The use of small molecule high-throughput screening to identify inhibitors of the proteinase 3-NB1 interaction.

Anti-neutrophil cytoplasmic antibodies (ANCA) to proteinase 3 (PR3) are found in patients with small-vessel vasculitis. PR3-ANCA bind strongly to membrane PR3 (mPR3) that is presented by the NB1 receptor. We performed high-throughput screening using a small molecule library to identify compounds that inhibit PR3-NB1 binding. We established a human embryonic kidney (HEK293) cell-based system, where approximately 95 +/- 2% of the NB1-transfected cells expressed the NB1 receptor on the cell surface. Addition of 0.1 microg/ml human PR3 to 10(4) NB1-expressing HEK293 cells resulted in PR3 binding that was detected by immunofluorescence using a fluorescence plate reader assay. We identified 13 of 20 000 molecules that inhibited PR3 binding by >70%. Seven of 13 substances showed reproducible inhibition in four additional validation experiments. Two selected compounds (27519 and 27549) demonstrated a dose-dependent inhibition over a range from 6.25 to 100 microM as measured by the plate reader assay. We used flow cytometry as a second assay, and found that both compounds reproducibly inhibited PR3 binding to NB1-transfected HEK293 cells at 50 microM (inhibition to 42 +/- 4% with compound 27519 and to 47 +/- 6% with compound 27549 compared to the dimethylsulphoxide control). Furthermore, compounds 27519 and 27549 also inhibited binding of exogenous PR3 to human neutrophils. In contrast, the compounds did not decrease mPR3 expression on resting neutrophils, but reduced the tumour necrosis factor-alpha-mediated mPR3 increase on NB1(pos) neutrophils when present continuously during the assay. The findings suggest that small inhibitory compounds provide a potential therapeutic tool to reduce mPR3 by preventing its binding to NB1.

Chronic obstructive pulmonary disease is missed in asthmatics in specialty care in Trinidad, West Indies.

SETTING: Underdiagnosis of chronic obstructive pulmonary disease (COPD) in asthmatics attending specialty care in Trinidad, West Indies. OBJECTIVE: To determine the prevalence of COPD in diagnosed asthmatics receiving specialty respiratory care. DESIGN: In a cross-sectional study, 258 asthmatics were screened for lung function measures to examine forced expiratory volume after 1 second (FEV1), forced vital capacity (FVC) and post-bronchodilator FEV1/FVC (COPD was defined as FEV1/FVC < 70 per cent). RESULTS: Of 165 patients evaluated (response rate 64.0 per cent), 53 (32.1 per cent, 95 per centCI 25.0-39.2) had a study diagnosis of COPD and a mean FEV1/FVC of 60.12 +/- 1.2. Proportionally, more males had COPD (50.9 per cent) than asthma (24.1 per cent, P < 0.001). Patients with COPD were 10 years older than asthmatics (P < 0.001). Persons with asthma who smoked were more likely to have COPD (56.0 per cent) (OR 3.26, 95 per cent CI 1.36-7.80, P = 0.006). In both sexes, FEV1/FVC was lower among older people (P < 0.001), with a greater effect (OR 2.75, 95 per cent CI 1.00-7.56, P < 0.01) seen among men in this cross-sectional study. CONCLUSIONS: One third of diagnosed asthmatics in specialty care also have COPD. Lung function was lower among older persons. Early spirometric evaluation of elderly asthmatics who smoke can determine the presence of COPD and facilitate appropriate management.

Chronic obstructive pulmonary disease is missed in asthmatics in specialty care in Trinidad, West Indies.

SETTING: Underdiagnosis of chronic obstructive pulmonary disease (COPD) in asthmatics attending specialty care in Trinidad, West Indies. OBJECTIVE: To determine the prevalence of COPD in diagnosed asthmatics receiving specialty respiratory care. DESIGN: In a cross-sectional study, 258 asthmatics were screened for lung function measures to examine forced expiratory volume after 1 second (FEV1), forced vital capacity (FVC) and post-bronchodilator FEV1/FVC (COPD was defined as FEV1/FVC < 70%). RESULTS: Of 165 patients evaluated (response rate 64.0%), 53 (32.1%, 95%CI 25.0-39.2) had a study diagnosis of COPD and a mean FEV1/FVC of 60.12 +/- 1.2. Proportionally, more males had COPD (50.9%) than asthma (24.1%, P < 0.001). Patients with COPD were 10 years older than asthmatics (P < 0.001). Persons with asthma who smoked were more likely to have COPD (56.0%) (OR 3.26, 95%CI 1.36-7.80, P = 0.006). In both sexes, FEV1/FVC was lower among older people (P < 0.001), with a greater effect (OR 2.75, 95%CI 1.00-7.56, P < 0.01) seen among men in this cross-sectional study. CONCLUSIONS: One third of diagnosed asthmatics in specialty care also have COPD. Lung function was lower among older persons. Early spirometric evaluation of elderly asthmatics who smoke can determine the presence of COPD and facilitate appropriate management.

Clinical presentation of acute chest syndrome in sickle cell disease.

In this study the records of 45 patients with sickle cell disease involved in 63 presentations of acute chest syndrome at the Princess Margaret Hospital in Nassau, the Bahamas, between 1997 and 2001 were examined. Patients were divided into three groups on the basis of age (<13 years, 13-18 years, >/=19 years) with a view to assessing clinical presentation. The incidence of symptoms, physical signs, and laboratory findings were enumerated and significant differences between age groups determined. The data were analysed using analysis of variance, t test, and chi(2) test and compared with existing knowledge on the subject. This study proposed to evaluate the clinical presentation of acute chest syndrome with emphasis on historical and physical findings, and to encourage the physician to maintain a high index of suspicion for the condition in susceptible patients. It was found that presentation varied significantly with age groups, children presenting most classically with fever and cough and adults, with chest pain. The 13-18 age group emerged as the group which presented most frequently with the typical symptoms of chest infection, thus potentially making diagnosis easier. Of note, the most frequent finding was a normal examination, while the second commonest physical finding was crepitations on auscultation of the chest.

The interferon-inducible 204 gene is transcriptionally activated by mouse cytomegalovirus and is required for its replication.

Infection of cells with viable or UV-inactivated murine cytomegalovirus (MCMV) increased the IFN-inducible 204 gene at both the mRNA and the protein levels. The activity of a reporter gene driven by the mouse Ifi204 promoter induced following virus infection showed that this increase was due to transcriptional activation. Moreover, FACS analysis of infected mouse embryo fibroblasts (MEF) stably transfected with a p204-dominant-negative mutant (p204dmMEF) revealed that they do not accumulate at the G1/S border in the same way as infected MEF transfected with the empty vector (neoMEF). MCMV DNA synthesis is significantly delayed (144 h in p204dmMEF vs 72 h in neoMEF), due to retarded expression of viral genes, namely, IE1 and DNA polymerase, as shown by Western blot comparison of p204dmMEF and neoMEF extracts. These results demonstrate that MCMV may exploit the Ifi204 gene to regulate the cell cycle and enhance its DNA synthesis.

Are routine chest X-rays for students entering university worthwhile?

Mass radiographic screening for tuberculosis has lost favour in many countries. The aim of this study was to determine whether the continued practice of such screening of prospective students at the University of the West Indies was warranted by assessing the yield and the cost of the programme in our setting. In a cross- sectional retrospective study, 12,662 chest X-ray reports collected over the period 1989-1997 were studied. No active case of tuberculosis was detected. Three students reported a previous history of tuberculosis and 10 students had a positive family history of tuberculosis. Three hundred and ninety-nine clinically insignificant abnormalities were reported, such as mild scoliosis and calcified foci. Routine radiological screening of prospective students at the University of the West Indies for tuberculosis has an extremely low yield, places the students at unnecessary risk of radiation exposure and should be discontinued.

The retinoblastoma protein is an essential mediator that links the interferon-inducible 204 gene to cell-cycle regulation.

We have previously demonstrated that overexpression of p204, a member of the Ifi 200 gene family, inhibits growth, delays G0/G1 progression into S phase, and impairs E2F-mediated transcriptional activity. In this study, we show that p204 directly binds the retinoblastoma protein (pRb) in vivo to exert its activity. Transient p204 overexpression in Rb+/+ mouse embryo fibroblasts (MEF) inhibits cell proliferation, but does not affect cell growth in MEF derived from Rb-/- mice. Two human cell lines, Saos2 and C33A, bearing an inactive pRb, but not primary human embryo fibroblasts, are resistant to the p204 antiproliferative activity. p204 contains two 200 amino acid motifs, designated as type a or b domains, each containing a canonical Rb binding motif (LXCXE). When dominant-negative mutants at the Rb binding motif were transfected in Rb+/+ MEF, p204 lost its ability to inhibit cell growth, delay cell transition from G1 to S phase, and impair DNA synthesis. Moreover p204 overexpression in Rb+/+ MEF led to a significant decrease of both DHFR and PCNA proteins, two S phase markers. By contrast, this effect was not observed when Rb+/+ MEF were transfected with a p204 mutated at both Rb binding sites. Finally, overexpression of the LXCXE p204 mutant rendered Rb+/+ MEF resistant to the IFN-alpha antiproliferative activity, in comparison to the untransfected Rb+/+ MEF. As expected, Rb-/- cells were unsensitive to the IFN-alpha induced growth inhibition. Taken as a whole, these results suggest that (i) p204 contributes to the IFN-alpha antiproliferative activity and (ii) the primary target of p204 leading to efficient G1 arrest as well as to blockade of DNA replication from G1 phase is the pRb regulatory system.