Faculty Bibliography

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

Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector

The cysteine protease inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) inhibited the growth of the Calu-1 lung carcinoma cells and induced a prolonged cell cycle arrest in the S phase. c-Jun N-terminal kinases (JNKs) participate in cellular responses to mitogenic stimuli, environmental stresses, and apoptotic signals but its role in cell cycle checkpoint control has not been elucidated. In this report, we examined the role of JNK in LLnL-induced S phase checkpoint by overexpression of a dominant-negative mutant of JNK1 (JNK1-APF) in Calu-1 cells. Expression of high levels of JNK1-APF blocked the growth-inhibitory effects of LLnL and abrogated S phase arrest induced by LLnL. These results support the role of JNK in the activation of cell cycle checkpoint induced by LLnL

BACKGROUND, Fine-needle aspiration biopsy (FNA) has been successful in diagnosing epithelial lesions of the breast. Its role in the evaluation of spindle cell and mesenchymal lesions of the breast, which include a variety of benign and malignant conditions, is less clear. This article discusses the cytologic features and differential diagnosis of these lesions, as well as the potential diagnostic pitfalls associated with them. METHODS, FNAs of the breast, in which a spindle cell or mesenchymal component was a key or dominant feature, were retrieved. Fibroadenomas without cellular stroma and typical lipomas were excluded. RESULTS. Forty-six aspirates (0<87%) in a series of 5306 breast FNAs contained a significant spindle cell or mesenchymal component. The aspirates were classified into 4 categories: 1) reactive conditions, including 2 diabetic mastopathies, 3 granulation tissue specimens, and 7 granulomatous lesions; 2) benign neoplastic conditions, including 1 mammary hamartoma, 1 dermatofibroma, 1 fibromatosis, 2 granular cell tumors, 2 angiolipomas, and 7 cellular fibroadenomas; 3) low grade malignant neoplastic lesions, including 10 low grade phyllodes tumors; and 4) high grade malignant neoplastic lesions, including 1 metaplastic carcinoma with chondroid stroma, 1 pleomorphic liposarcoma, 2 malignant fibrous histiocytomas, 2 osteosarcomas, and 4 metastatic melanomas. A specific diagnosis was rendered in 38 cases (82<6%). The mammary hamartoma was diagnosed as fibrocystic changes; the dermatofibroma as benign spindle cell lesion, not otherwise specified (NOS); and the primary osteosarcoma as an atypical spindle cell proliferation, NOS. The reactive ductal epithelial cells in one of the granulomatous mastitis specimens, as well as the hyperplastic ductal epithelial cells in one of the phyllodes tumors, were interpreted as atypical ductal proliferation. The marked cytologic atypia displayed by one granular cell tumor was interpreted as low grade adenocarcinoma and the primary liposarcoma as poorly differentiated carcinoma. CONCLUSIONS. Breast lesions with a significant spindle cell or mesenchymal component are rarely encountered in FNA and constitute a heterogeneous group that may pose a diagnostic dilemma FNA should be the initial diagnostic procedure for investigating these lesions, as a specific diagnosis was rendered in the majority of cases. Cancer (Cancer Cytopathol) 1999;87:359-71. (C) 1999 American Cancer Society

Overexpression or activation of insulin-like growth factor I receptor (IGF-IR) has been observed in many human cancers including breast, lung, colon and gastric carcinomas. We demonstrate that inhibition of the endogenous insulin-like growth factor I receptor by stable expression of a dominant-negative IGF-IR represses the transforming activity in vitro and tumorigenicity of human lung carcinoma cells A549 in vivo. The suppression of tumorigenicity in nude mice is correlated with the induction of glandular differentiation. In addition, functional inhibition of the endogenous receptor dramatically increases the sensitivity of A549 cells to a variety of apoptotic signals including UV irradiation and proteasome inhibitors. These effects are due to the formation of a stable heterocomplex of the dominant-negative receptor with the endogenous wild type receptor which reduces the kinase activity of the latter by twofold. Thus, inhibition of the IGF-IR signaling pathway not only suppresses tumorigenicity but also enhances sensitivity to apoptosis-inducing agents. Antagonizing IGF-IR signaling by promoting tumor differentiation and enhancing sensitivity to apoptotic death are potential cancer therapeutic approaches