Variables related to chronic immune thrombocytopenia: experience from a single center and comparison to a meta-analysis.

Classically, several variables have been related to the disease course of chronic primary immune thrombocytopenia (cITP), though to date, there is no consensus on their clinical relevance. In a recent systematic review, a meta-analysis was made and confirmed the existence of certain cITP-related variables that may be related to prognosis in pediatric patients. We retrospectively analyzed a cohort of patients diagnosed with ITP, identified prognostic variables, and compared our results to the variables described by the authors. A multivariate study revealed that older age at diagnosis and higher platelet count were the only independent variables related to cITP. Children up to age 4 years and those with lower platelet counts (below 20 × 109/L) were at lower risk for cITP.Conclusion: We therefore concluded that only age and platelet count at diagnosis are independent variables that should be considered when evaluating the risk of developing cITP. What is Known: • Around 20% of patients with immune thrombocytopenia progress to chronic disease as determined by a sustained platelet count below 100×109/L for more than 12 months. • A number of variables potentially related to the development of cITP are being studied, such as age, sex, cell count, and previous treatment. What is New: • This is a new group of patients diagnosed with ITP in which the platelet count and age at diagnosis are the only independent variables closely related to cITP. • In this new series, we could not confirm other variables previously related to cITP such as total leukocyte count or the absence of treatment at diagnosis.

Malnutrition and obesity: influence in mortality and readmissions in chronic obstructive pulmonary disease patients.

BACKGROUND: The present study aimed to assess the association of obesity and malnutrition with the mortality of hospitalised patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) and the risk of readmission in <30 days. METHODS: A retrospective chart review of consecutive patients admitted with COPD as the primary reason for discharge in Spain between 1 January 2006 and 31 December 2007 was performed. Patients with a diagnosis of obesity or malnutrition in the hospital discharge clinical report were identified. The in-hospital mortality and re-admittance 30 days after discharge indices of obese and malnourished patients were compared against the subpopulation without these diagnoses. RESULTS: Of the 313 233 COPD admittances analysed, there were 22 582 (7.2%) diagnoses of obesity and 6354 (2.0%) diagnoses of malnutrition. In-hospital global mortality and the re-admittance risk were 12.0% and 16.7%, respectively. Obese patients showed a lower in-hospital mortality risk [odds ratio (OR) = 0.52; 95% confidence interval (CI) = 0.49-0.55] and early re-admittance risk (OR = 0.87; 95% CI = 0.85-0.92) compared to non-obese patients. Malnourished patients had a much higher risk of death when in hospital (OR = 1.73; 95% CI = 1.62-1.85) or of being re-admitted within 30 days after discharge (OR = 1.29; 95% CI = 1.22-1.38), even after adjusting for possible confounding factors. CONCLUSIONS: Obesity in patients hospitalised for COPD substantially reduces in-hospital mortality risk and the possibility of early re-admittance. Malnutrition is associated with an important increase in in-hospital mortality and risk of re-admittance in the 30 days following discharge.

The impact of weekends on outcome for acute exacerbations of COPD.

Differences in hospital staffing may influence outcomes for patients with acute conditions, including acute exacerbations of chronic obstructive pulmonary disease (COPD), depending on which day of the week the patients are admitted. This study was conducted to determine whether weekend admission increases the risk of dying in hospital. We analysed the clinical data of 289,077 adults with acute exacerbations of COPD admitted to the hospital at any public centre in Spain, during 2006 and 2007. We analysed the following factors for their association with death rate: day of admission, demographics, medical history and comorbidity. During the study period, there were 35,544 (12.4%) deaths during admission in COPD patients. Weekend admissions were associated with a significantly higher in-hospital mortality (12.9%) than weekday admissions (12.1%) among COPD patients (OR 1.07 (95% CI 1.04-1.10)). The differences in mortality persisted after adjustment for age, sex and coexisting disorders (OR 1.05 (95% CI 1.02-1.08)). Analyses of deaths within 2 days after admission showed larger relative differences in mortality between the weekend and weekday admissions (OR 1.17 (95% CI 1.11-1.23)). We conclude that patients with acute exacerbations of COPD are more likely to die in the hospital if they are admitted on a weekend compared with a weekday.

[Invasive procedures in patients hospitalized in Spanish internal medicine departments]. / Procedimientos invasivos en pacientes ingresados en los servicios de Medicina Interna españoles.

AIMS: Invasive procedures (IP) have become routine techniques that benefit an important number of patients on improving their quality of life or avoiding more aggressive treatments. We have conducted a study on the IPs performed in Spanish Internal Medicine (IM) Departments between 2005 and 2009. PATIENTS AND METHODS: IP performed to patients admitted to Spanish Internal Medicine departments were analyzed based on the information obtained from the Minimum Basis Data Set (CMBD). IP was defined as the following: filter placement in the inferior vena cava, chest tube placement, biliary, esophageal and colon prosthesis placement, pleurodesis, nephrostomy, external biliary drain placement, gastrostomy tube placement, thoracocentesis and peritoneal catheter placement. RESULTS: During the study period, a total of 75,853 invasive procedures on 70,239 admittances were performed in 2,766,673 patients (2.5%). IP subjects were younger (68.1 vs 71.4; P<.001), predominantly male (61.9 vs 53.2%; P<.001), with higher mortality (14.6 vs 9.9%; P<.001) and longer stay (18.4 vs 9.6 days; P<0.001). Cost of admittance was clearly higher than the rest of the patients (5,600€ vs 3,835€; P<.001). CONCLUSIONS: IPs are performed on a low percentage of IM Department hospitalized patients. They are costly, entail high mortality and a longer stay period compared to the mean population admitted to IM. A considerable proportion of the patients receiving IP suffer from neoplastic diseases, frequently in advances stages, which justifies the high inhospital mortality of this population.

The indication of tracheotomy conditions the predictors of time to decannulation in critical patients.

OBJECTIVE: Variables predicting optimal timing for tracheostomy decannulation remain unknown. We aimed to determine whether classifying patients into two groups according to their indications for tracheostomy could identify variables associated with time to decannulation. DESIGN: A prospective, observational cohort study was carried out. LOCATION: Two medical-surgical ICUs. PATIENTS: We included all patients tracheostomized during ICU stay, excluding patients with do-not-resuscitate orders, tracheostomies for long-term airway control, neuromuscular disease, or neurological damage. Patients were classified into two groups: patients tracheostomized due to prolonged weaning and/or prolonged mechanical ventilation (Group 1), and patients tracheostomized due to low level of consciousness or inability to manage secretions (Group 2). INTERVENTIONS: Patients were weaned and decannulated according to established protocols. MAIN VARIABLES: We recorded the following variables: time to tracheostomy, forced vital capacity, peak flow, suctioning requirements, Glasgow Coma Score (GCS), characteristics of respiratory secretions, and swallowing function. Statistical analyses included Cox-proportional multivariate analysis with time to decannulation as the dependent variable. RESULTS: A total of 227 patients were tracheostomized in the ICUs; of these, 151 were finally included in the study. In the multivariate analysis, time to decannulation in Group 1 was associated with the male gender (HR 1.74 (1.04-2.89), p= 0.03), age>60 years (HR 0.58 (0.36-0.91), p= 0.02), high suctioning frequency (HR 0.81 (0.67-0.97), p= 0.02), low forced vital capacity (HR 0.48 (0.28-0.82), p<0.01), and low peak flow (HR 0.25 (0.14-0.46), p<0.01). In Group 2 time to decannulation was associated to GCS >13 (HR 2.73 (1.51-4.91), p<0.01), high suctioning frequency (HR 0.7 (0.54-0.91), p<0.01), and inadequate swallowing (HR 1.97 (1.11-3.52), p=0.02). CONCLUSION: Variables associated with longer time to decannulation in ICU-tracheostomized patients differ with the indications for tracheostomy.

[Survey of Spanish Society of Internal Medicine on health care provision by internists in the public health system]. / Encuesta SEMI sobre la oferta asistencial de los internistas en los hospitales del Sistema Público de Salud.

AIMS: To know the organization of internal medicine departments (IMD) and the opinion of their heads of department. METHODS: In 2008, a survey was mailed to 410 heads of department of the IMD of 313 Spanish public hospitals. It included a standardized structured questionnaire on staff, hospitalization, outpatients, consultation, research and teaching. The heads of departments were also asked for their opinion and suggestions on management, projects and future. RESULTS: Sixty-eight surveys (22%) were filled out. Internists are on call an average of 3 times a month and perform 200 discharges, 500 outpatient visits and 40 consultations in a year. The average IMD consists of 10 internists with one-fifth of the hospital beds. One third of hospitals have alternatives to inpatient care, the most frequent being palliative care. Infectious diseases accounts for the most common monographic outpatient visit, one-third of IMD lack a structured relationship with primary care and the emergency department is independent of IMD. Half of the IMD have at least one IM resident and 6 residents in other specialties; half are involved in at least two clinical trials and one-third train medical students. The heads of the IMD identify problems in their relationship with hospital managers, other specialties and local population. Excessive workload, aging and discouragement of staff and patients' social problems have negative effects. Even so, they want to initiate projects, are optimistic about the future and take an interest in clinical epidemiology research. CONCLUSIONS: Although the sample is small and heterogeneous, it permits a valuable panoramic view of the structure and standard operation of a Spanish IMD as well as their expectations and areas of improvement.

Possible association between NOD2 variants and joint surgery in psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) has been inconsistently associated with common NOD2 gene variants, although some of these studies did not include patient stratification by clinical phenotype. OBJECTIVES: To analyse the association between the three common NOD2 variants (R702W, G908R and L1007fs) and clinical phenotypes of PsA, particularly with surrogate markers of severe joint destruction. PATIENTS AND METHODS: A total of 183 unrelated PsA patients and 187 controls were included. Demographic, clinical, biological and immunological characteristics were collected. Genotypes for the three common NOD2 gene variants were obtained by PCR and direct sequencing. RESULTS: NOD2 variants in PsA patients (7.6%) are just as prevalent as in healthy controls (7.5%). 18.5% of PsA patients carrying at least one NOD2 variant underwent joint surgery compared with 4.5% of those without these variants (p=0.019). Multivariate analysis confirmed this finding (OR 8.82, CI 1.7-46.3). There was no requirement for early surgery in patients carrying the NOD2 variants but there was an increased possibility of requiring surgery at similar times of disease duration. No other association with clinical features and NOD2 status carrier was found. CONCLUSIONS: Common NOD2 gene variants are not associated with PsA, but might increase the risk of undergoing joint replacement surgery, suggesting that this autoinflammatory-associated gene could act as a phenotypic modifier gene in PsA patients by increasing the risk of joint destruction. Given the small number of PsA patients with joint surgery included, we consider our findings a new hypothesis that will need further testing.

Deafness on the island of Providencia - Colombia: different etiology, different genetic counseling.

Providencia is a small island located in the Caribbean Ocean, northwest of Colombia with an unusually high frequency of individuals with hearing loss (5 in 1,000) is present. The hearing loss in the island was characterized as non-syndromic autosomal recessive deafness accounting for 47% (8/17) of the deaf population, Waardenburg Syndrome (deafness associated with pigmentary anomalies) for 29% (5/17), and the remaining 24% (4/17) are cases of sporadic non-syndromic deafness. For appropriate genetic counseling a complete pedigree of families with deaf individuals was constructed. The 35delG mutation in GJB2 gene, which encodes connexin 26 (Cx26), is responsible for the deafness observed in the 8 individuals with autosomal recessive non-syndromic hearing loss. The deaf individuals with Waardenburg Syndrome and the sporadic cases did not have this mutation. Therefore, we present here an atypical case of an isolated community with at least two different genetic etiologies for deafness: non-syndromic genetic deafness caused by the 35delG mutation in the GJB2 gene and deafness associated with Waardenburg Syndrome not related to GJB2. In a small and isolated population, it is feasible to assume that the deafness is caused by the same factor; however, Providencia is an atypical case. Therefore, it is extremely important to define the exact etiology of deafness in each case, since different etiologies require different genetic counseling.

Venous thromboembolism in patients with liver diseases.

Essentials Emerging evidence shows that patients with liver disease are not protected from thrombotic events. We assessed the risk of venous thromboembolism (VTE) in patients with liver disease. The presence of VTE resulted in an increase in mortality for patients with liver disease. Hospitalized patients with moderate-severe liver disease had low risk of VTE during admission. SUMMARY: Background and Aims Patients with liver disease were traditionally believed to be protected against development of blood clots, but some studies have shown a potential increased risk of venous thrombotic complications. We assessed the risk of venous thromboembolism (VTE) in patients with liver disease. Methods Data in discharge reports of patients with liver disease and control patients without liver disease were analyzed from the national inpatient sample. Incidence of VTE was compared in patients with mild, moderate-severe or no liver disease, and the impact on in-hospital mortality and length of stay was calculated. Results The overall incidence of VTE for patients with no liver disease, mild liver disease and moderate-severe liver disease was 2.7, 2.4 and 0.9 per 100 patient discharges, respectively. In the presence of VTE, in-hospital mortality was 10.8%, 5.8%, and 21.7% for the no liver disease, mild disease and moderate-severe liver disease, respectively. The presence of VTE resulted in an increase in mortality for patients with no liver disease (OR, 1.16; 95% CI, 1.14-1.18) and moderate-severe liver disease (OR, 1.63; CI 95%, 1.42-1.88). Conclusions Patients with moderate-severe liver disease have a lower risk of VTE than those without liver disease. Development of thrombosis during admission increased the risk of in-hospital mortality.

Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.

Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.

Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.

During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation.

Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

Comparison of CT during arterial portography and MR during arterial portography in the detection of liver metastases.

OBJECTIVE: This study compared MR during arterial portography (MRAP) with CT during arterial portography (CTAP) with regard to the detection and differentiation of liver metastases before surgery. MATERIALS AND METHODS: Fifteen patients with liver metastases were enrolled before surgery according to the guidelines of our institutional review board and good clinical practice. After mesentericography, unenhanced scans (Volume Zoom) were performed initially. For CTAP, the contrast medium was injected through the superior mesenteric artery. Images were acquired in portal and delayed enhancement. The MR protocol (1.5 T; Magnetom Symphony) started with T1-weighted fast low-angle shot (FLASH) T2-weighted turbo spin echo (TSE). MRAP followed with gadolinium-enhanced dynamic T1-weighted 3D FLASH. Delayed-phase T1-weighted 2D FLASH axial images were performed 2 min after IV injection of the contrast medium. Qualitative and quantitative evaluation of CTAP and MRAP was performed by three blinded radiologists regarding the number of lesions and their size, localization, and differential diagnosis. RESULTS: The overall sensitivity in detecting liver metastases was 97% with MRAP and 93% with CTAP (p > 0.05, not significant [n.s.]). The specificity was calculated to be 97% for MRAP and 82% for CTAP (p < 0.0001, statistically significant [s.s.]). The differences in sensitivity were more accentuated if only lesions 10 mm or smaller were considered (95% vs 88%, p > 0.05, n.s.), for which the respective specificities were 95% and 80% (p < 0.0014, s.s.). Improvements in sensitivity and specificity were associated with a higher lesion-to-liver contrast-to-noise ratio (59.4 +/- 51.0 for MRAP vs 10.4 +/- 7.3 for CTAP) and resulted in higher diagnostic confidence in the differential diagnosis of liver lesions (p < 0.001, s.s.) and better interobserver agreement (median kappa value, 0.88 vs 0.63). CONCLUSION: MRAP proved to be a reliable method in the preoperative detection of small liver metastases in particular, with a higher sensitivity and specificity than CTAP. If organizational difficulties of MRAP can be overcome, MRAP could be considered instead of CTAP in the preoperative invasive evaluation of metastatic liver disease.

A new cis element is involved in the HER2 gene overexpression in human breast cancer cells.

The HER2 proto-oncogene product is overexpressed in 30% of breast cancers, and this correlates with poor prognosis. Increased levels of HER2 mRNA in breast cancer cell lines result from increased gene transcription. We report the identification of a new 17-bp-long cis sequence located between positions -506 and -489 from the transcription start site. This sequence is recognized by a trans-activating factor that we tentatively named HER2 transcription factor (HTF). This factor, involved in the increased transcription of the HER2 gene in the BT-474 mammary tumor cells, has a molecular weight of about Mr 50,000. HTF can also bind, but with a lower affinity, to a related cis sequence present in the epidermal growth factor receptor promoter. Interestingly, the HTF binding activity is high in nuclear extracts from several mammary tumor cells overexpressing the HER2 gene.

Fos expression induces growth arrest in tumorigenic neuroretina cells.

E26-infected chicken neuroretina (CNR) cells expressing P135gag-myb-ets are non-transformed and growth can be stimulated by basic fibroblast growth factor (bFGF). In contrast, MHE226-infected CNR cells, which express p61/63myc in addition to the P135gag-myb-ets of E26, are transformed, tumorigenic cells and are growth inhibited by bFGF. We looked for differences in both cells types which could help to understand the molecular basis of the bFGF response. We found marked differences in c-fos expression. In MHE226-CNR cells c-fos mRNA was induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) and bFGF, both inhibitors of MHE226-infected cell growth. In contrast, serum, a strong growth inducer, did not stimulate c-fos expression. In E26-infected cells all of these factors induced cell growth and c-fos expression. MHE226-CNR cells superinfected with v-fosFBJ-expressing retrovirus were inhibited in their growth and unresponsive to bFGF. Introduction of v-fosFBJ in E26 CNR cells transformed them but they remained sensitive to bFGF. These results suggest that fos is involved in the induced growth arrest of MHE226-CNR cells.

New human erg isoforms generated by alternative splicing are transcriptional activators.

The erg gene is a member of the ets gene family. ETS proteins have been shown to bind specifically the (GGA-A/T) motif and to transactivate via this consensus sequence. The human erg products exhibit approximately 70% homology with ETS proteins in their DNA-binding domain. We have isolated three erg cDNAs from a human fetal liver library. Two of them are different from the previously described erg-1 and erg-2 cDNAs (Rao et al., Science, 1987, 237, 635-639), in the middle of their coding sequence and in their 5' part where a novel initiation codon is introduced. These isoforms are generated by alternative RNA splicing from a single gene that leads to the inclusion or exclusion of different exon sequences. The three cDNAs expressed by an in vitro transcription-translation system direct the synthesis of proteins of approximately 38, 49 and 55 kDa. These in vitro erg products were tested for their DNA-binding activity by gel mobility-shift assays with different probes containing the ETS-specific binding site. The results indicated that all these erg isoforms are able to bind the ETS binding site in a specific manner. Our data using transient transfection assays indicate that erg protein isoforms function as transcriptional activators.

Back-mutation of the V-Ets to the C-Ets carboxy-terminal amino acids in the P135gag-myb-ets results in chicken neuroretina cells transformation and loss of basic fibroblast growth factor responsiveness.

The v-Myb, v-Ets containing E26 retrovirus (called in this work E26ABC) induces the proliferation of chicken neuroretina (CNR) cells in minimal medium, strongly stimulated by basic Fibroblast Growth Factor (bFGF) which confers on them the ability to form colonies in soft agar. V-Ets differs from its cellular counterpart c-Ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids by 16 unrelated residues as a consequence of DNA segment inversion in the viral sequence. It has been documented that this different C-terminal sequence influences DNA binding activity and specificity. Replacement in E26ABC virus of the sequence encoding the 16 v-Ets last C-terminal amino acids by the sequence encoding the 13 c-Ets-1 derived C-terminus (virus E26ABO), results in the production of a P135gag-myb-ets with modified biological properties on CNR cells. E26ABO infected CNR cells proliferate in minimal medium more efficiently than E26ABC, are unresponsive to bFGF and able to grow in soft agar. In contrast, CNR cells infected by viruses encoding Myb and Ets proteins either in the E26ABO or in the E26ABC configuration are bFGF responsive. Since Myb alone is sufficient to induce bFGF responsiveness on CNR cells, these results suggest that the c-Ets-1 C-terminus interferes with the Myb activity of the E26ABO P135gag-myb-ets protein in CNR cells.

The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors.

The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions -158 and -138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.

C-Myb acts as transcriptional activator of the quail PAX6 (PAX-QNR) promoter through two different mechanisms.

To understand the regulation of the Pax-6 gene, which plays an important role in eye development, we have characterized the promoter region of the quail Pax-6(Pax-QNR) gene. In addition to TATA and CAAT boxes, sequence analysis revealed several putative cis-regulatory elements among which three myb-responsive elements (MRE). C-myb encodes a nuclear, DNA-binding phosphoprotein that functions as transcriptional regulator. Co-transfection in quail embryo cells of the Pax-QNR/pax-6 promoter with a vector expressing the 75 kDa c-myb protein resulted in an increase in Pax-QNR promoter activity. By footprinting experiments we identified multiple binding sites for the myb protein within the promoter region. Protein containing the myb DNA-binding domain fused to the VP16-transactivation domain was fully efficient in Pax-QNR promoter transactivation, demonstrating that myb can transactivate through a direct binding on DNA. However, a myb truncated protein devoid of DNA-binding domain was also able to transactivate the Pax-QNR promoter. These results show that this promoter can be transactivated by the myb protein directly as well as indirectly. Finally we show by in situ hybridization that c-myb is strongly expressed in the developing neuroretina, simultaneously with Pax-QNR. These observations suggest that the c-myb protein may be a regulator of Pax-QNR/pax-6.

Involvement of poly (ADP-ribose)-polymerase in the Pax-6 gene regulation in neuroretina.

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.