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Biomedical subjects

W N Rom

Publications and source records attributed to W N Rom.

At least 91 records · Page 5Linked to original sources
PubMed1995

The polymerase chain reaction in the diagnosis and evaluation of pulmonary infections.

One of the most heralded developments in basic science to reach clinical application in recent years has been the polymerase chain reaction (PCR). This technique of DNA amplification, which has already had an enormous effect on the way in which molecular biology research is done (and whose inventor, Kary Mullis, was awarded the Nobel Prize in Chemistry in 1993 in recognition of the extraordinary impact of PCR technology on scientific research generally), was quickly appreciated by clinical investigators as having potentially widespread utility in the early diagnosis of a wide range of disorders, such as inherited illnesses and infectious diseases (1). This Commentary will review the application of PCR to the diagnosis and evaluation of respiratory infections. The underlying principle guiding this Commentary is that the ideal diagnostic test for respiratory infectious disease should have the following characteristics: high sensitivity and specificity, high positive and negative predictive value, rapid turnaround time, ease of performance, reliability across samples (the same sample tested repeatedly should give the same result) and across those performing the assay (different laboratories performing the assay on the same specimen will report the same result), and low cost. It is against these criteria that PCR will be measured in this review.

AIDS-Related Opportunistic Infections↗
PubMed1995

Enhanced interleukin-8 release and gene expression in macrophages after exposure to Mycobacterium tuberculosis and its components.

Mycobacterium tuberculosis infection is accompanied by acute and chronic inflammatory infiltrates associated with necrotizing granulomas in lung tissue. The cellular infiltrate is characterized by inflammatory cells which include neutrophils, lymphocytes, and macrophages. In animal and in vitro models of mycobacterial infection, cytokines including tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin-1 beta (IL-1 beta) participate in granulomatous inflammation. We hypothesized that interleukin-3, a potent chemoattractant for neutrophils and lymphocytes, could be released by activated alveolar macrophages after exposure to M. tuberculosis or its components and contribute to granulomatous lung inflammation. A quantitative immunoassay revealed that IL-8 protein release was significantly elevated in supernatants of macrophages and in lavage fluid obtained from patients with pulmonary tuberculosis compared to normal controls. In addition, Northern blots demonstrated striking up-regulation of IL-8 mRNA in macrophages from these patients. M. tuberculosis and its cell wall components lipoarabinomannan (LAM), lipomannan (LM), and phosphoinositolmannoside (PIM) stimulated IL-8 protein release and mRNA expression in vitro from alveolar macrophages, but deacylated LAM did not. Neutralizing antibodies to TNF-alpha and/or IL-1-alpha and beta blocked 83% of the stimulation. IL-8 synthesis and release is an early response of macrophages after phagocytosis of M. tuberculosis. Its production serves to attract both acute and chronic inflammatory cells of active infection and thus participates in the process of containment of the pathogen.

Adult↗
PubMed1995

Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat.

Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to 208-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription 20-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-IL6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and IL-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals.

AIDS-Related Opportunistic Infections↗
PubMed1995

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.

Adult↗
PubMed1994

Endemic tuberculosis among homeless men in New York City.

OBJECTIVES: The purpose of the study was to describe demographic and clinical characteristics of patients at the only long-term care facility for homeless men with tuberculosis in New York City, and to evaluate the outcome of a directly observed therapy program for these men. METHODS: The study population included residents at the "tuberculosis unit" for men in the New York City municipal shelter system. A cross-sectional survey described the characteristics of 76 men in the unit during November 1991. A retrospective cohort study evaluated 104 consecutive admissions to the facility from October 1, 1990, through March 30, 1991, and determined the outcome of directly observed therapy. RESULTS: Cross-sectional survey (n = 76). The median age was 43 years (range, 25 to 60 years); 67 patients (88%) had pulmonary tuberculosis. Among 58 isolates of Mycobacterium tuberculosis, eight were resistant to one drug (14%) and an additional nine were resistant to at least two drugs (16%). A history of previous treatment was associated with an odds ratio of 5.1 for having multiple drug-resistant tuberculosis (exact 95% confidence interval, 0.8 to 53.5). Retrospective cohort (n = 104). Excluding 21 men whose care was transferred to other agencies or institutions, 39 (47%) of 83 subjects completed or were still receiving treatment after 12 months and 44 (53%) of 83 subjects failed to complete the program. CONCLUSIONS: As expected, previous treatment for tuberculosis among homeless men is associated with an increased risk of having multiple-drug resistance. A directly observed therapy program successfully treated less than half of the enrolled subjects. Increased efforts are needed to control the spread of tuberculosis among homeless individuals.

Adult↗
PubMed1994

Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis.

Sputum examination for rapid diagnosis of pulmonary tuberculosis is not always satisfactory. We examined peripheral blood with the polymerase chain reaction (PCR). Blood samples were collected from 8 consecutive patients with suspected pulmonary tuberculosis and from 18 healthy controls, half of whom were tuberculin skin-test positive. All 8 patients had evidence of circulating Mycobacterium tuberculosis DNA in the lymphocyte fraction of peripheral blood, and positive sputum cultures indicating active pulmonary tuberculosis. None of the healthy controls had positive PCR results. This PCR technique may prove useful for the rapid diagnosis of tuberculosis.

DNA, Bacterial↗
PubMed1994

Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B.

The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms. Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guérin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins. In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection. We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner. LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes. Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay. Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment. Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner. Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS. We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements.

Base Sequence↗
PubMed1994

Mycobacterium tuberculosis alters expression of adhesion molecules on monocytic cells.

The host response to Mycobacterium tuberculosis is characterized by interactions between mononuclear cells, with recruitment and fusion of these cells culminating in granuloma formation. In addition, the host response to M. tuberculosis requires CD4+ T-cell reactivity, mediated by antigen-independent as well as antigen-dependent mechanisms. Thus, we hypothesized that cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1; CD54) would participate in the response to infection with M. tuberculosis. Exposure of THP-1 cells derived from a monocyte/macrophage cell line to M. tuberculosis (1:1 bacterium/cell ratio) elicited a sustained increase (660% +/- 49% above resting level) in the expression of ICAM-1 that continued for at least 72 h. Neither the expression of vascular cell adhesion molecule 1 (VCAM-1; CD106) nor that of the integrins lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) or CR3 (CD11b/CD18) was increased to a similar extent at corresponding time points. The increase in ICAM-1 protein expression was accompanied by an increase in steady-state mRNA (Northern [RNA] analysis). Neutralizing monoclonal antibodies directed against tumor necrosis factor alpha but not interleukin 1 alpha or interleukin 1 beta substantially abrogated the response to M. tuberculosis consistent with a paracrine or autocrine response. Continuous upregulation of the expression of ICAM-1 on mononuclear phagocytes induced by M. tuberculosis may mediate the recruitment of monocytes and enhance the antigen presentation of M. tuberculosis, thus permitting the generation and maintenance of the host response.

Cell Adhesion Molecules↗
PubMed1994

Isolation of the gene for the beta subunit of RNA polymerase from rifampicin-resistant Mycobacterium tuberculosis and identification of new mutations.

Tuberculosis (TB) is one of the most important infections worldwide, with an estimated incidence of 10 million active cases per year. Rifampicin is a key component of the first-line therapy used in the treatment of tuberculosis. In Escherichia coli and Mycobacterium leprae, rifampicin has been shown to inhibit the beta subunit of RNA polymerase. The gene (rpoB) encoding this enzyme has been described in both species. We report the isolation of the homologous functional rifampicin resistance gene from M. tuberculosis. A library was constructed with 15 to 25 kb BamHI-digested DNA fragments from a rifampicin-resistant M. tuberculosis clinical isolate that was ligated into an E. coli-mycobacterial shuttle plasmid. Southern analysis of BamHI-digested DNA from 200 recombinant plasmids was performed and filters were hybridized to a 411 bp fragment of the beta subunit of RNA polymerase from M. tuberculosis. Only DNA from one plasmid (#86) hybridized, which suggested that the gene is found as a single copy per genome. This plasmid was able to transfer rifampicin resistance to sensitive M. smegmatis and thus codes for a functional genetic unit. Sequence analysis in the expected "hotspot" region in eight rifampicin-resistant M. tuberculosis strains (including one multidrug-resistant strain) revealed two novel mutations as well as others previously described.

Cloning, Molecular↗
PubMed1994

The third epidemic--multidrug-resistant tuberculosis.

We recently observed a striking increase in multidrug-resistant tuberculosis (MDR-TB) among patients admitted to the Chest Service at Bellevue Hospital Center in New York. We reviewed the laboratory susceptibility test results of 4,681 tuberculosis (TB) cases over the past 20 years, Combined resistance to isoniazid and rifampin increased from 2.5 percent in 1971 to 16 percent in 1991 with higher rates noted for individual drugs. We reviewed the medical records of 100 patients with drug-resistant TB, finding that these individuals were predominantly less than 40 years of age, minority, male, jobless, undomiciled, with a high percentage of drug abuse and human immunodeficiency virus infection. We conclude that the epidemics of AIDS and TB are complicated by a third epidemic of MDR-TB. This third epidemic requires urgent attention to achieve more rapid diagnosis, to develop new therapeutic regimens, and to address the social and hospital environment ot care for these individuals.

Adult↗
PubMed1994

Clinical utility of the polymerase chain reaction in the diagnosis of infections due to Mycobacterium tuberculosis.

OBJECTIVE: To evaluate the clinical utility of the polymerase chain reaction (PCR) in the diagnosis of infections due to Mycobacterium tuberculosis. DESIGN: Clinical specimens were assayed by PCR for the presence of the insertion element IS6110, a DNA sequence unique to the M tuberculosis complex of organisms. The PCR results were then correlated with acid-fast bacilli (AFB) smears, cultures, pathology, and clinical histories. SETTING: Bellevue Hospital, a large municipal teaching hospital. PATIENTS: Inpatients on the Bellevue Chest Service. MEASUREMENTS AND RESULTS: Sixty-five patients were evaluated. The PCR for M tuberculosis was positive in 37 patients and negative in 28. When correlated with smears, cultures, pathology, and clinical history, the sensitivity of PCR for a diagnosis of active tuberculosis (TB) was 100 percent. However, the specificity for a diagnosis of active TB was only 70 percent, as the PCR assay was positive in a number of patients with only prior, treated TB, or asymptomatic tuberculous infection. For a diagnosis of any TB infection (active, treated, or asymptomatic), sensitivity of PCR was 87.5 percent and specificity was 90 percent. CONCLUSIONS: The PCR assay for TB is extremely sensitive, but it lacks specificity for a diagnosis of active TB. Its role in clinical practice will likely be limited to well-defined situations, such as HIV-positive patients with intrathoracic adenopathy, and it may be most useful in excluding active TB from consideration in selected patients. Given the cost of the assay and the labor intensity it requires, it should not be part of the routine initial evaluation of patients with suspected pulmonary TB.

DNA, Bacterial↗
PubMed1994

Hemoptysis: prospective high-resolution CT/bronchoscopic correlation.

The precise roles of fiberoptic bronchoscopy (FOB) and computed tomography (CT) of the chest in the evaluation of patients presenting with hemoptysis have not been clearly defined. On the assumption that both procedures would likely provide unique and complementary information, a prospective study with blinded interpreters using a modified high-resolution CT technique (HRCT) and FOB was designed to evaluate 57 consecutive patients admitted to Bellevue Hospital with hemoptysis. Etiologies included bronchiectasis (25 percent), tuberculosis (16 percent), lung cancer (12 percent), aspergilloma (12 percent), and bronchitis (5 percent): in an additional 5 percent of cases, hemoptysis proved to be due miscellaneous causes, while in 19 percent hemoptysis proved to be cryptogenic. Patients with lung cancer all were at least 50 years old, smoked an average of 78 pack-years, and had less severe hemoptysis but of longer duration. All had conditions diagnosed both by HRCT and FOB. High-resolution CT proved of particular value in diagnosing bronchiectasis and aspergillomas, while FOB was diagnostic of bronchitis and mucosal lesions such as Kaposi's sarcoma. Fiberoptic bronchoscopy localized bleeding in only 51 percent of cases. The high sensitivity of CT in identifying both the intraluminal and extraluminal extent of central lung cancers in conjunction with its value in diagnosing bronchiectasis suggest that CT should be obtained prior to bronchoscopy in all patients presenting with hemoptysis.

Adult↗
PubMed1994

Pleural tuberculosis and HIV infection.

Human immunodeficiency virus infection changes the clinical presentation of tuberculosis infection with atypical radiographs and more common extra-pulmonary involvement. We retrospectively studied pleural tuberculosis in HIV-positive patients over a 5-year period. We identified 70 patients with pleural tuberculosis by positive Mycobacterium tuberculosis cultures of pleural fluid and/or pleural tissue, including 43 HIV-positive and 27 HIV-negative patients. The HIV-positive patients were significantly younger (mean age, 38 +/- 1 years in HIV-positive vs 52 +/- 3 years in HIV-negative patients, p < 0.05). There were more intravenous drug abusers in the HIV-positive group (74 vs 30 percent, p < 0.01). The HIV-positive group had significantly fewer positive tuberculin skin tests (41 percent vs 76 percent, p < 0.03). Both groups had similar pleural fluid cellularity and pleural biopsy histologic conditions, but the HIV-positive patients demonstrated significantly more acid-fast bacteria identifiable in pleural tissue (69 percent vs 21 percent, p < 0.01), and a higher incidence of positive M tuberculosis cultures of sputum (53 percent vs 23 percent, p = 0.02). Pleural tuberculosis in HIV-positive patients presented more often as a manifestation of a greater burden of microorganisms and impaired host response.

AIDS-Related Opportunistic Infections↗
PubMed1993

Normal human mesothelial cells and mesothelioma cell lines express insulin-like growth factor I and associated molecules.

Insulin-like growth factor (IGF) I has important growth regulatory functions in normal growth and development. IGF-I is also a mitogen for a number of cancer cell lines; however, its autocrine effect has not been well established. In this study, the expression of IGF-I, its receptor, and its major serum-binding protein were examined in 5 normal human mesothelial (NHM) cell samples and 11 pleural mesothelioma cell lines. All NHM cells and mesothelioma cell lines expressed IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-I receptor mRNA by either Northern blot or reverse transcription polymerase chain reaction analysis. IGF-I (0.136 +/- 0.024 ng/ml, mean +/- SEM) and IGFBP-3 (18.5 +/- 3.2 ng/ml) proteins were readily detected in the conditioned medium of mesothelioma cell lines but were not greater than corresponding measurements in that of NHM cells (IGF-I, 0.120 +/- 0.080 ng/ml; IGFBP-3, 15.9 +/- 1.3 ng/ml). Exogenous recombinant IGF-I stimulated cell proliferation of NHM cells, demonstrating the presence of a functional IGF-I receptor. Our results suggest that IGF-I may function as an autocrine growth stimulus in normal proliferating mesothelial cells, which may contribute to their malignant transformation.

Carrier Proteins↗
PubMed1993

Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure.

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.

Adult↗
PubMed1993

Cytokine gene activation and modified responsiveness to interleukin-2 in the blood of tuberculosis patients.

Selected parameters of cellular immunity relating to cytokine gene activation and responsiveness to interleukin-2 (IL-2) were analyzed in 27 patients with active pulmonary tuberculosis and no human immunodeficiency virus type 1 infection. Cytokine mRNAs were not expressed by peripheral blood mononuclear cells (PBMC) of normal controls. In PBMC of tuberculosis patients, messages for IL-1, IL-8, and tumor necrosis factor-alpha were uniformly expressed, whereas PBMC of only 5 of 18 patients expressed IL-6. PBMC of 7 patients (all of those with systemic symptoms) expressed interferon-gamma mRNA and none expressed IL-2 mRNA. Most patients' cells demonstrated IL-4 mRNA. Limiting dilution analysis of IL-2-responsive cells in PBMC revealed that tuberculosis patients had 10-fold fewer IL-2-responsive cells than did controls.

Cytokines↗
PubMed1993

Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs.

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.

Base Sequence↗