ex vivo monocyte adhesion to the endothelium, increased the production of superoxide anions, and activated the NF-kappa B system. In concert with this modification of the redox state of the vascular wall in L-NAME-treated rats, the expression of proinflammatory cytokines interleukin-6, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor was increased. In parallel, expressions of both TIMP-1 and HO-1 were increased. All these changes were prevented by treatment with an angiotensin-converting enzyme inhibitor (Zofenopril). Hypertension associated with a proinflammatory phenotype of the vascular wall induced by blockade of NO production could be due to an increase in oxidative stress, which, in turn, activates the NF-kappa B system and increases gene expression. In parallel, the arterial wall overexpresses factors such as TIMP-1 and HO-1, which could participate in the resistance to cell migration and oxidative stress.

A functional NF-kappaB binding site in the human papillomavirus type 16 long control region.

By computer search, we identified one potential NF-kappaB binding site in the HPV16 long control region (LCR) at position 7554-7563 having two mismatches in comparison to the consensus NF-kappaB binding site of the Igkappa L promoter. Bandshift experiments with nuclear extracts from HeLa cells or purified glutathione S-transferase-p65 fusion protein clearly demonstrated that NF-kappaB is able to bind to this region of the LCR. However, in comparison to NF-kappaB binding on a consensus probe, the affinity of NF-kappaB for this site is about 250-fold reduced. When mutations were introduced into this NF-kappaB binding site, the activity of the LCR was increased, strongly suggesting that NF-kappaB was acting as a transcriptional repressor in the context of the HPV16 LCR. In addition, overexpression of NF-kappaB p65 repressed the activity of the HPV16 LCR, strengthening this conclusion.

Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBPbeta isoforms.

During genital human papillomavirus (HPV) infection several cytokines are released, such as interleukin-1 (IL-1), tumor necrosis factoralpha (TNFalpha), IL-6, and IL-8. These cytokines may play a role in the immune surveillance against viral infection. Two of these cytokines, IL-1 and TNFalpha, suppress the transcription of the HPV16 early genes. CAATT/ enhancer binding protein, (C/EBPbeta), which is activated by IL-1 and TNFalpha, has been suggested to act as a mediator of this transcriptional downregulation. C/EBPbeta contains three different translation initiation sites that can lead probably by leaky ribosome scanning to the generation of three isoforms of C/EBPbeta, namely full-length C/EBPbeta, liver enriched transcriptional activator protein (LAP), and liver enriched inhibitory protein (LIP). When transiently expressed in C33A and HeLa cells, the first two C/EBPbeta isoforms activate the HPV16 long control region (LCR). LIP, which acts as an antagonist of C/EBPbeta, represses the HPV16 LCR activity. Our observation that treatment of HeLa cells with IL-1 leads to induction of LIP supports the hypothesis that the LCR downregulation by IL-1 is mediated by LIP.

Differential distribution of dystrophins in rat retina.

PURPOSE: Duchenne muscular dystrophy is frequently associated with a reduced amplitude of b-wave under scotopic conditions in the electroretinogram. This suggests that the dystrophin gene-encoded proteins play a role in retinal neurotransmission. The abnormal neurotransmission has been attributed to altered expressions of C-terminal products of the dystrophin gene in the outer plexiform layer, where photoreceptor cells form synapses with secondary neurons. The present study was undertaken to determine the cellular distribution of each member of the dystrophin superfamily in rat retina. METHODS: Examined in the study were the developmental pattern of dystrophins in rat retinae that exhibit inherited progressive photoreceptor degeneration; dystrophins messengers expression in the outer and the inner retina of normal rats, prepared by mechanical fractionation through the outer plexiform layer; and immunolocalization of dystrophin proteins and utrophin in normal and degenerated adult rat retinae, with several antibodies prepared against specific regions of the dystrophin superfamily. RESULTS: The results showed that Dp260 is exclusively localized in photoreceptor cells; Dp140 seems to be present in perivascular astrocytes; the exon 78 spliced isoform of Dp71 and the unspliced form are located in Müller glial cells and in perivascular astrocytes, respectively. Müller glial cells also contain utrophin. CONCLUSIONS: Although the role of these membrane cytoskeletal proteins remains to be elucidated in retina, the results support the hypothesis that b-wave reduction may be caused by molecular anomalies of C-terminal products of the dystrophin gene expressed in both neuron and glial cells.

[Epidemiology of childhood asthma in the department of Calvados]. / Epidémiologie de l'asthme infantile dans le département du Calvados.

A survey was conducted in 1707 sixth grade school children in the Calvados department of France. A self-administered questionnaire was filled out by the children in the presence of a school nurse. The cumulative prevalence of asthma was 14.9%. There was no significant difference between children living in urban or rural areas. There was however a significant difference by sex: 18% of the boys had asthma and 11% of the girls. The cumulative prevalence of wheezing was 25.4% (current prevalence 12.9%). The current prevalence of dry nocturnal cough, respiratory infections excluded, was 33.3%; that of exercise-induced asthma, 27%. Severity was evaluated on the basis of the number of wheezing episodes since the beginning of the school year (> 3 episodes: 4.5%), the number of awakenings at night per week (several per week: 2.2%), and aggravations severe enough to bother speech (4.3%). The rate of missed school days was 18.5% and that of asthma-related hospitalization 1.8%. One asthmatic child out of 4 had undergone pulmonary function tests and 1 out of 2 had a specific treatment for asthma. The high prevalence of childhood asthma in Calvados, with high morbidity, a significant number of missed school days, the exceptional nature of satisfactory pulmonary function testing, and the inconsistency of specific treatments, emphasizes the need for educational programs for parents and their family and improved physician training.

Evolution of the human immunodeficiency virus type 1 long terminal repeat promoter by conversion of an NF-kappaB enhancer element into a GABP binding site.

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-kappaB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-kappaB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-kappaB sequence and is present in the nuclear extract of unstimulated cells that lack NF-kappaB. These results suggest that inactivation of the NF-kappaB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-kappaB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-kappaB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

[Acute respiratory Chlamydia pneumoniae infections in adults. Value of direct gene amplification diagnosis]. / Infections respiratoires aiguës de l'adulte à Chlamydia pneumoniae. Intérêt du diagnostic direct par amplification génique.

Chlamydia pneumoniae has been established recently as an important human respiratory pathogen. The aim of this study was to define the incidence of Chlamydia pneumoniae in acute respiratory infections by evaluating its presence in posterior nasopharyngeal aspirates or broncho-alveolar lavage specimens by polymerase chain reaction-hybridization (PCR-EIA) as well as the titres of specific antibodies in serum by a rELISA test and a micro-immunofluorescence (MIF) test. 68 adults patients were investigated. Eight patients (11.8%) were positive by either rELISA or PCR-EIA or both, with an infection rate of 5 patients with community-acquired pneumonia, 2 asthmatic patients and 1 patients with an exacerbation of chronic obstructive pulmonary disease. Serological evidence of acute infection was found in four of these patients with rELISA test and in three others with MIF test. PCR-EIA detected Chlamydia pneumoniae DNA in four patients, but there were concordant results with rELISA and PCR-EIA in only one patient. In conclusion, Chlamydia pneumoniae appears to be a common etiologic agent of acute respiratory infections in adults. The discrepancy between serological test and PCR-EIA results reflects the difficulties in routine laboratory diagnosis of Chlamydia pneumoniae. The ambiguous results of serological tests from a single serum sample assess the utility of PCR for prompt diagnosis. When PCR is negative or no feasible, a second serology to 15/21 days of interval is necessary. Further studies with optimised techniques must be developed.

Survival of purified rat photoreceptors in vitro is stimulated directly by fibroblast growth factor-2.

Basic fibroblast growth factor (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-15 rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-15 retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal growth factor (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor-glia co-culture model.

1. Edge contrast enhancement is an integrated visual function based on the complex centre-surround organization of the cone photoreceptor light response. While centre responses result from direct light activation, surround responses are thought to result from lateral inhibition mediated by horizontal cells. This feedback signal has been attributed to GABAA receptors which have been found in lower vertebrate cones. 2. In order to study the GABA response of adult mammalian photoreceptors, we designed a culture system consisting of isolated photoreceptors seeded on a layer of retinal glial cells. Mature rods and cones required the presence of Muller glial cells to survive and develop neurites; they degenerated in the absence of glial cells. 3. Cone photoreceptors generated large GABA responses whereas rod photoreceptors did not respond to GABA applications. 4. Cone GABA responses consisted of two distinct components, one suppressed by the GABAA receptor blockers bicuculline and SR95531, and the second by the GABAC receptor antagonists TPMPA and imidazole-4-acetic acid (I4AA). Pentobarbital greatly increased the GABAA receptor component whereas it did not affect, or even reduced, the GABAC receptor component. During long GABA applications, GABAA receptor currents desensitized by 78%, contrasting with the sustained GABAC response. 5. Expression of GABAC receptors in cone photoreceptors was confirmed by anti-rho-subunit immunolabelling of porcine retinal sections. 6. These results indicate that both GABAA and GABAC receptors may participate in the feedback synapse from horizontal cells to cone photoreceptors in the mammalian retina.

Gangliosides and neurotrophic growth factors in the retina. Molecular interactions and applications as neuroprotective agents.

Polypeptide growth factors and gangliosides can both be considered as trophic agents involved in almost all stages of neural cell development, differentiation, survival, and pathology. In most cases their physiological roles are still not clear due to the considerable complexity in their regulation. Several growth factors [e.g., basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)] and one species of ganglioside (GM1) have been shown to exert interactions with each other and also to exhibit neuroprotective effects against retinal ischemia in vivo and cerebral excitotoxicity in vitro. Different experimental models are used to investigate their relevance to ischemic and excitotoxic conditions in the retina, and it is shown that (1) both bFGF and EGF show very effective neuroprotection for rat retinal neurones exposed to toxic levels of glutamate or its nonphysiological agonist kainate in vitro; (2) GM1 (10(-5M) used under the same conditions does not afford protection; (3) retinal glial cells also suffer morphological perturbations following glutamate or kainate treatment, but this effect is dependent on neuron-glial interactions, indicating the existence of intermediate neuron-derived messenger molecules; (4) these glial changes can be corrected by posttreatment with either bFGF or EGF in vitro; (5) using an in vivo animal model involving anterior chamber pressure-induced ischemia in adult rats, it is shown that either pretreatment by intraperitoneal injection of GM1, or posttreatment by intraocular injection of the same ganglioside, reduces significantly histological damage to inner nuclear regions; and (6) in cultured retinal Müller glial cells the existence of molecular and metabolic interactions between both types of trophic factors is demonstrated. Hence both these groups of trophic molecules show interesting features for retinal ischemic treatment.