Biomedical subjects
Publications and source records attributed to W J Long.
The authors extend the process of market positioning in the health care sector by focusing on the simultaneous utilization of traditional research methods and emerging new computer-based adaptive perceptual mapping technologies and techniques.
The diversity of infectious agents capable of inducing meningitis and blood-brain barrier (BBB) injury suggests the potential for a common host mediator. The inflammatory polypeptides, IL-1 and TNF, were tested in an experimental rat model as candidate mediators for induction of meningitis and BBB injury. Intracisternal challenge of rIL-1 beta into rats induced neutrophil emigration into cerebrospinal fluid (CSF) and significantly increased BBB permeability to systemically administered 125I-BSA as early as 3 h later (P less than 0.05). This injury was reversible, dose dependent and significantly inhibited by prior induction of systemic neutropenia (via intraperitoneal cyclophosphamide) or preincubation of the rIL-1 beta inoculum (50 U) with an IgG monoclonal antibody to rIL-1 beta. Similar kinetics and reversibility of CSF inflammation and BSA permeability were observed using equivalent dose inocula of rIL-1 alpha. rTNF-alpha was less effective as an independent inducer of meningitis or BBB injury over an inoculum range of 10(1) U (0.0016 micrograms/kg)-10(6) U (160 micrograms/kg) when injected intracisternally, but inoculum combinations of low concentrations of rTNF alpha (10(3) U) and rIL-1 beta (0.0005-5.0 U) were synergistic in inducing both meningitis and BBB permeability to systemic 125I-BSA. These data suggest that in situ generation of interleukin-1 within CSF (with or without TNF) is capable of mediating both meningeal inflammation and BBB injury seen in various central nervous system infections.
This paper addresses the nature of the prior probabilities of diseases for probabilistic diagnostic reasoning. Because diseases differ in their chronicity, occurrence, reoccurrence, and likelihood of becoming part of the patient population, reasoning in terms of the frequency of disease episodes is necessary to capture the important distinctions. Even with these complexities, it is possible to formulate a reasonably accurate, computationally tractable, frequency estimation method for combinations of diseases. This method also suggests ways in which the needed numbers can be estimated from patient data.
Different cell culture media were compared for their ability to support and promote the growth of stable hybridoma cell lines derived from three commonly used parental murine myelomas. Supplemented Dulbecco's modified Eagle's media (DMEM) and RPMI 1640 media were studied. The DMEM-based media were found to support greater numbers of cells for longer time periods than were the RPMI 1640-based media. Aminopterin supplemented medium was shown to be significantly less effective in supporting hybridoma reproduction and viability than medium without aminopterin. Antibody levels were directly related to cell concentration and viability regardless of the medium used for the hybridoma culture. An optimally formulated DMEM-based medium is suggested as the medium of choice for hybridoma propagation and maintenance.
Eight different cell feeder layers and 2 conditioned media were compared for their ability to increase the establishment efficiency of newly formed hybridoma cells. Enhancement was noted particularly with all 3 irradiated human lung diploid fibroblasts which were tested. Hybridomas established in the presence of the feeder layers were found to be stable upon propagation even in the absence of the feeder cells. MRC-5 diploid fibroblasts are suggested as the hybridoma feeder layer cell of choice due to this cell's ease of culture and handling.
A computerized system for the management of hybridoma cell growth data using voice input is described. The system permits the storage, retrieval, and analysis of data for hybridoma cells secreting monoclonal antibodies. It consists of a local system residing on a professional computer interfaced to a mainframe processing system. The local system uses voice-input to facilitate data entry while data flagged for retention is stored on the mainframe. The system permits the investigator to monitor thousands of candidate cell lines until selection for specific antibody production is completed. All record-keeping aspects of hybridoma cell culture from establishment through freezing and storage of selected cell lines can be accomplished by personnel without computer expertise.
Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage.
The cerebral capillary endothelium is unique and functions as an effective blood-brain barrier (BBB) owing to its intercellular tight junctions and rare pinocytotic vesicles. To assess how bacterial meningitis alters the BBB, rats were inoculated intracisternally with three encapsulated meningeal pathogens (Escherichia coli K1+, Streptococcus pneumoniae type III, Haemophilus influenzae type b) and an unencapsulated mutant strain (H. influenzae Rd). After defined infection durations, the morphologic alterations of the cerebral capillary endothelium were quantitatively assessed by transmission electron microscopy. Results revealed a significant increase in pinocytotic vesicle formation (P less than 0.001) early after meningitis induction (4 h) that was sustained with longer infection durations (10 h, 18 h) for all encapsulated strains tested. In addition, there was a progressive increase in completely separated intercellular junctions with increasing infection duration, (P less than 0.05). 4 h after induction of meningitis with H. influenzae Rd, cerebrospinal fluid (CSF) bacterial concentrations, cerebral capillary morphologic changes, and functional BBB permeability to circulating 125I-albumin were similar to those observed with H. influenzae type b. However, prolonging the H. influenzae Rd infection to 18 h allowed for CSF clearance of the organism, thereby precluding the significant increase in separated junctions or progression of functional BBB permeability seen with the encapsulated H. influenzae type b. These data suggest a uniform morphologic explanation for altered BBB permeability in meningitis with a reproducible temporal sequence. Encapsulation does not appear essential for BBB injury, but may facilitate its progression by allowing the organism to evade host clearance.
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BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.
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Cryostat sections from 160 rectal suction biopsies were stained for cholinesterases by the method of Karnovsky and Roots (1964) in an attempt to facilitate the diagnosis of Hirschsprung's disease. The method proved at least as reliable as experienced assessment of paraffin haematoxylin-eosin sections, and appeared to offer the advantages of reduced scanning fatigue and superior demonstration of the increased cholinesterase-positive nerves in Hirschprung's disease. Contrary to the findings of Meier-Ruge (1971) it was not possible to base a diagnosis on mucosal cholinesterase activity.
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OBJECTIVE: Evaluate the accuracy of the detailed diagnostic reasoning of the Heart Failure Program incorporating a new mechanism to handle temporal relationships and severity constraints. DESIGN: Tools were developed to summarize diagnoses and automatically generate evaluation forms. Five expert cardiologists were asked to review the reasoning of the program, with two analyzing each case. Cases were gathered retrospectively for diversity and difficulty and 26 randomly selected cases were evaluated. The underlying issues were identified and classified. RESULTS: Both reviewers rated the first diagnosis correct in 25% of the cases and at least one rated it wrong in 10%. Analyzing the detailed reasoning, 137 issues were raised, about 5.3 per case. Of these, 53% were possible concerns raised by one reviewer. Of the 5.3 issues per case, 2.5 were attributable to controversies, misunderstandings, or mistakes; 1 was due to the overly simplistic representation of the summaries; and 1.8 were issues related to the program. CONCLUSION: Overall, the program is capable of providing high-quality detailed diagnostic hypotheses for complex cardiovascular cases. The results highlight several issues: 1) the difficulty of effectively summarizing hypotheses, 2) the nature of a physician's causal explanation, and 3) some problems in evaluating detailed diagnostic reasoning. The mistakes the program made imply that some additional refinement is needed but that the reasoning mechanisms developed can support the appropriate reasoning. The appropriate next step is a prospective evaluation addressing the program's usefulness.