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Biomedical subjects

P A Johnson

Publications and source records attributed to P A Johnson.

At least 19 recordsLinked to original sources
PubMed2001

Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma.

Replication-competent, attenuated herpes simplex viruses (HSV) have been demonstrated to be effective oncolytic agents in a variety of malignant tumors. Cytokine gene transfer has also been used as immunomodulatory therapy for cancer. To test the utility of combining these two approaches, two oncolytic HSV vectors (NV1034 and NV1042) were designed to express the murine GM-CSF and murine IL-12 genes, respectively. These cytokine-carrying variants were compared with the analogous non-cytokine-carrying control virus (NV1023) in the treatment of murine SCC VII squamous cell carcinoma. All three viruses demonstrated similar infection efficiency, viral replication, and cytotoxicity in vitro. SCC VII cells infected by NV1034 and NV1042 effectively produced GM-CSF and IL-12, respectively. In an SCC VII subcutaneous flank tumor model in immunocompetent C3H/HeJ mice, intratumoral injection with each virus caused a significant reduction in tumor volume compared with saline injections. The NV1042-treated tumors showed a striking reduction in tumor volume compared with the NV1023- and NV1034-treated tumors. On subsequent rechallenge in the contralateral flank with SCC VII cells, 57% of animals treated with NV1042 failed to develop tumors, in comparison with 14% of animals treated with NV1023 or NV1034, and 0% of naive animals. The increased antitumor efficacy seen with NV1042 in comparison with NV1023 and NV1034 was abrogated by CD4(+) and CD8(+) lymphocyte depletion. NV1042 is a novel, attenuated, oncolytic herpesvirus that effectively expresses IL-12 and elicits a T lymphocyte-mediated antitumor immune response against murine squamous cell carcinoma. Such combined oncolytic and immunomodulatory strategies hold promise in the treatment of cancer.

Animals↗
PubMed2001

Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells.

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.

Animals↗
PubMed2000

The mitotic spindle and DNA damage-induced apoptosis.

EM9 Chinese hamster ovary cells cannot rejoin DNA strand breaks induced by alkylating agents. Ethyl methanesulphonate (EMS)-treated EM9cells underwent G2 arrest for a prolonged period followed by entry into mitosis and apoptosis. EM9 cells treated with EMS in G1 entered mitosis 24-36 h after release from synchrony, approximately 12 h after untreated control cells, but the mitoses were morphologically abnormal. The spindle-poison nocodazole reduced apoptosis by greater than 60%, and allowed some cells to complete a second round of DNA replication. We conclude that the assembly of a mitotic spindle, or progression beyond the mitotic checkpoint, is important for apoptosis following DNA strand breakage.

Animals↗
PubMed2000

Estradiol regulation of follistatin and inhibin alpha- and beta(B)-subunit mRNA in avian granulosa cells.

Estradiol modulation of granulosa cell growth and regulation of follistatin and inhibin alpha- and beta(B)-subunit mRNA were investigated in cultured chicken granulosa cells. Granulosa cells were isolated and pooled according to size from the F(4) + F(5), small yellow (SYF), and large white (LWF) follicles. Isolated and dispersed granulosa cells were then cultured in the absence or presence of 1 x 10(-5) M 17 beta-estradiol. In Experiment 1 (n = 4 replications) the effect of estradiol on the growth of granulosa cells from the different-sized follicles was examined at 24 and 48 h of culture. Untreated and treated granulosa cells from all three follicle sizes proliferated during culture, and cell viability for all cultures was over 95% throughout the experiment. After 48 h the untreated cultures for all follicle types had 1.6 to 2.2 times (P < 0.05) more cells than the estradiol-treated cultures. In Experiment 2 (n = 3 replications), the cultures were terminated at 4 and 24 h after plating. Follistatin mRNA levels were higher in estradiol-treated cells at 24 h in F(4) + F(5) follicles, at 4 and 24 h in the SYF, and at 4 h in the LWF. beta(B)-subunit mRNA levels were also increased by estradiol at 4 h in F(4) + F(5) cells and at 4 and 24 h in the LWF. Steady state mRNA levels for the alpha-subunit were higher (P < 0.05) in estradiol-treated cultures at 4 and 24 h in F(4) + F(5) follicles and at 24 h in the SYF. Immunoreactive alpha-subunit protein, however, was not increased by estradiol treatment. Thus, whereas estradiol inhibited granulosa cell growth, it exerted a generally stimulatory effect on the expression of FS and the inhibin alpha- and beta(B)-subunit mRNA.

Animals↗
PubMed2000

Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in aotus.

G207 is a multimutated, conditionally replicating herpes simplex virus type 1 (HSV-1) that is currently in clinical trial for patients with malignant glioma. G207 exhibits an efficient oncolytic activity in tumor cells, yet minimal toxicity in normal tissue when injected into the brains of HSV-susceptible mice or nonhuman primates. In this study, we evaluated the shedding and biodistribution of clinical-grade G207 after intracerebral inoculation (3 x 10(7) pfu) in four New World owl monkeys (Aotus nancymae). Using PCR analyses and viral cultures, neither infectious virus nor viral DNA was detected from tear, saliva, or vaginal secretion samples at any time point up to 1 month postinoculation. Analyses of tissues obtained at necropsy at 1 month from two of the four monkeys, plus one monkey inoculated with laboratory-grade G207 (10(9) pfu) 2 years earlier, showed the distribution of G207 DNA restricted to the brain, although infectious virus was not isolated. Histopathology revealed normal brain tissues including the sites of inoculation. A measurable increase of serum anti-HSV antibody titer was observed in all monkeys, as early as 21 days postinoculation. The results ascertain the safety of G207 in the brain and indicate that strict biohazard management may not be required for G207-treated patients.

Animals↗
PubMed2000

Chronic lymphocytic leukemia B cells are highly sensitive to infection by herpes simplex virus-1 via herpesvirus-entry-mediator A.

We found that chronic lymphocytic leukemic (CLL) B cells are highly sensitive to infection with vectors derived from replication-defective herpes simplex virus-1 (rdHSV-1). CLL B cells were found to express high levels of herpes virus entry mediator (Hve) A, but not HveC, the other known receptor for HSV-1. An HveA cDNA from CLL cells was found to encode Arg-->Lys and Val-->Iso substitutions at amino acids 17 and 241, respectively. Nevertheless, this cDNA encoded a functional receptor for HSV-1 when transfected into Chinese hamster ovarian (CHO) cells. Antibodies to HveA could block rdHSV-1 infection of CLL cells and HveA-transfected CHO cells with similar efficiencies in vitro. In contrast to B cells of normal donors, CLL B cells were resistant to the cytopathic effects of infection by rdHSV-1 and maintained high-level expression of the transgene for several days in vitro. We propose that this is due to the expression by CLL cells of the anti-apoptotic protein, bcl-2. Consistent with this, we found that transduction of HeLa cells with a retrovirus expression vector encoding bcl-2 rendered HeLa cells resistant to the cytopathic effects of rdHSV-1. HSV-1-derived vectors should be excellent vehicles for gene transfer into CLL B cells, allowing for its potential use in gene therapy for this disease. Gene Therapy (2000) 7, 1210-1216.

Animals↗
PubMed2000

Molecular cloning and expression analysis of the complementary deoxyribonucleic acid for chicken inhibin/activin beta(B) subunit.

Inhibins and activins are dimeric peptide hormones that play an integral role in the intraovarian regulation of folliculogenesis. The domestic hen, with its well-defined follicular hierarchy, provides a unique model in which to study the role of these hormones in follicular development. In the present study, the complete coding sequence and deduced amino acid sequence for the chicken inhibin/activin beta(B) subunit has been determined from cDNA clones isolated from a chicken ovarian granulosa cell library. This beta(B)-subunit cDNA predicts a precursor protein of 392 amino acids containing the mature C-terminal 115 amino acid beta(B) subunit. When compared to the beta(B) subunit isolated from a variety of species, the chicken cDNA clone showed high nucleotide identity in the full-length coding region (>70%) and in the mature coding region (>80%). In addition, the deduced amino acid sequence of chicken beta(B) subunit showed greater than 95% identity compared to other species in the mature peptide region. Expression of the beta(B)-subunit mRNA was detected by reverse transcription-polymerase chain reaction in both gonadal and extragonadal tissues. Northern blot analysis detected expression in the gonadal tissues only, specifically in the granulosa tissue from the F3-F5 follicles, small yellow follicles (SYF), large white follicles, and immature and mature rooster testes. A major transcript of approximately 4.1 kilobases (kb) and three minor transcripts of approximately 8.4 kb, 6.5 kb, and 1.7 kb were detected in the SYF granulosa samples. To examine the expression pattern of the beta(B) subunit around the stage of follicle selection, the SYF granulosa was subdivided into two groups: 6-8 mm and 9-12 mm. Quantification of RNA expression (n = 3) showed that expression of the beta(B) subunit was maximal in the 6-8 mm SYF. Activin B, as well as other intraovarian signals, may regulate early follicle selection and/or development in the chicken.

Activins↗
PubMed2000

Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses.

Direct intramuscular injection of plasmid DNA can generate immune responses against encoded antigens. However, the relative ability of DNA vaccines to induce cellular and humoral immunity after a single or booster immunization and the persistence of this response have not been fully elucidated. In this study, induction and maintenance of antibody and T cell subtypes with different doses of naked DNA encoding the haemagglutinin (HA) gene of influenza virus were examined and compared to the immune responses and protection induced by respiratory tract infection and immunization with a killed virus vaccine. Like natural infection, immunization with HA DNA induced potent Th1 responses. Spleen cells from mice immunized once with HA DNA in the dose range 10 ng to 100 microgram secreted significant levels of IFN-gamma, but low or undetectable IL-5, in response to influenza virus in vitro. Furthermore, CD4(+) HA-specific Th1 clones were generated from spleens of immunized mice. Although T cell responses waned 12 weeks after a single immunization, antigen-specific Th1 cells persisted in the spleen for at least 6 months after two booster immunizations. In contrast, influenza virus-specific ELISA IgG titres were low after a single immunization and required two booster immunizations to reach significant levels. Furthermore, haemagglutination inhibition (HI) antibodies were weak or undetectable after two immunizations. Nevertheless, two doses of HA DNA conferred almost complete protection against respiratory challenge with live virus. Thus, despite the limited ability to induce antibodies, DNA vaccines confer protective immunity against influenza virus infection, which appears to be mediated by Th1 cells.

Animals↗
PubMed1999

Emergency department triage strategies for acute chest pain using creatine kinase-MB and troponin I assays: a cost-effectiveness analysis.

BACKGROUND: Evaluation of acute chest pain is highly variable. OBJECTIVE: To evaluate the cost-effectiveness of strategies using cardiac markers and noninvasive tests for myocardial ischemia. DESIGN: Cost-effectiveness analysis. DATA SOURCES: Prospective data from 1066 patients with chest pain and from the published literature. TARGET POPULATION: Patients admitted with acute chest pain. TIME HORIZON: Lifetime. PERSPECTIVE: Societal. INTERVENTIONS: Creatine kinase (CK)-MB mass assay alone; CK-MB mass assay followed by cardiac troponin I assay if the CK-MB value is normal; CK-MB mass assay followed by troponin I assay if the CK-MB value is normal and electrocardiography shows ischemic changes; both CK-MB mass and troponin I assays; and troponin I assay alone. These strategies were evaluated alone or in combination with early exercise testing. OUTCOME MEASURES: Lifetime cost, life expectancy (in years), and incremental cost-effectiveness. RESULTS OF BASE-CASE ANALYSIS: For patients 55 to 64 years of age, measurement of CK-MB mass followed by exercise testing in appropriate patients was the most competitive strategy ($43000 per year of life saved). Measurement of CK-MB mass followed by troponin I measurement had an incremental cost-effectiveness ratio of $47400 per year of life saved for patients 65 to 74 years of age; it was also the most cost-effective strategy when early exercise testing could not be performed, CK-MB values were normal, and ischemic changes were seen on electrocardiography. RESULTS OF SENSITIVITY ANALYSIS: Results were influenced by age, probability of myocardial infarction, and medical costs. CONCLUSIONS: Measurement of CK-MB mass plus early exercise testing is a cost-effective initial strategy for younger patients and those with a low to moderate probability of myocardial infarction. Troponin I measurement can be a cost-effective second test in higher-risk subsets of patients if the CK-MB level is normal and early exercise testing is not an option.

Biomarkers↗
PubMed1999

A mitotic spindle requirement for DNA damage-induced apoptosis in Chinese hamster ovary cells.

Promiscuously reactive electrophilic agents induce DNA and other cellular damage. DNA repair-defective cells, when compared with genetically matched, repair-proficient parental cells, provide a means to distinguish cellular responses triggered by individual genetic lesions from other macromolecular damage. The Chinese hamster ovary (CHO) cell line EM9 is hypersensitive to the alkylating agent ethyl methanesulfonate (EMS) and is unable efficiently to repair DNA single strand breaks in contrast to parental AA8 cells. EM9 was used to examine how CHO cells couple unrepaired DNA strand breaks to loss of viability. Flow cytometry revealed that EMS-treated EM9 cells underwent prolonged cell cycle arrest in G2, followed by entry into mitosis, micronucleation, and apoptosis. EM9 cells synchronized in G1 prior to EMS treatment entered mitosis 24-36 h after release from synchrony, approximately 12 h after untreated control cells. Mitoses in EMS-treated cells were abnormal, involving multipolar mitotic spindles and elongated and/or incompletely condensed chromosomes. The mitotic spindle poison nocodazole reduced DNA damage-induced apoptosis by >60%, whereas the frequency of micronucleation was similar in the presence or absence of nocodazole. Flow cytometry revealed that nocodazole-treated cells sustained a second round of DNA replication without intervening mitosis. These results demonstrate that nuclear fragmentation and inappropriate DNA replication are insufficient to trigger apoptosis following DNA strand breakage and demonstrate a requirement for mitotic spindle assembly for this process in CHO cells.

Animals↗
PubMed1999

A proposed strategy for utilization of creatine kinase-MB and troponin I in the evaluation of acute chest pain.

In recent years, cardiac troponins have attracted great interest as a marker for myocardial injury. However, there are limited data on strategies for use of creatine kinase (CK)-MB and troponin I (cTnI) in clinical practice. We sought to develop a testing strategy using prospectively collected clinical data including serial CK-MB and cTnI levels from 1,051 patients aged > or = 30 years admitted to a teaching hospital for acute chest pain. Diagnostic performance was evaluated for peak values of CK-MB and cTnI obtained during the first 24 hours for the combined end point of acute myocardial infarction and/or major cardiac events within 72 hours. The overall diagnostic accuracy was similar for both cardiac markers alone, and for the combination of cTnI and CK-MB (receiver-operating characteristic curve 0.84, 0.86, and 0.87, respectively). In the multivariate analysis, models including cardiac markers showed that both CK-MB and cTnI added information to clinical data to predict the combined end point, but cTnI added significantly less. Using recursive partitioning analysis, we developed a strategy that would restrict routine cTnI use to patients with normal CK-MB results and findings on the electrocardiogram consistent with ischemia. This strategy would divide patients with suspected myocardial ischemia into 4 groups with risks for the combined end point of 4%, 13%, 26%, and 85%. Thus, cTnI adds information to CK-MB mass and clinical data for predicting major cardiac events, but this contribution is mainly in patients with evidence of myocardial ischemia on their electrocardiograms.

Acute Disease↗
PubMed1999

Value of additional two-hour myoglobin for the diagnosis of myocardial infarction in the emergency department.

Myoglobin has been described as an early marker of myocardial injury. It increases within 1 to 3 hours of myocardial injury, and falls back to normal early after the event. Few data suggest that myoglobin can be used to triage patients with chest pain. To assess the diagnostic utility of second myoglobin levels drawn within 2 to 3 hours after presentation to the emergency department for detecting myocardial infarction, we prospectively collected myoglobin levels in 368 patients aged > or = 30 years who were admitted with chest pain. Myoglobin levels were measured at admission and 2 to 3 hours later. Sensitivity and specificity for detecting acute myocardial infarction for a twofold increase in myoglobin level from baseline were 39% and 98%, respectively. The early diagnostic performance of myoglobin at admission, the second level drawn 2 to 3 hours later, and creatine kinase-MB mass drawn at admission were similar (receiver-operating characteristic curves 0.80, 0.86, and 0.85). The diagnostic performance of each of these markers was significantly superior to the absolute change from baseline to second myoglobin (receiver-operating characteristic curve 0.77). In patients who presented within 4 hours of symptom onset, myoglobin drawn 2 to 3 hours later had the highest yield for detecting myocardial infarction. These results suggest that serial myoglobin measurement aiming to detect changes over time is not superior to single marker determinations. Myoglobin measured in 2 to 3 hours from admission may be helpful in triaging patients who present within 4 hours from onset of symptoms.

Creatine Kinase↗
PubMed1999

Gonadotropin regulation of inhibin alpha-subunit mRNA and immunoreactive protein in cultured chicken granulosa cells.

Gonadotropin regulation of the inhibin alpha-subunit was investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F(1) and F(3) + F(4) follicles from three to four hens, pooled according to size, dispersed, and cultured (n = 3 replications for each experiment). In Experiments 1 and 2 either ovine LH or FSH was added to the cultures at doses of 0, 5, and 25 ng/ml. The cultures were terminated at 4, 24, and 48 h after plating. For both follicle sizes the expression of mRNA for the inhibin alpha-subunit was less (P < 0.05) at 24 and 48 h in untreated cells than in those treated with both doses of LH. Expression of the mRNA for the inhibin alpha-subunit was significantly increased only by the 25 ng/ml dose of FSH and only in the F(1) follicle at 24 and 48 h compared to the untreated cells. After 48 h of culture, immunoreactive alpha-subunit protein accumulation was greater for both follicle types in the media of cells treated with the highest dose of LH and FSH than in the media from untreated cells. In Experiment 3, doses of 0, 5, 25, or 50 ng/ml of either LH or FSH were added to F(1) and F(3) + F(4) granulosa cells. All cultures were terminated at 48 h. LH and FSH increased the expression of the mRNA and immunoreactive protein for the inhibin alpha-subunit equally in a time-dependent manner. These experiments indicate that gonadotropins enhance the expression of both the mRNA and the protein for the inhibin alpha-subunit in chicken granulosa cells.

Animals↗
PubMed1999

Cardiac troponin T as a marker for myocardial ischemia in patients seen at the emergency department for acute chest pain.

BACKGROUND: Identification of patients with acute chest pain at high risk for cardiovascular complications is a common and difficult challenge for clinicians and must be based initially on data from the history, physical examination, electrocardiogram, and chest radiograph. Some data suggest that elevations in cardiac troponin T (cTnT) may be useful for detection of less severe degrees of myocardial injury that may occur in some patients with unstable angina. Therefore we designed a prospective follow-up study to assess the diagnostic performance and prognostic value of cTnT in a population of patients presenting to the emergency department with acute chest pain. METHODS: The patient population included all 1477 admitted patients aged 30 years or more who presented to the emergency department of an urban teaching hospital from October 1992, through February 1994, with a chief symptom of acute chest pain not explained by trauma or chest radiograph abnormalities. The 1303 patients (88%) who had 2 or more measurements of cTnT during the first 24 hours after presentation comprised the final study population. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operator characteristics curve (ROC) were determined for cTnT and creatine kinase-MB (CK-MB) (measured using activity and mass assays) data from the first 24 hours after admission for the outcomes of acute myocardial infarction (AMI) and major cardiac events during the first 72 hours of hospitalization. RESULTS: The sensitivity and specificity of cTnT (threshold of 0.1 ng/mL) for detecting AMI during the first 24 hours after presentation were 99% and 86%, respectively. The CK-MB activity and mass assays had diagnostic performance for detecting AMI similar to cTnT. Among patients who did not meet study criteria for AMI, cTnT was elevated during the first 24 hours in 31% of patients who had major complications, compared with a 17% rate for the CK-MB activity assay and a 3% rate for the CK-MB mass assay. In these patients, the cTnT assay had superior diagnostic performance compared with the CK-MB mass assay as a marker for cardiac complications as assessed with ROC analysis (P <.0004). CONCLUSIONS: In a heterogeneous population of patients seen in the emergency department with acute chest pain, cTnT was similar to CK-MB (activity and mass assays) for detection of AMI and superior to the CK-MB mass assay as a marker for major cardiac events early in the hospital course among those who were ruled out for an AMI. Further study is required to determine how this assay can be used to provide more appropriate, cost-effective care.

Acute Disease↗
PubMed1999

Double gloving and a system for identifying glove perforations in maxillofacial trauma surgery.

The treatment of some maxillofacial fractures has an incidence of glove perforation as high as 50%, with over 80% going unnoticed at the time of operation. We investigated double gloving and a glove perforation indication system to ascertain whether the latter offered any additional protection. 1061 gloves used for 113 patients were examined. The outer glove perforation rate was significantly higher than the inner glove (0.48 compared with 0.10, P < 0.0001). There were fewer unnoticed perforations in the glove perforation indication group than the standard surgical glove group (19% compared with 79%, P < 0.0001). The indication system was most effective in wet operating fields. The perforation rate varied with the type of fracture and treatment. Mandibular fractures that were 'hand-held', while bone-plated had a lower mean number of outer glove perforations/operation than fractures treated with temporary intermaxillary fixation (0.43 compared with 4.62, P < 0.0001).

Chi-Square Distribution↗