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.

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.

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.

GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28.

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The evolution of health administration education for public health: responding to a changing environment.

Health administration education in schools of public health has undergone a steady but remarkable evolution over the last five decades. What was once taught was simply an enumeration of statutory requirements and programs managed by public health agencies. This changed dramatically in the 1960s with the incorporation of both theoretical concepts and skills from the fields of public administration and business administration. In the 1990s, the differentiation between training required for public health administration and for health services administration has become increasingly blurred as institutional responsibility for the health of defined populations has necessitated the adoption of the community epidemiology perspective, long the centerpiece of public health programs, by all health services administration programs. The future challenge for programs located in schools of public health is to identify the unique characteristics of public health practice and to prepare graduates to assure that core public health functions are met adequately in the communities in which they will serve.

Characterization of human Gadd45, a p53-regulated protein.

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.

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.

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.

Inpatients with AIDS and AIDS-related complex: economic inpact on hospitals in North Carolina.

To determine the economic impact of acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) cases on North Carolina hospitals, we collected inpatient data from all North Carolina hospitals on charges and number of patients discharged with these diagnoses. More than 97% of the state's hospitals responded to the survey for the study year (1987-1988). There were 540 AIDS/ARC discharges from 58 North Carolina general hospitals and 125 AIDS/ARC discharges from 13 other types of hospitals, for a statewide total of 665 patients. The total general hospital charges for AIDS/ARC inpatients in North Carolina were approximately $7.7 million per year, and almost $2 million of these charges were uncompensated by any insurance. The greatest burden of cost for this care was borne disproportionately by 15 of the 58 general hospitals, accounting for 82% of the discharges.