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Biomedical subjects

J A Roth

Publications and source records attributed to J A Roth.

At least 271 records · Page 15Linked to original sources
PubMed1993

Characterization of protective antigens and the protective immune response.

The protective immune response to a vaccine may be due to the presence of circulating antibody (humoral immunity), the actions of sensitized T-lymphocytes (cell-mediated immunity), the presence of secretory IgA on mucosal surfaces (mucosal immunity), or a combination of these factors. In general, humoral immunity is felt to be particularly important in protection against extracellular phases of systemic viral and bacterial infections and in protection against toxin-induced diseases. Cell-mediated immunity is particularly important in protection from facultative intracellular bacterial pathogens, intracellular viral infections, fungal diseases, and protozoal diseases. Secretory IgA is important in protecting against those bacterial and viral diseases where the organism must attach to epithelial surfaces in order to produce disease and against diseases induced by toxins produced at mucosal surfaces. To characterize the protective immune response it is necessary to characterize specific protective antigens, to determine the onset and duration of the response, and to demonstrate which aspects of the immune response are responsible for protection. This information is needed to be sure that an in vitro potency assay is relevant to in vivo protection and to be sure that the relevant immune response is being measured when requalifying reference antigens. Determining which component(s) of the immune response is actually responsible for protection may be difficult, particularly for diseases where protection is only partial or where multiple components of the immune response contribute to protection.

Animals↗
PubMed1993

Targetted phototherapy with sensitizer-monoclonal antibody conjugate and light.

Photodynamic therapy (PDT) was performed in vitro and in vivo using monoclonal antibody conjugated to hematoporphyrin (HP). The antibody (45-2D9) recognized a cell surface glycoprotein on cells derived from NIH 3T3 cells which were transformed with the ras oncogene (45-342). Radionuclide imaging with either In111 or I125 chelated to 45-2D9 or the isotype identical (IgG1) antibody MOPPC-21 revealed selectivity of 45-2D9 for 45-342 flank tumours in nude mice, and minimal targetting for a 45-342 clone which did not express the cell surface glycoprotein. The 45-2D9-HP conjugate resulted in selective killing of the 45-342 line compared with the parent line in vitro. At HP concentrations of 76 micrograms ml-1, the 45-2D9-HP conjugate resulted in significantly more long-term cures of PDT treated flank tumours compared with free HP at the same concentration. 45-2D9 alone had no effect on tumour growth. The antibody-HP conjugate resulted in significantly less local toxicity compared with standard Photofrin II PDT, and also achieved a greater number of long-term cures. This 'photoimmunotherapy' demonstrates the ability to treat established tumours with greater efficacy and decreased morbidity, probably due to specific sensitizer targetting which allows normal surrounding tissue to be spared upon illumination.

3T3 Cells↗
PubMed1993

Retroviral vector-mediated transduction of K-ras antisense RNA into human lung cancer cells inhibits expression of the malignant phenotype.

A retroviral vector system was developed to transduce a K-ras antisense construct efficiently into human cancer cells. A 2-kb fragment of K-ras gene DNA in antisense orientation was linked to a beta-actin promoter and inserted into retroviral vector LNSX in two different orientations. The constructs were transfected into amphotropic packaging cell line GP+envAm12 followed by alternating transduction between the ecotropic packaging cell line psi-2 and GP+envAm12. Titers up to 9.7 x 10(7) colony-forming units (cfu)/ml were achieved without detectable replication-competent virus. The human large cell lung carcinoma cell line H460a, which has a homozygous codon 61 K-ras mutation, was transduced with an efficiency of 95% after five to seven repeated transductions. DNA polymerase chain reaction (PCR) and genomic DNA Southern blot analysis showed that the retroviral construct was integrated into the genome of H460a cells. K-ras antisense RNA expression was detected in the cells by Northern analysis, slot blot hybridization, and reverse transcriptase-PCR. Translation of the mutated K-ras p21 protein RNA was specifically inhibited, whereas expression of other p21 species was unchanged. Proliferation of H460a cells was suppressed 10-fold following transduction by the antisense construct. Colony formation in soft agarose and tumorigenicity in an orthotopic lung cancer model in nu/nu mice were dramatically reduced in H460a cells expressing antisense K-ras. We conclude that an antisense construct for K-ras can be expressed effectively in a retroviral vector that can efficiently transduce human cancer cells.

3T3 Cells↗
PubMed1993

Stable expression of the wild-type p53 gene in human lung cancer cells after retrovirus-mediated gene transfer.

A retroviral vector-mediated system was established to allow efficient transduction of the wild-type p53 gene into human lung cancer cell lines H358a (deleted p53) and H322a (mutant p53). LNSX/p53 constructs incorporating p53 cDNA driven by a beta-actin promoter mediated stable integration of p53. p53 mRNA and protein were detected in these cell lines 6 months after transduction by Northern and Western blot analyses. Restoration of the wild-type p53 gene suppressed growth in the two transduced cell lines but had no effect in another transduced tumor cell line, H460a, which has an endogenous wild-type p53 gene. A high transduction efficiency was obtained in cell lines H460a, H322a, and H358a after five cycles of transduction in vitro. Mixing experiments showed that transduced cells could reduce the growth rate of nontransduced cells; this reduction may have been mediated by factors shed into the supernatant of the transduced cell cultures.

Base Sequence↗
PubMed1993

Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.

The polymerase chain reaction was used to detect and characterize low-abundance bovine leukemia virus (BLV) mRNAs. In infected cattle we could detect spliced mRNA with a splice pattern consistent with a Tax/Rex mRNA, as well as at least four alternatively spliced RNAs. Two of the alternatively spliced mRNAs encoded hitherto unrecognized BLV proteins, designated RIII and GIV. The Tax/Rex and alternatively spliced mRNAs could be detected at their highest levels in BLV-infected cell cultures; the next highest levels were found in samples from calves experimentally infected at 6 weeks postinoculation. Alternatively spliced mRNAs were also expressed, albeit at lower levels, in naturally infected animals; they were detected by a nested polymerase chain reaction. Interestingly, the GIV mRNA was specifically detected in naturally infected cows with persistent lymphocytosis and in two of five calves at 6 months after experimental infection with BLV. Furthermore, the calf with the strongest signal for GIV had the highest lymphocyte counts. These data may suggest a correlation between expression of the GIV product and development of persistent lymphocytosis. Some of the donor and acceptor sites in the alternatively spliced mRNAs were highly unusual. The biological mechanisms and significance of such a choice of unexpected splice sites are currently unknown.

Alternative Splicing↗
PubMed1993

Identification of transactivation-response sequences in the long terminal repeat of bovine immunodeficiency-like virus.

Transient expression assays using the reporter gene that encodes chloramphenicol acetyltransferase were used to identify cis-acting sequences necessary for bovine immunodeficiency-like virus (BIV) transactivation. Computer analyses identified two RNA stem-loop structures located immediately downstream of the transcription start site in the long terminal repeat. Deletion analysis of the long terminal repeat indicated that sequences containing the proximal stem-loop structure located between +4 and +31 are required for virus-specific transactivation. Therefore, BIV likely utilizes a mechanism of transactivation similar to that of the human and simian lentiviruses.

Animals↗
PubMed1993

Evaluation of the influence of potential toxins on neutrophil function.

Neutrophils undergo a complex series of events when functioning to control bacterial, viral, and fungal infections. Any factor that interferes with neutrophil production or suppresses neutrophil function will rapidly make the animal more susceptible to infectious diseases. In vitro assays are used to detect and characterize defects in neutrophil function that may be induced by exposure to potential toxins. This paper focuses on important considerations in designing experiments for the evaluation of neutrophil function and interpretation of data.

Animals↗
PubMed1993

Intensive preoperative chemotherapy with colony-stimulating factor for resectable adenocarcinoma of the esophagus or gastroesophageal junction.

PURPOSE: The curative resection rate in patients with potentially resectable carcinoma of the esophagus is approximately 55% and their median survival time is 11 months. Preoperative chemotherapy with high doses of chemotherapeutic agents was used to evaluate clinical and pathologic responses, curative resection rate, toxicity, and survival. Colony-stimulating factor (CSF) was added to reduce the severity of myelosuppression. PATIENTS AND METHODS: Twenty-six consecutive assessable patients with potentially resectable adenocarcinoma of the esophagus or gastroesophageal junction were treated with two preoperative courses of intensive chemotherapy (etoposide, doxorubicin, and cisplatin [EAP]) with granulocyte-macrophage CSF (GM-CSF). Additional three conventional-dose postoperative chemotherapy courses without GM-CSF were given to patients who responded to preoperative chemotherapy. RESULTS: A median of three courses (range, one to six), were administered. Of 27 patients, 26 were assessable for response to preoperative EAP; 13 (50%) achieved a major response. Among 23 patients who underwent surgery, 15 (65%) had a curative resection (58% of 26 assessable patients); none of the patients had a pathologic complete response, but two patients had only microscopic carcinoma in the resected specimen. Six patients had carcinoma present at the resection margins and received postoperative radiotherapy. Two patients were found to have liver metastases at exploration. At a median follow-up of 22 months, the median survival of 26 patients was 12.5 months (range, 2 to 32 +). Fourteen patients died of their carcinoma; two patients died of treatment-related causes; one died of an unrelated CNS arterial malformation; and the causes of death in two patients remain unknown. Seven patients are alive with no evidence of relapse. Major toxicities of this regimen included severe myelosuppression, nausea and vomiting, infections, and severe constitutional symptoms related to GM-CSF. However, subcutaneous injection of GM-CSF was well tolerated. CONCLUSION: High-dose EAP is active against locoregional adenocarcinoma of the esophagus and gastroesophageal junction but can be associated with significant toxicity. Although this strategy remains attractive and needs to be developed further, less toxic and more effective regimens need to be identified.

Adenocarcinoma↗
PubMed1993

Binding of cellular proteins to a conformational domain of tumor suppressor protein p53.

The genes regulated by p53, as well as the factors modulating its function, need to be identified before the mechanism of action of p53 in control of cell growth can be adequately understood. Binding of the SV40 large T-antigen protein to an evolutionally conserved (conformational) domain of p53 inhibits p53's DNA-binding and transcription activation activities. Cellular proteins might also bind to this same region of p53 to regulate its function. A hybrid protein composed of protein A fused to the conformational domain (amino acids 115-295) of p53 was expressed in Escherichia coli and used as an affinity probe for binding proteins in detergent lysates of non-small cell lung carcinoma (NSCLC) cells. The wild-type p53 hybrid protein associated with several major proteins of molecular weights 45 K, 56 K, and 70 K, as well as other minor species ranging in molecular weight from 30 K to 90 K. These proteins bound specifically to the p53 sequence of the hybrid protein. Protein A did not associate with these proteins and the two p53 hybrid proteins containing missense mutations at codons 273 and 175 exhibited a 40-80% weaker association. In addition, T antigen competed with the cellular proteins for binding to the conformational domain. The conditions of cell growth had a profound effect on the expression of the p53 binding proteins. Considerably more p53 binding proteins were expressed in actively growing cells than in cultures maintained under conditions for slow growth. Quantitative differences in expression of p53-binding proteins were observed among different NSCLC cell lines. The expression of p53-binding proteins was not restricted to NSCLC cell lines; detergent extracts of an osteosarcoma cell line yielded similar p53-binding proteins.

Antigens, Polyomavirus Transforming↗
PubMed1993

Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis.

A simplified method for generating recombinant adenovirus was developed by using liposome-mediated co-transfection and by directly observing for the cytopathic effect caused by recombinant adenovirus in transfected 293 cells. This approach avoided difficulties associated with calcium-phosphate precipitation and agarose overlays for plaque assays. The ease of generating recombinant adenovirus was considerably improved. Analysis by PCR of DNA samples from the supernatant of the cell cultures with the cytopathic effect was also developed, which made identification of any newly generated recombinant virus rapid and specific.

Adenoviruses, Human↗
PubMed1992

Comparison of left ventricular cryolesions created by liquid nitrogen and nitrous oxide.

OBJECTIVES: This study was designed to compare the cryosurgical lesions produced by liquid nitrogen (-196 degrees C) and nitrous oxide (-76 degrees C). BACKGROUND: Cryosurgical ablation is a useful method of arrhythmia surgery, but information on the dimensions of cardiac lesions produced by modifying cryoprobe temperature is limited. METHODS: We compared the dimensions, volumes and electrophysiologic effects of cryolesions created by a liquid nitrogen cryoprobe (Group I) and a nitrous oxide cryoprobe (Group II) on the left ventricular myocardium in the beating canine heart. Exposure time was compared at 1, 2, 3 and 4 min. In each of 18 dogs, two to four lesions were created on the left ventricle and analyzed: 35 lesions created with use of the nitrous oxide cryoprobe and 30 lesions created with the liquid nitrogen cryoprobe. Lesions were measured at the time of induced death 6 weeks postoperatively and assessed by tissue staining with the Masson trichrome technique. RESULTS: The volumes (mm3) of the cryolesions created by the liquid nitrogen cryoprobe were significantly larger (p < 0.05) than those of lesions created by nitrous oxide: 826 +/- 163 versus 493 +/- 197 at 1 min; 1,101 +/- 327 versus 666 +/- 185 at 2 min; 1,356 +/- 318 versus 787 +/- 258 at 3 min and 1,735 +/- 534 versus 923 +/- 376 at 4 min. CONCLUSIONS: Decreasing the temperature of the cryoprobe by using liquid nitrogen increases the volume of the lesions. Programmed electrical stimulation before and 6 weeks after cryoablation indicated no arrhythmogenicity.

Animals↗
PubMed1992

Rapid suppression of spontaneous ventricular arrhythmias during oral amiodarone loading.

OBJECTIVE: To determine the time course of effects of amiodarone during an oral loading period. DESIGN: A prospective, nonrandomized study. SETTING: Arrhythmia referral center at a university hospital. PATIENTS: Fifty patients with refractory sustained ventricular tachycardia (n = 44) or ventricular fibrillation (n = 6) and frequent (greater than or equal to 30/h) ventricular premature complexes. INTERVENTION: Oral amiodarone, 1200 mg/d for 14 days and 400 mg/d thereafter. MEASUREMENTS: Ambulatory electrocardiographic monitorings, 12-lead electrocardiograms, and amiodarone blood levels on days 3, 5, 7, 9, 11, 13, and 28. RESULTS: Dramatic reductions of ventricular arrhythmias were noted during the first 72 hours of the therapy. Average ventricular premature complexes/h, couplets/h, and nonsustained ventricular tachycardias/24 h were 524 +/- 1224/h, 16 +/- 61/h, and 167 +/- 611/24 h, respectively, at baseline, and reduced to 140 +/- 243/h, 11 +/- 50/h, and 33 +/- 117/24 h, respectively, on day 3 (P less than 0.05 for all). Subsequent reductions of ventricular arrhythmias from day 3 to day 13 were more gradual but were still significant (P less than 0.05). A significant reduction of ventricular arrhythmias (greater than or equal to 70% reduction of ventricular premature complexes and greater than or equal to 90% reduction of nonsustained ventricular tachycardias) was noted in 50% of patients on day 3, in 65% on day 7, and in 83% on day 13. Prolongation of the QT interval exhibited a similar time course. There were no further differences in reduction of ventricular premature complexes or QT intervals between day 13 and day 28. CONCLUSIONS: Oral amiodarone given in loading doses produces rapid and dramatic reductions in spontaneous ventricular arrhythmias within 72 hours. Subsequent reductions of spontaneous arrhythmia were gradual and less dramatic.

Administration, Oral↗
PubMed1992

Reproducibility of electrophysiologic testing during antiarrhythmic therapy for ventricular arrhythmias secondary to coronary artery disease.

Although electrophysiologic studies are often used to assess antiarrhythmic drug efficacy in patients with ventricular tachycardia (VT), the reproducibility of these studies during therapy has not been definitively established. Confirmation studies were performed during drug therapy in 64 patients (51 men, mean age 63 years) with sustained ventricular arrhythmias induced during initial study to assess the reproducibility of drug effect. All patients had coronary artery disease. The stimulation protocol used included the serial introduction of up to 3 premature ventricular stimuli during sinus rhythm and with ventricular pacing at 2 pacing rates. Rapid ventricular pacing techniques were also used. Antiarrhythmic drug efficacy was confirmed in 77% of patients. Sustained VT was induced at repeat electrophysiologic study in 19% of patients during antiarrhythmic therapy that was previously thought to be effective. In summary, electrophysiologic study results during antiarrhythmic therapy exhibit significant day-to-day variability. Sustained VT can be induced during antiarrhythmic therapy that was previously defined as effective by programmed stimulation in a substantial number of patients.

Adult↗
PubMed1992

New approaches to treating early lung cancer.

The survival from lung cancer has not changed over the past 15 years, despite the intensive application of combined modality therapy for advanced disease. Prevention and early diagnosis appear to be the most promising strategies for reduction of lung cancer mortality. A Keystone Colloquium was held April 1 to 7, 1991, on the Biology of and Novel Therapeutic Approaches for Epithelial Cancers of the Aerodigestive Tract, to discuss recent basic and clinical research in this area. This paper summarizes the presentations relevant to lung cancer, including genetic mechanisms, growth factors, neuropeptides, growth and differentiation, carcinogenesis, chemoprevention, multidrug resistance, and immunology.

Humans↗
PubMed1992

Recognition of substrates by tyrosylprotein sulfotransferase. Determination of affinity by acidic amino acids near the target sites.

The sulfation of proteins by tyrosylprotein sulfotransferase (TPST) is highly site-specific. In this study, we examined the sequence specificity of the target site for TPST by determining the kinetics of rat liver TPST with peptides related to the sequence of the C4 component of complement. The data obtained from this study demonstrate that selective elimination of negative charges from the -5 to +5 region of the acceptor tyrosine, either by removal or by isosteric substitution or the acidic amino acids in the region, produced a substantial change in the Km value, with relatively little effect on Vmax. Substitutions at -1 and +1 positions increase the Km value by 22- and 4-fold, respectively, whereas removal of the acidic amino acids from the -5, -4, or +4 positions increased the Km values by a factor of 2-4. The effect of elimination of an acidic amino acid on the Km value was constant and specific for its particular position in relation to tyrosine, and the effect of modification of more than one amino acid was multiplicative. This study provides evidence that: 1) acidic residues near tyrosines promote sulfation by increasing the affinity of enzyme-substrate binding and have little effect on catalytic rate; 2) the contribution of each acidic residue to affinity for TPST is independent and varies according to position relative to the acceptor tyrosine; and 3) the enzyme interacts with a segment of at least 4-5 residues on each side of the tyrosine, with the residues on the -1 and +1 positions being the most important determinants. In general, residues on the NH2-terminal side of the tyrosine have a greater effect on affinity for TPST.

Amino Acid Sequence↗
PubMed1992

Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma.

Determinants of 5-year survival were evaluated after complete resection of pulmonary metastases from adult soft-tissue sarcomas. Fifty-eight patients had complete resection (median survival 25 months, P = 0.0002), with a 25.8% absolute 5-year survival (15 of 58 patients); six patients had unresectable disease (median survival 6 months) and were excluded from additional analysis. Eleven patients remain disease free, with a median follow-up of 76 months. Significant independent prognostic indicators associated with improved survival (P less than 0.05) included metastasis doubling time of 40 days or greater (median survival 37 months versus 15 months if less than 40 days); unilateral disease on preoperative radiography (33 months versus 15 months if bilateral disease); three or fewer nodules on preoperative computed tomography (40 months versus 14 months if 4 or more nodules); two nodules or fewer resected (40 months versus 17 months if 3 or more nodules resected), and tumor histology (33 months for malignant fibrous histiocytoma versus 17 months for all others). Multivariate analysis identified the number of nodules detected by computed tomography preoperatively as having significant prognostic value.

Adult↗
PubMed1992

Molecular surgery for cancer.

Advances in the understanding of the process of carcinogenesis may allow prevention, diagnosis, and treatment of cancer to be approached at the molecular level. Studies in our laboratory show that growth factors (transforming growth factor alpha), dominant oncogenes (HER-2/erb B2 and K-ras), and tumor suppressor genes (p53) are functionally important in the maintenance of the malignant phenotype of human non-small-cell lung cancer cells. Application of these findings to clinical problems include the identification of p53 mutations as markers for malignant change in Barrett's epithelium, the use of discordant p53 mutations to diagnose second primary malignant neoplasms in patients with head and neck cancer, and the potential for therapy by the reversal of genetic lesions.

Adenocarcinoma↗