TNF alpha-mediated inhibition and reversal of adipocyte differentiation is accompanied by suppressed expression of PPARgamma without effects on Pref-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide hormone with pleiotropic effects on cellular proliferation and differentiation. To investigate how TNF alpha inhibits and reverses adipocyte differentiation, we studied the expression of two factors involved in the adipocyte differentiation process. Peroxisome proliferator-activated receptor gamma (PPARgamma) is a positive regulator of adipogenesis, whereas preadipocyte factor 1 (Pref-1) inhibits adipocyte differentiation. The expression patterns of both PPARgamma and Pref-1 change during early stages of adipocyte differentiation. Decreased expression of Pref-1 and increased expression of PPARgamma occur 1 day and 2 days, respectively, after 3T3-L1 cells reach confluence. During TNF alpha-mediated inhibition of adipocyte differentiation, PPARgamma messenger RNA (mRNA) expression stays at low levels. In contrast, TNF alpha treatment has no effect on the normal decrease in Pref-1 gene expression that occurs during adipogenesis. We observed that certain cytokine and growth factors [such as TNF alpha, basic fibroblast growth factor, transforming growth factor beta, and protein kinase C-activating agents plus calcium ionophore], when added to differentiated adipocytes, cause rapid down-regulation of PPARgamma mRNA expression with concomitant decrease in adipocyte-specific gene expression but fail to increase Pref-1 mRNA expression. Moreover, addition of TNF alpha to fully differentiated adipocytes results in the rapid disappearance of PPARgamma protein expression and the rapid loss of PPARgamma DNA-binding activity. Therefore, Pref-1 seems to function as a nonreversible molecular checkpoint whose expression is insensitive to TNF alpha-generated signals, whereas PPARgamma expression remains sensitive to TNF alpha at all stages of the adipogenesis program. Our results support the notion that dedifferentiated adipocytes and preadipocytes are not identical, though they share many similar morphological and gene expression patterns.

Production of a urokinase plasminogen activator-IgG fusion protein (uPA-IgG) in the baculovirus expression system.

Numerous studies have demonstrated the importance of urokinase plasminogen activator (uPA) and its receptor, uPAR, in the processes of tumor progression and metastasis. Thus, the uPA/uPAR interaction may represent an important target for inhibiting metastatic disease. The baculovirus expression system was used to produce high levels of a secreted uPA-Immunoglobulin G fusion protein (uPA-IgG) which could then be used for displacing uPA from the surface of tumor cells. The recombinant uPA-IgG fusion protein was placed under the control of either the viral polyhedrin promoter or a copy of the viral basic protein promoter. Recombinant viruses were then used to infect Sf9 and BTI-Tn-5B1-4 cells. Infection of both cell types resulted in the production of secreted uPA-IgG. The molecular mass of the secreted protein as determined by SDS-PAGE was approximately 40 kDa. The highest level of secreted uPA-IgG, 444 microg/ml, was found in the culture medium of BTI-Tn-5B1-4 cells 72 h post-infection with the basic protein promoter-uPA-IgG virus. In the case of Sf9 cells, the highest level of secreted protein was 195 microg/ml. The amount of cell-associated uPA-IgG in infected BTI-Tn-5B1-4 cells was significantly less than that of infected Sf9 cells, reflecting the superior secretory capability of the BTI-Tn-5B1-4 cells. The uPA-IgG was readily purified using a combination of zinc chelate and sephacryl S-100 column chromatography. Routinely, greater than 100 mg of greater than 95% pure protein could be obtained per liter of culture medium collected at 72 h post-infection of BTI-Tn-5B1-4 cells with the basic protein promoter virus. BIAcore analysis and competition binding assays using LOX human malignant melanoma cells expressing uPAR indicated that the purified recombinant protein possessed similar ligand binding characteristics to that of human uPA.

Fluorescent microtiter screening assay for immunocytochemically reactive antibodies.

We have developed a rapid in situ screening procedure that enables prescreening of hundreds of hybridomas in a 96-well format. The procedure involves fluorescence immunostaining of cells cultured in 96-well plates and the use of a fluorescence plate reader to detect reactive antibodies. Positive immunostaining in individual well, as denoted by elevated readings, is then confirmed by fluorescence microscopy. Using the method described here, we have successfully identified monoclonal antibodies that are specific to the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma). This assay is readily applicable for screening hybridomas raised against cell surface or intracellular antigens to aid in the initial identification of antibodies reactive in immunocytochemical procedures.

Future requirements for and supply of ophthalmologists. What do the forecasts show?

Forecasts of the requirements for and supply of ophthalmologists in 1990 have produced conflicting results because of varying assumptions about the future utilization of eye care services, incidence and prevalence of ophthalmic disease, physician productivity, and availability of residency training positions. A typical "utilization-based" models, founded on present consumer behavior, predicts a substantial 1990 surplus of ophthalmologists at current rates of residency training. Two "need-based" models, founded on ideal rather than actual use, reach different conclusions because of varying use of a fragile data base and the need to rely heavily on the subjective judgment of experts with regard to norms of care. The 1980 Graduate Medical Educational National Advisory Committee forecasted a surplus, while the 1978 American Academy of Ophthalmology predicted a deficit. Utilization-based models may slightly underestimate future ophthalmologist requirements. However, analysis of the factors that will influence future use suggests that need-based models are likely to overestimate the requirements. It is risky to accept the need-based model projections because of the high cost of a surplus, which include not only the expenses of training unneeded ophthalmologists but also the cost of their decreased exposure to disease and of declining physician morale, acumen, and thresholds for surgical procedures. Because free market mechanisms are ineffective in governing the supply of health providers, it will be necessary for the profession itself to review the current and projected supply and to set limits on the number of persons in training.

Ophthalmology manpower: shortfall or windfall?

Forecasts of ophthalmology manpower depend on assumptions about future supply and requirements. The two factors that influence supply are the number of residents trained and the attrition of ophthalmologists in practice. The factors that influence requirements are the estimated amount of future ophthalmic services the public will demand and the productivity of ophthalmologists. Previous forecasts have produced widely differing results because of an inadequate data base and varying theoretical assumptions. A conservative approach is to use past ophthalmologist to population ratios as a guide, and to tailor the output of ophthalmologists accordingly. A yearly reduction from 1982 to 1990 between 5% and 10% in the number of residency positions will be necessary to bring supply in line with requirements within the next thirty years.

Computer content analysis of applied biological knowledge in dentistry.

The study suggests that the Inquirer II System used by computers in content analysis of (textual) specific written material has value for longitudinal studies. The research findings indicate that the application of the computer in content analysis requires considerable effort in the preparation of materials and specifications of directions to the computer; standardizes data analysis, thus reducing subjective error in replication of studies; reduced measurement error in longitudinal studies, giving greater stability and power to statistical comparison; classified data as it is collected, quickly and reliably, while tabulating statistical information; reduced costs compared to human coders when used with large amounts of data; and competes effectively with human coders in terms of reliability and validity.

Uncompensated care provided by private practice physicians in Florida.

While a great deal of attention has been paid in recent years to establishing the magnitude and characteristics of uncompensated care in hospitals, comparatively little research has been undertaken to study physician uncompensated care. This article reports the results of a prospective patient-specific study of uncompensated care in Florida. Of 4,042 cases examined, 26.2 percent had charges voluntarily reduced below the usual and customary charge at the time of service. However, only 13.5 percent of those reductions were attributed to charity. Overall, 10.4 percent of the total billed amount was left unresolved. When payment source was considered, it was found that self-pay patients accounted for 30.6 percent of the cases but accounted for 52.0 percent of the unresolved amounts. Further analysis indicated that the self-pay patients were 35.5 times more likely to leave an outstanding balance than individuals with some type of insurance coverage. Odds of unresolved balances were also calculated as a function of income, specialty type, practice size, and type of visit.

Determining VA physician requirements through empirically based models.

OBJECTIVE: As part of a project to estimate physician requirements for the Department of Veterans Affairs, the Institute of Medicine (IOM) developed and tested empirically based models of physician staffing, by specialty, that could be applied to each VA facility. DATA SOURCE/STUDY SETTING: These analyses used selected data on all patient encounters and all facilities in VA's management information systems for FY 1989. STUDY DESIGN: Production functions (PFs), with patient workload dependent on physicians, other providers, and nonpersonnel factors, were estimated for each of 14 patient care areas in a VA medical center. Inverse production functions (IPFs), with physician staffing levels dependent on workload and other factors, were estimated for each of 11 specialty groupings. These models provide complementary approaches to deriving VA physician requirements for patient care and medical education. DATA COLLECTION/EXTRACTION METHODS: All data were assembled by VA and put in analyzable SAS data sets containing FY 1989 workload and staffing variables used in the PFs and IPFs. All statistical analyses reported here were conducted by the IOM. PRINCIPAL FINDINGS: Existing VA data can be used to develop statistically strong, clinically plausible, empirically based models for calculating physician requirements, by specialty. These models can (1) compare current physician staffing in a given setting with systemwide norms and (2) yield estimates of future staffing requirements conditional on future workload. CONCLUSIONS: Empirically based models can play an important role in determining VA physician staffing requirements. VA should test, evaluate, and revise these models on an ongoing basis.

Implementation of a model for census prediction and control.

A model is described that predicts hospital census and computes, for each day, the number of elective admissions that will maximize the census over the short run, subject to constraints on the probability of overflow. Where a computer is available the model provides detailed predictions of census in units as small as 10 beds; used with manual computation the model allows production of tables of the recommended numbers of elective admissions to the hospital as a whole. The model has been tested in five hospitals and is part of the admissions system in two of them; implementation is described, and the results obtained are discussed.

Who cares what it costs to dispense a Medicaid prescription?

Results of a 1992 Medicaid cost-of-dispensing study among North Carolina pharmacies are presented. The estimated statewide weighted average cost incurred by pharmacies to dispense a prescription was $5.37 in 1991. The variation in dispensing costs found among pharmacies of various sizes, organizational types, and locations is identified. Higher average dispensing costs were reported for large chain pharmacies and those pharmacies in urban areas. Considering the potential for expanded prescription drug benefits under a reformed health care system, the implications of the study's findings for pharmacy payment policy are discussed.

A resident-based reimbursement system for intermediate care facilities for the mentally retarded.

In this article, the authors present a resident-based reimbursement system for intermediate care facilities for the mentally retarded (ICFs-MR), which represent a large and growing proportion of the medicaid budget. The statistical relationship between resident disability level and the expected cost of caring for the individual is estimated, allowing for the prediction of expected resource use across the population of ICF-MR residents. The system incorporates an indirect cost rate, a base direct care rate (constant across all providers), and an individual-specific direct care rate, based on the expected cost of care.

Beneficiary survey-based feedback on new Medicare informational materials.

In response to the Balanced Budget Act (BBA) of 1997, the Center for Medicare & Medicaid Services (CMS) initiated a massive information and education campaign to promote effective health plan decision-making. Early results suggest that the pilot version of the Medicare & You handbook and other new Medicare informational materials were viewed favorably overall. Despite their limitations, most beneficiaries found the information useful. The longer, more comprehensive materials were not perceived to be more useful than the shorter, less complicated version. Additional research is needed to determine which subgroups of beneficiaries may need more and, possibly less, information.

Shorter development of immunoassay for drugs: application of the novel RIMMS technique enables rapid production of monoclonal antibodies to ranitidine.

High affinity, specific murine monoclonal antibodies have been produced for ranitidine using the novel RIMMS (repetitive immunizations, multiple sites) technique. We demonstrate that this technique can be employed to produce high affinity monoclonal antibodies to drug haptens in approximately 1 month; whereas, conventional techniques typically require 3-9 months. Polyclonal antiserum development typically requires at least 6 months. Consequently, RIMMS has a clear impact allowing reagent antibodies to be available earlier in the drug development process. Isotyping studies demonstrated that the developed antibodies are either IgG1 or IgG2b immunoglobulins which confirms that the technique produces class-switched, affinity matured reagent antibodies. The most promising monoclonal antibody for quantitative applications afforded similar sensitivity, by competitive ELISA, to the established sheep polyclonal anti-ranitidine sera. The calibration range, estimated as the limits between the asymptotic regions of calibration graphs, is 0.5-41.2 ng ranitidine per well. Specificity studies indicated that the monoclonal antibody afforded superior selectivity, yielding only 4.1% cross-reactivity with the ranitidine sulphoxide metabolite; the corresponding value for the antiserum was 8.6%. Both reagents had similar cross-reactivities with the N-oxide metabolite.

Identification of tyrosine phosphorylated adhesion proteins in human cancer cells.

Tyrosine phosphorylation is a form of signal transduction that regulates cell growth, differentiation, migration, and survival. This knowledge has promoted much interest in the role of tyrosine kinases and phosphatases in regulating cell behavior during development and tumorigenesis. However, it is generally less well appreciated that tyrosine phosphorylated proteins are enriched within sites of cell adhesion, particularly in transformed cells. To identify these, we developed a panel of monoclonal antibodies specific for tyrosine phosphorylated proteins in breast cancer cells, using extensive modifications of existing technologies for immunization, somatic fusion, and antibody screening. Mice were immunized with a complex mixture of phosphotyrosine containing proteins using the newly developed RIMMS method. By increasing the sensitivity of antigen recognition, we isolated reagents specific for a wide diversity of tyrosine phosphorylated adhesion proteins in breast cancer cells.

High-affinity monoclonal antibodies to PED/PEA-15 generated using 5 microg of DNA.

Class-switched, affinity-matured murine monoclonal antibody (MAb) producing cell lines reactive with PED/PEA-15 were generated and isolated in less than 4 weeks following polynucleotide immunizations using only 5 microg of DNA in conjunction with the Powderject gene gun. Somatic fusions of peripheral lymph node cells were performed 13 days after initiating delivery of DNA encoding the target antigen. The data presented demonstrates the rapid production, identification, and characterization of class-switched, affinity-matured MAbs that bind PED/PEA-15. The reported strategy enabled the rapid development of MAbs that are useful in enzyme-linked immunoadsorbent assay (ELISA), Western blotting, and immunoprecipitations.

Development of class-switched, affinity-matured monoclonal antibodies following a 7-day immunization schedule.

Previously we reported making high-affinity monoclonal antibodies (MAbs) 13 days after the onset of Repetitive Immunizations Multiple Sites (RIMMS) strategy. The Ig subclass variety and affinity of these antibodies suggested that maturational processes had already begun within draining lymph nodes. We now demonstrate that this diversity can in fact be captured as early as Day 7. In the work reported here, somatic fusion of immune lymphocytes isolated from peripheral lymph nodes resulted in the isolation of affinity-matured MAbs reactive with cytosine deaminase. This model further demonstrates and substantiates at a cellular level the rapid development and maturation of T-cell-dependent B-cell responses occurring within draining lymph nodes following antigen challenge.

Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor.

Class-switched, affinity-matured murine monoclonal antibody (MAb)-producing cell lines were generated against the Flt-3 receptor in less than 4 weeks following polynucleotide immunizations, used in conjunction with repetitive immunizations, multiple sites (RIMMS). Plasmid DNA encoding Flt-3/Fc was coated onto gold particles, which were subsequently propelled into the epidermis of mice using biolistic particle bombardment using the Accell gene gun. Pools of immune peripheral lymph node cells were somatically fused 13 days after the onset of delivery of DNA encoding the target antigen. To determine if early responses could be augmented, DNA-encoding murine GM-CSF was delivered 3 days prior to the Flt-3/Fc DNA immunizations. The data presented demonstrates the successful identification and characterization of class-switched, affinity-matured MAbs that bind to the Flt-3 receptor. When compared to conventional methodologies or intramuscular targeted DNA-based immunization for the generation of MAbs, use of the gene gun in conjunction with RIMMS allows for a more rapid production of affinity-matured MAb-producing cell lines.

Monoclonal antibodies generated against recombinant ATM support kinase activity.

We report on the rapid generation of two monoclonal antibodies, ATM A16.35 and ATM D16.11, that bind to the kinase domain of mutated ataxia telangiectasia (ATM). These antibodies were generated against E. coli-expressed recombinant protein using the RIMMS strategy. We show that ATM A16.35 binds ATM by Western blot analysis, and ATM D16.11 forms immune complexes with native ATM in immunoprecipitations without neutralizing kinase activity.

Rapid development of affinity matured monoclonal antibodies using RIMMS.

Affinity matured murine monoclonal antibody producing cell lines can now be rapidly generated using a novel repetitive, multiple site immunization strategy designated RIMMS. RIMMS capitalizes on rapid hypermutation and affinity maturation events which occur in B cell populations localized within secondary lymphatic tissue early in response to antigenic challenges. A murine myeloma cell line, P3XBcl-2-13, stably transfected with Bcl-2, enhances the outgrowth of hybridomas following somatic fusion with immune lymphocytes isolated from pooled peripheral lymph nodes (PLN) 8-14 days after the initial immunization. Immunizations somatic fusion, screening and isolation of affinity matured IgG secreting monoclonal antibody cell lines occur within a one month time period. By using RIMMS, we have been able to expedite the isolation of affinity matured monoclonal antibodies to numerous antigens, including a drug hapten.

The production and characterization of murine monoclonal antibodies to human Gadd45 raised against a recombinant protein.

The production of two different murine monoclonal antibodies to human Gadd45, a protein that is induced in response to DNA damage, is reported. Antibodies were generated in a SJL mouse using a recombinant form of the human Gadd45 protein. Monoclonal antibody 4TCYA1, which recognizes the denatured form of human Gadd45 in Western blots, was selected based upon the recognition of Gadd45 induced by functional p53 in the human myeloid leukemia cell line, ML-1. A second monoclonal antibody, designated 30T.14, immunoprecipitates native human Gadd45 in lysates produced from RKO cells, a colorectal carcinoma cell line that expresses relatively high basal levels of Gadd45, as well as from cell lysates made from ML-1 cells after exposure to ionizing irradiation (IR). Since 4TCYA1 fails to immunoprecipitate Gadd45, and 30T.14 fails to bind to IR-induced Gadd45 in immunoblotting, these two monoclonal antibodies probably recognize different epitopes.