Faculty Bibliography

Idiopathic pulmonary fibrosis (IPF) is characterized by activated alveolar macrophages (AM), alveolar epithelial cell proliferation and interstitial matrix, and immune complex deposition. Spontaneous release of competence and progression-type growth factors and their associated binding proteins may contribute to the pathologic features of IPF. To study the role of insulin-like growth factor (IGF) molecules in IPF we evaluated spontaneous release of IGF-I and IGFBP-3 in bronchoalveolar lavage cells from control subjects and from patients with IPF. IGF-I levels were similar compared with those in control subjects. In contrast, IGFBP-3 was significantly increased in IPF. In situ hybridization of open lung biopsies showed IGF-I to be abundant in IPF lung tissue in alveolar macrophages, interstitial mesenchymal cells, and epithelial cells. Northern, Western ligand blotting, reverse transcription PCR, and radioimmunoassay suggested that immune complexes stimulate expression of IGFBP-3 in mononuclear phagocytes in a time- and dose-dependent manner bearing strong similarities to stimulation by LPS. These data are compatible with the hypothesis that IGFBP-3 increases the bioactivity of IGF-I derived from a variety of lung tissues contributing to the fibrosis and remodeling seen in IPF

The current tuberculosis epidemic in the United States is marked, in many areas, by high rates of noncompliance with antituberculous regimens. In response to this, a comprehensive program of medical, nursing, social services, and supervised therapy was developed at Bellevue Hospital. Most patients were referred to the on-site directly observed therapy program (DOT) located in the hospital. Patients on DOT received daily or twice weekly therapy, and were given incentives to enhance compliance. Outreach was used to track patients who missed appointments. From November 1992 through July 1993, 113 patients were referred. HIV infection, homelessness, illicit drug use, and alcoholism were common. Follow-up revealed that 11 patients were noncompliant and completely lost to follow-up; of the remaining 102, 99% achieved bacteriologic cure. Of the 102 patients who received therapy, 74 attended the Bellevue DOT clinic, 16 attended other DOT programs in the city or received medication at home, and three died of HIV-related, nontuberculous illness. Nine patients were self-medicated and judged treatment successes. We conclude that a comprehensive hospital-based tuberculosis control program is capable of achieving a high degree of success, even in a population at high risk for noncompliance

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

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

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

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