Faculty Bibliography

Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses

To evaluate the role of the NF-kappaB signaling pathway in oncogenic transformation, we expressed IkappaBbeta, a specific inhibitor of NF-kappaB, in two human lung adenocarcinoma cell lines, A549 and H441. Expression of IkappaBbeta significantly reduced NF-kappaB activation induced by cotransfection with p65/RelA or TNF-alpha and abrogated the basal NF-kappaB activity in A549 cells. Transfection of IkappaBbeta into A549, H441 and K-ras-transformed NIH3T3 cells suppressed anchorage-independent growth as measured by colony formation in soft agar. Anchorage-independent growth of vector-transfected A549 cells in reduced serum could be enhanced by both EGF and IGF-I. In contrast, only EGF but not IGF-I could induce anchorage-independent growth of IkappaBbeta-expressing A549 cells, suggesting that the IGF-I signaling pathway regulating growth and survival may be blocked by IkappaBbeta. Interestingly, expression of IkappaBbeta suppressed growth of A549 cells in low serum in vitro without affecting in vivo growth subcutaneously in nude mice. However, metastatic growth of IkappaBbeta-expressing A549 cells in the lungs of nude mice was significantly inhibited. These results provide evidence that NFkappaB plays an important role in anchorage-independent growth and metastatic growth of lung carcinoma cells

The two-step polymerase chain reaction (PCR) and sequencing analysis was used to analyze the immunoglobulin heavy chain variable (Ig V(H)) genes of open-chest biopsy or autopsy samples from five patients with Epstein-Barr virus-negative human immunodeficiency virus (HIV)-related lymphoid interstitial pneumonia (LIP), and the results were compared with those for Ig V(H) genes from five HIV-negative LIP patients. The findings of this study are consistent with the different immunological situations of HIV-related and HIV-negative LIP. (a) The Ig V(H)3 subgroup was underexpressed in three of five cases of HIV-related LIP. In contrast, none of the HIV-negative cases showed this abnormality. Because the Ig V(H)3 subgroup encodes the largest portion of Ig V(H) genes, a depletion of B cells expressing Ig V(H)3 genes reflects a major alteration in the B-cell compartment. (b) All HIV-related LIP cases demonstrated two or three oligoclonal populations. HIV-negative cases showed minor monoclonal or polyclonal populations, but not oligoclonal ones. These oligoclonal populations suggest the coexistence of several occult clonal B-cell populations in HIV-related LIP. (c) Some oligoclonal clones in HIV-related LIP showed mutated framework regions not demonstrated in HIV-negative clones. This degree of variation exceeds the usual mutation rate for frameworks, suggesting a role for framework residues in antigen binding. (d) The frequency of D-D fusions of minor oligoclonal clones (HIV-related LIP) is higher than that of minor monoclonal clones (HIV-negative LIP). Such D-D fusions may enhance the probability of expression of antibodies capable of binding HIV glycoproteins

Study objectives: To investigate the dramatic rise in number of Mycobacterium xenopi isolates identified in our mycobacteriology laboratory, and to determine if this increase was due to emerging clinical pathology or to changes in culture technique. DESIGN: Retrospective chart and laboratory review. SETTING: University-affiliated tertiary-care city hospital. PATIENTS: Eighty-one patients with a single culture positive for M xenopi from 1975 to 1994 (period 1), and 47 patients with two or more cultures positive from 1994 to 1998 (period 2). INTERVENTIONS: The Bellevue mycobacteriology laboratory changed the culture medium from solid Lowenstein-Jensen medium (used from 1975 to 1990) to the Septi-Check AFB System (Becton-Dickinson; Glencoe, MD; used from 1991 to 1994), to the Mycobacteria Growth Indication Tube (MGIT; Becton-Dickinson; used from 1995 to 1998). Measurements and results: We recovered 29 M xenopi isolates from 1975 to 1990, 12 isolates from 1991 to 1994, and 381 isolates from 1995 to 1998. We subsequently identified and reviewed the medical records of all 81 patients who were culture positive for M xenopi from 1975 to 1994 (period 1), and 46 patients who had two or more isolates culture positive for M xenopi from 1995 to 1998 (period 2). For period 1, 75% of the subjects were male, 80% were minority, and at least 43% were HIV positive. Only one patient had clinical M xenopi lung disease during this period. For period 2, 79% of the subjects were male, 83% were minority, and at least 58% were HIV positive; two additional patients were identified who had clinical M xenopi lung disease. CONCLUSIONS: The dramatic increase in M xenopi isolates noted in our hospital was due to a more sensitive laboratory isolation technique, rather than a true increase in clinical disease. Other hospitals utilizing MGIT systems for mycobacterial recovery should interpret positive M xenopi cultures with caution

Inherited genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in the autosomal recessive glycogen storage disease type II (GSD II). To investigate whether we could generate a functional recombinant human GAA (rhGAA) for enzyme replacement therapy, we subcloned the cDNAs for human GAA and mouse dihydrofolate reductase (DHFR) into DHFR(neg) Chinese hamster ovary cells and established a stable cotransformant that expressed rhGAA. We cultured the recombinant cells in media with progressively increasing concentrations of methotrexate and found that human GAA enzyme activity increased to over 2,000 IU per gram protein. Importantly, the human GAA enzyme activity correlated to equivalent amounts of human GAA protein by rocketimmunoelectrophoresis. We confirmed that the human GAA enzyme activity corresponded to an amplification in human GAA mRNA by Northern analysis and human GAA cDNA copy number by Southern analysis. Exposing the rhGAA to human GSDII fibroblast cells or patient's lymphocytes or monocytes resulted in uptake of the rhGAA and reversal of the enzymatic defect. Mannose-6-phosphate in the media blocked uptake. GAA -/- mice were treated with the rhGAA at 1 mg/kg, which resulted in heterozygous levels of GAA in tissues, most notably skeletal muscle, heart and diaphragm after two infusions. More importantly, after multiple infusions, hind, and fore-limb muscle weakness was reversed. This rhGAA would be ideal for enzyme replacement therapy in GSD II.

We report an illustrative case of advanced 'hut lung,' or domestically acquired particulate lung disease (DAPLD), in a recently emigrated nonsmoking Bangladeshi woman with a history of 171 hour-years of exposure to biomass smoke. She presented with symptoms of chronic cough, dyspnea, and early parenchymal lung disease. High-resolution computed tomography (CT) of the chest demonstrated numerous 2- to 3-mm nodules, sparing the pleural surface. To our knowledge, this is the first such report of CT findings in the literature. Bronchoscopy yielded typical anthracotic plaques and diffuse anthracosis with interstitial inflammation on histopathologic examination of biopsy specimens. DAPLD is potentially the largest environmentally attributable disorder in the world, with an estimated 3 billion people at risk. Caused by the inhalation of particles liberated from the combustion of biomass fuel, DAPLD results in significant morbidity from infancy to adulthood. Clinically, DAPLD manifests a broad range of disorders from chronic bronchitis and dyspnea to advanced interstitial lung disease and malignancy. While a detailed environmental history is essential for making the diagnosis in most individuals, for patients with advanced DAPLD, invasive modalities such as bronchoscopy with transbronchial biopsy and examination of bronchoalveolar lavage fluid help differentiate it from other diseases. Recognition of this syndrome and removal of the patient from the environment is the only treatment. The development of well-controlled interventional trials and the commitment of sufficient resources to educate local populaces and develop alternative fuel sources, stove designs, and ventilation are essential toward reducing the magnitude of DAPLD

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein beta (C/EBPbeta) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPbeta transcription factor. IFN-beta induced inhibitory 16-kDa C/EBPbeta in macrophages, but had no effect on C/EBPbeta expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-kappaB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPbeta, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis

New modalities of treatment for small-cell lung cancer (SCLC) are needed, because the majority of patients continue to die of disseminated disease despite an initial response to conventional chemotherapy. Abnormal surface expression of the neural-cell adhesion molecule (NCAM) has been noted to be highly associated with SCLC. We examined the ability and efficiency of a streptavidin-Protein A (ST-PA) fusion protein complexed with an anti-NCAM monoclonal antibody (Mab) to transfer biotinylated beta-galactosidase into human SCLC cell lines NCI-H69, NCI-H526, and NCI-H446. When the surface molecule NCAM was targeted with this system, more than 99% of the targeted cells internalized and exhibited beta-galactosidase activity. In addition, we evaluated cytotoxic activity against SCLC lines NCI-H69 and NCI-H526 by efficient delivery of biotinylated glucose oxidase using the same ST-PA/anti-NCAM Mab complex. Cytotoxicity of the transduced cells (SCLC) was 10-fold and 100-fold greater, respectively, than the glucose oxidase control. This system could be widely applied for specific therapy of cancer cells by targeting unique surface molecules (antigens) using the corresponding Mab/ST-PA complex to transfer a variety of effector molecules; e.g., immunotoxic compounds, into target cells with a high degree of efficiency and specificity