Biomedical subjects
Publications and source records attributed to J A Roth.
Gene therapy, in the future, may offer new strategies to treat various malignancies in lung cancer. Several phase I trials have demonstrated the safety of these approaches. The limitation of current gene transfer techniques include transient gene expression, locoregional delivery, and the ability to target tumor tissue in a safe manner.
The identification of genetic lesions that lead a normal cell to become malignant presents us with the opportunity of targeting those lesions as a means of therapy. Given the key role played by the tumor suppressor gene p53 in cell cycle regulation and apoptosis, and the evidence linking p53 mutations with non-small cell lung cancer, attempts at p53 replacement are a logical approach to therapy in this disease. In a phase I study, administration of an adenoviral p53 vector (Adp53) to 21 patients with advanced non-small cell lung cancer produced little toxicity. Up to six intratumoral injections at monthly intervals were well-tolerated. Expression of the p53 transgene was evident, along with potentially useful clinical responses. Time to disease progression in the indicator lesion treated with Adp53 appears to be enhanced by higher doses of vector, concomitant cisplatin therapy, and evidence of apoptosis on tumor biopsy specimens. Phase II trials should now be undertaken to determine the response rate to Adp53.
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SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirus-expressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 x 10(5), with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors.
Alteration of the wild-type (wt) p53 gene by mutation, deletion or re-arrangement is a major factor in the development of human colon cancer. Recent studies have demonstrated that p53 might be an essential component of the apoptotic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We examined the anti-tumor effects of adenovirus-mediated wt-p53 gene transfer in combination with a chemotherapeutic drug on the human colon cancer cell line WiDr, which is homozygous for a mutation in the p53 gene. Treatment with the chemotherapeutic drug cisplatin following infection with a replication-deficient, recombinant adenoviral vector expressing wt-p53 (termed AdCMVp53) significantly suppressed the growth of WiDr cells compared to single treatments alone. To evaluate the in vivo efficacy of AdCMVp53 and cisplatin given sequentially, WiDr cells were inoculated s.c. in nu/nu mice. After 3 days, AdCMVp53 was injected s.c. into the area where tumor cells were implanted, followed by i.p. administration of cisplatin. Analysis of initial growth inhibition at 21 days demonstrated a profound therapeutic cooperativity, though administration of either AdCMVp53 or cisplatin alone was followed only by a slowing of growth. Our results suggest that gene therapy using wt-p53-expressing adenovirus in combination with a chemotherapeutic DNA-damaging drug could be a useful strategy for treating human colon cancer.
The p21WAF1/CIP1 gene, which encodes a cyclin-dependent kinase inhibitor, may be critical for tumor suppressor gene p53-induced cell cycle arrest. The p53 gene is known to regulate G1 checkpoint, which can either induce G1 arrest or initiate apoptosis. To directly examine the role of p21WAF1/CIP1 in the control of p53 function, we have introduced human p21WAF1/CIP1 gene into a p53-deficient human non-small cell lung cancer cell line H1299 using a p21WAF1/CIP1-expressing adenoviral vector (AdCMVp21). Infection with AdCMVp21 resulted in high levels of p21WAF1/CIP1 expression and significantly suppressed the growth of H1299 cells through the G1 arrest of the cell cycle. In contrast, transient expression of the wild-type p53 gene by a recombinant adenoviral vector (AdCMVp53) in H1299 cells induced apoptotic cell death and resulted in a rapid loss of cell viability. We then examined the effects of combined infection with AdCMVp21 and AdCMVp53 on H1299 cells to explore the dominant function of these molecules. Interestingly, introduction of exogenous p53 overcame p21WAF1/CIP1-mediated cell cycle arrest at G1 and induced apoptosis, although viral-transduced p21WAF1/CIP1 expression level was unaffected. These observations suggest that p53 expression converts a p21WAF1/CIP1-mediated growth arrest into apoptosis. The result was repeated with two additional human colon adenocarcinoma cell lines with the different p53 status, mutant p53-expressing DLD-1 and wild-type p53-expressing LoVo, suggesting that this phemonenon is a general event among human cancer cells. Thus, p53-mediated apoptotic pathway is dominant over the growth arrest pathway, indicating that p53 may be an essential upstream mediator of p21WAF1/CIP1 in the regulation of a cell process leading either to growth arrest or to apoptotic suicide.
A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with bovine neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3',5,5'-tetramethylbenzidine as a substrate. The assay was validated by evaluating the effects of various stimulants and inhibitors of degranulation, the kinetics of primary granule exocytosis, and comparing the total myeloperoxidase content of neutrophils obtained from calves and adults. The results demonstrate that the assay is capable of detecting important differences that may occur in degranulation of bovine neutrophil primary granules and in total neutrophil myeloperoxidase content.
Melatonin has previously been reported to influence cell differentiation and growth in a number of cell culture systems in vitro. In this paper, we describe the effects of high pharmacological and low physiological concentrations of melatonin on cell growth in rat pheochromocytoma cells (PC12 cells). Melatonin produced a biphasic response with respect to cell growth in PC12 cells. At low concentrations (1-10 nM) melatonin suppressed PC12 cell growth whereas at higher concentration (10 microM) it prevented cell death. Cultures treated with high concentrations of melatonin displayed an increase in cell number and a decreased release of lactic acid dehydrogenase (LDH) into the culture media, indicating that melatonin was enhancing cell survival as opposed to stimulating cell proliferation. Inhibition of cell death by high concentrations of melatonin was both time and concentration-dependent and did not require the continued presence of melatonin throughout the entire time of incubation. These studies suggest melatonin is preventing either apoptosis or programmed cell death. In contrast, concentrations of melatonin (1-10 nM) at or near the binding affinity for the nuclear receptor, RZRbeta, suppressed PC12 cell growth. At these concentrations, melatonin failed to inhibit forskolin-induced cAMP formation and process outgrowth as well as prevent forskolin suppression of cell growth. These data indicate that PC12 cells probably lack functionally active cell surface receptors for melatonin and suggest the interaction of melatonin with the nuclear receptor may be responsible for suppression of PC12 cell growth.
2-Methoxyestradiol (2-MeOE2) treatment caused significant growth inhibition of H460 and A549 human lung cancer cell lines which contain wild-type p53. However, 2-MeOE2 had a little effect on the p53 negative H358 and p53 mutated H322 cell lines. Western blot analysis indicated that 2-MeOE2 treatment resulted in an eightfold increase in the endogenous wild-type p53 protein, while the level of the mutant p53 protein remained unchanged. TdT staining indicated that following 2-MeOE2-mediated increases in wildtype p53 protein, cells bypass the G1-S checkpoint of the cell cycle with 30 to 40% undergoing apoptosis. Introduction of anti-sense wt-p53 into wt-p53 cells abrogated the 2-MeOE2 effect. A significant portion of lung cancer retains the wild-type p53 gene therefore, 2-MeOE2 may have therapeutic application.
Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.
Supernatants from bovine immunodeficiency-like virus (BIV)-infected cells have been previously shown to affect monocyte random migration, chemotaxis, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. The experiments in this report demonstrate that the BIV Gag (core) proteins can enhance monocyte random migration, chemotaxis, and phagocytosis. Supernatants from BIV-infected cells contained 10-30 and 30-50 kDa proteins, which significantly (p < 0.05) increased monocyte chemotaxis. The 30-50 kDa protein(s) could be cleaved by limited proteolysis into 10-30 kDa active components. Affinity purification with monoclonal anti-p26 (capsid) antibodies yielded preparations that were active in the random migration, chemotaxis, and phagocytosis assays, but did not affect ADCC. Furthermore, activity of the affinity purified preparation could be specifically neutralized by hyperimmune rabbit serum against BIV Gag proteins. A recombinant Gag protein, consisting primarily of BIV p26, also enhanced monocyte random and chemotactic migration. It appears, therefore, that direct treatment with affinity-purified BIV Gag proteins or a recombinant Gag protein, is able to significantly affect the function of normal monocytes in vitro. Factors affecting monocyte migration and phagocytosis appear to be one or more breakdown products of the BIV Gag precursor, particularly those containing the p26 (capsid) protein.
Sotalol's usefulness in treatment of atrial fibrillation and atrial flutter is unproven. This study evaluated (1) the efficacy of sotalol in preventing recurrences of paroxysmal atrial fibrillation or atrial flutter and controlling ventricular rate (in chronic atrial fibrillation or relapse of paroxysmal atrial arrhythmias), (2) the safety of sotalol, and (3) predictors of sotalol efficacy. Thirty-three patients, 28 with paroxysmal and five with chronic atrial fibrillation or atrial flutter, received an average dose of 265 +/- 119 mg of oral sotalol per day. During a 10 +/- 12 month follow-up, recurrence rate for paroxysmal arrhythmia was 64%, with a 50% recurrence at 4.6 months. For patients with chronic atrial fibrillation, ventricular rates were well controlled with sotalol administration (136 +/- 33 beats/min versus 88 +/- 23 beats/min; p = 0.04). No patient with chronic atrial fibrillation converted to sinus rhythm during the study. Side effects necessitated sotalol discontinuation in three patients. By multivariate analysis, younger age, higher ejection fraction, and absence of hypertension independently predicted sotalol efficacy.
BACKGROUND: Thoracic surgeons typically perform fiberoptic bronchoscopy (FOB) before thoracotomy, usually on the day of the operation after intubation with a single-lumen endotracheal tube (ETT) and before insertion of a double-lumen ETT. This routine requires two laryngoscopies and two intubations. The laryngeal mask airway (LMA) is an airway device developed in England and approved by the Food and Drug Administration in 1991 for clinical use in the United States. It requires neither mask ventilation nor laryngoscopy and allows FOB visualization of the epiglottis, larynx, and entire trachea. We assessed the LMA as an alternative to a single-lumen ETT for FOB before thoracotomy. METHODS: Through prospective assessment, 50 patients underwent FOB after insertion of an LMA before thoracotomy. Pulse rate, blood pressure, ease of insertion of the LMA, quality of FOB, and complications of LMA insertion were assessed. RESULTS: During LMA insertion, blood pressure and pulse rate increased less than 5% from baseline in all patients. The LMA was inserted successfully in all patients within 10 seconds. No complications occurred as a result of LMA insertion. CONCLUSIONS: Insertion of the LMA causes minimal hemodynamic response. From the time of induction of general anesthesia, insertion of the LMA is quick, simple, and safe and eliminates the need for endotracheal intubation with a single-lumen ETT before double-lumen tube insertion. The LMA, in contrast to the ETT, allows a complete survey of the larynx and trachea. The LMA is autoclavable, reusable, and cost effective. Therefore, in patients who require FOB immediately before thoracotomy, LMA use should be the standard for airway evaluation.
BACKGROUND: Multidisciplinary surgical teams enable an aggressive approach to tumors involving the thoracic spine. METHODS: From February 1994 to July 1996, 61 patients underwent anterior resections of thoracic spine tumors. Their median age was 56 years. The indications for operation were curative in intent in 7 of 61 and palliative in 54 of 61 (to relieve intractable metastatic bone pain with neurologic compromise [n = 38] and pain alone [n = 16]). Sixteen patients came to our institution unable to ambulate with impending paraplegia. RESULTS: Anterior approaches included combined left side of the neck and median sternotomy for lesions involving vertebrae T-1 through T-3 (n = 9), posterolateral thoracotomy for T-3 through T-10 (n = 39), and thoracoabdominal approach at T-11 and T-12 (n = 13). Median hospital stay was 9.0 days (range, 4 to 57 days). Complications occurred in 18 of 61 (29.5%). In 55 of 61 (90%), pain was significantly improved after the operation. Twelve of the 16 patients who initially presented in wheelchairs regained ambulatory function. There were five perioperative deaths (8.2%). The 1-year cumulative survival for the entire group was 60%. CONCLUSIONS: An aggressive surgical approach in cancer patients with locally advanced or metastatic disease in the thoracic spine was associated with acceptable morbidity and mortality. There was significant improvement in their quality of life by control of intractable pain in 90% and recovery of ambulatory function in 75% of patients who presented with critical spinal cord compromise.
The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.
Conventional programmed electrical stimulation (PES) of the ventricle is useful for establishing inducibility or noninducibility of clinical ventricular arrhythmias (VA) but is complex and time consuming. The present study was designed to compare a standard PES protocol with an alternative method using ultrarapid train stimulation in patients with VA and coronary artery disease (CAD). A prospective, randomized, crossover design was used. During each session in the electrophysiology laboratory, patients were studied using both the trains and PES protocols in randomized order. In 82 matched pairs of comparisons in 50 patients, results were concordant in 85% (p < 0.0001). There were no differences related to type of clinical arrhythmia or to the presence of antiarrhythmic drugs. There were no significant differences in the induction of nonclinical arrhythmias with the two methods (p < 0.0001 for concordance). There were no significant differences related to the cycle length of the trains (10, 20, or 30 ms, equivalent to 100, 50, or 33 Hz). The number of drive-extrastimuli sequences and the time required to complete the trains protocol was significantly shorter (p < 0.0001) using trains versus PES. Ultrarapid train stimulation provides results in CAD patients that are comparable with those of conventional PES protocols. There is a significant savings in time, adding practical value to intrinsic electrophysiologic interest. Trains may be useful when multiple inductions are desirable, for example, in the setting of antitachycardia pacing parameters in an implantable defibrillator (ICD), during ICD implantation, or in other circumstances where the main question is inducibility of ventricular arrhythmias.
Pectoral ICD implantation, although feasible with the release of smaller devices, can be cosmetically disturbing to some patients due to the device protruding under the skin. An ICD was implanted using the retromammary approach in a 25-year-old female patient. Retromammary implantation of an ICD is feasible and offers an alternative approach in women.
The adenovirus E4 promoter was replaced by a synthetic promoter composed of a minimal TATA box and five consensus 17-mer yeast GAL4-binding-site elements. The viral vectors, which also contained human factor IX (hFIX) cDNA driven by Rous sarcoma virus long terminal repeat in the E1 region, were then constructed and expanded in 293 cells permanently expressing GAL4/VP16 fusion protein. Viral replication and expression of adenovirus E4 genes and late genes (hexon and fiber) were evaluated in vitro in the human lung carcinoma cell line H1299. Viral replication and viral gene expression were dramatically reduced in the cells transduced by vectors with a replaced E4 promoter compared to the levels in the cells transduced by vectors with the wild-type E4 promoter. The levels of transgene (hFIX) expression remained similar between vectors with or without E4 promoter replacement. These results indicate that diminution of viral gene expression and viral replication is achievable by promoter replacement.