Characterisation of an antibody coated microcantilever as a potential immuno-based biosensor.

In this study, we investigated the activity, stability, lifetime and re-usability of monoclonal antibodies to myoglobin covalently immobilised onto microfabricated cantilever surfaces. These sensing surfaces are of interest to us in the development of novel cantilever-based immunosensors. For such sensors the antibody layer represents the sensing element while the microcantilever acts as a mechanical transducer. A procedure for producing re-usable biological coatings has been tested with different independent techniques. An Enzyme Linked Immunosorbent Assay (ELISA) was used to determine the presence of an active antibody coating, and to monitor the lifetime and stability of the immobilised antibody. Through this analysis, the activity of the immobilised antibody layer was found to be more stable with the introduction of sucrose, as a stabilising agent. Sucrose was applied to the immobilised antibody layer after each regeneration step. The immobilised antibody was found to have a stable active lifetime for up to 7 weeks. Fluorescence microscopy was used to give information on the distribution of the coating on the gold and silicon nitride sides of the cantilever. Atomic Force Microscopy was used to determine the presence of the biological coating on the cantilever and to obtain information on the surface morphology of the biological element of the sensor. The combined results provide valuable information on the development of an optimised sensing element and demonstrate a set of methods to use for future sensor-to-sensor characterisation. Preliminary experimental results showing the antibody activity against myoglobin, detected with a microcantilever based sensor prototype confirmed the motivations and potentialities of the proposed immunosensing technique.

Sendai virus wild-type and mutant C proteins show a direct correlation between L polymerase binding and inhibition of viral RNA synthesis.

The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of four independently initiated carboxy-coterminal proteins encoded on the P mRNA from an alternate reading frame. Together the C proteins have been shown to inhibit viral transcription and replication in vivo and in vitro and C' binds the Sendai virus L protein, the presumed catalytic subunit of the viral RNA polymerase. To identify amino acids within the C' protein that are important for binding L, site-directed mutagenesis of the gstC' gene was used to change conserved charged amino acids to alanine, generating nine mutants. Additionally, a tenth natural mutant, gstF170S, was also constructed. Six of the gstC' mutants, primarily in the C-terminal half of C', exhibited a defect in the ability to bind L protein. The mutants were assayed for their effect on in vitro transcription and replication from the antigenomic promoter, and the data suggest in all but one case a direct correlation between the ability of C to bind L and to inhibit these steps in RNA synthesis. Further studies with two nonfusion C mutants showed that this correlation was specifically due to the C' portion, and not the gst portion, of the fusion proteins. To study their individual functions, each of the four C proteins was fused downstream of glutathione S-transferase. The gstC', gstC, gstY1, and gstY1 fusion proteins were all able to bind L protein and to inhibit viral mRNA and (+)-leader RNA synthesis, and antigenome replication in vitro. In addition, the nonfusion C, Y1, and Y2 proteins all inhibited transcription. The inhibition of (+)-leader RNA and mRNA synthesis by wt C proteins (nonfusion) showed nearly identical dose-response curves, suggesting that inhibition occurs early in RNA synthesis.

Mutations in the measles virus C protein that up regulate viral RNA synthesis.

The measles virus RNA-dependent RNA polymerase consists of two virus-encoded subunits, the phosphoprotein (P) and the large (L) protein. The P mRNA also codes for a C protein in the +1 reading frame relative to P. The activities of the measles P and C proteins from the vaccine strain, EdB, a wild-type CM strain, and an SSPE P4 strain were investigated using a CAT reporter minigenome assay. CAT is synthesized following replication and transcription of a DI-CAT minigenome supported by individual P, L, and N plasmids expressed in a mammalian expression system. As measured by CAT activity, CMP1 and P4P1 stimulate transcription and replication four- to six- and six- to eightfold, respectively, better than EdP. There are 10 and 16 amino acid changes in the P protein and three and four changes in C in CMP1 and P4P1, respectively, relative to EdP. By constructing chimeric P genes we showed that mutations throughout P4P1 were required for enhanced polymerase activity, while only mutations in the 5'-terminal portion, encompassing the C ORF, of the CMP1 gene mediated stimulation. Abrogation of C expression from the Ed and CM P genes resulted in an increase in RNA synthesis of twofold for CMP1S and four- to fivefold for EdPS. With the addition of C protein expressed from a separate plasmid that contains only the C ORF, EdC reduces viral RNA synthesis more strongly than CMC. These data suggest that EdC and CMC proteins give a differential inhibition that accounts for most of the differences in RNA synthesis by EdP and CMP1.

Body composition of children with myelomeningocele, determined by 40K, urinary creatinine and anthropometric measures.

OBJECTIVE: The purpose of this study was to develop a method to prevent obesity in myelomeningocele (MM) children by determining the effects of growth, calories and physical activity on body composition and to apply this data to clinical practice, specifically, to correlate lean body mass (LBM) with calorie needs and to correlate body fatness with anthropometry measures. METHODS: The body compositions of 14 children (four males and ten females) with MM were estimated from potassium content (40K), urinary creatinine and anthropometry measurements at the beginning and end of a six-month period. Three subjects who did not have MM were also evaluated as controls. Dietary and physical activity and dietary-goals estimates were taken from records kept by the patients after initial discussion of physical activity and dietary goals with the families. RESULTS: The study showed potassium content (40K), or LBM, in children with MM to be approximately 50% of the potassium content (40K) of children without MM. The percentage of LBM correlated with physical activity, but not with the location of neurological lesion. Lean body mass and creatinine excretion also were significantly correlated, indicating that creatinine excretion is a good measure of lean body tissue in these children. Thorax and abdominal skinfolds, as well as total circumferential measurements (waist being highest), showed significant correlations with the percent of body fat. The caloric intake requirement to maintain the growth of a child with MM was found to be approximately 50% of the recommended daily allowance (RDA) for a child without MM of the same age. Changes in LBM were observed in children with MM who increased physical activity over a six-month interval and were greater than in those who reduced calories alone, however not significantly, presumably due to the short duration of the study. CONCLUSION: To prevent obesity, physical activity should begin in infancy; this will increase LBM and thereby calorie needs. Skinfolds and circumferences (abdominal and thorax skinfolds and waist circumference) which significantly correlated with body fat should be used clinically. Calorie needs according to LBM should be reduced to 50% of the RDA or approximately nine cal/cm height for maintenance or seven cal/cm height for weight loss in MM children after age six (possibly at three to four years of age if nonambulatory).

Monitoring clinical quality in Medicaid managed care.

With the recent exponential growth in managed-care plan enrollment by Medicaid beneficiaries, a variety of quality monitoring systems have emerged. This paper argues that existing quality monitoring systems, originally designed for use by employers in the private sector, are a useful starting point for quality monitoring in the Medicaid program, but that substantial enhancements are needed to provide adequate quality assurance for Medicaid beneficiaries. This paper describes the development of a range of indicators for monitoring the clinical aspect of the quality of care for Medicaid beneficiaries and offers a framework for adapting existing monitoring systems to the needs of the Medicaid population. This work is part of a larger collaborative effort by the Connecticut Department of Public Health and the Department of Epidemiology and Public Health of Yale University School of Medicine to develop a blueprint for state government's role in quality assessment and performance management for managed care.

The Medicaid managed care policy consensus for welfare recipients: a reflection of traditional welfare concerns.

An Aid to Families with Dependent Children (AFDC)-Medicaid managed care policy consensus has emerged in the American states. Although there are two main organizational forms--primary care case management and risk-based capitation models--states are converging on the risk-based approach for their AFDC recipients. Risk-based Medicaid managed care for AFDC recipients assumes a distinct purpose and meaning. The reform is not just about cost control and improving access but about enduring welfare concerns: deservingness, need, and empowerment. Despite recent federal policies that have essentially severed the eligibility link between AFDC and Medicaid, state policy elites still conceive of poor families on Medicaid as a "welfare" group. Assumptions about the need for behavior modification and the need to integrate this group into "mainstream" America shape perceptions about why Medicaid managed care is appropriate for AFDC-Medicaid recipients.

Emphasizing patient participation: the key to determining appropriate end-of-life care?

Traditional approaches to end-of-life care have been criticized for both their high cost and their assumption that the physicians knows what is best for the patient. Both fiscal and ethical concerns can be addressed by increased patient participation in treatment choices during end-of-life care.

Urban economic reform and access to health care coverage in the People's Republic of China.

While the economic reforms since 1978 have brought about substantial changes in the rural health care system, there have been many and far-reaching implications for the urban health insurance system as well. This paper describes the evolving urban economy and examines the implications of these changes for access to health care coverage. Provided first is a brief description of the Chinese urban health care system. Three major areas of urban economic reform since 1984 are outlined and the possible effects of these reforms on health care coverage are discussed. The analysis reveals, first, the emergence of insurance coverage inequities under employment-based health insurance as China moves toward a open market economy. Second, the process of focusing on health services in rural and urban areas as separate systems is unnecessary and even counter-productive in light of the emerging integration of China's urban and rural-economies. Finally, some reasons for why China continues to search for employment-based health financing approaches are offered.

Hope in federalism? What can the states do and what are they likely to do?

Although ERISA is a barrier to state health care reform, its hurdle is not as formidable as the financial and political constraints that state governments face. As the states themselves confess, they cannot solve this problem alone (National Governors Association 1993); but if they must, they will focus on the incremental, "low-cost" reform options--insurance reforms and Medicaid managed care. Connecticut Representative Courtney perhaps summarized state sentiment best: "Maybe we shouldn't be using the word 'reform'; it raises expectations" (Spencer-Molloy 1994b). Indeed, states will proceed more cautiously now than just a year ago because the federal failure accentuated the political gamble of health care reform and made the future prospect of transferring federal funds to state governments appear very bleak. Unfortunately, such cautious reforms will not move us toward the goal of universal (or statewide) coverage, a goal that seemed within our grasp only two years ago.

How will we use clinical guidelines? The experience of Medicare carriers.

While support for the development of clinical guidelines is widespread, there is little agreement about how they should be used. Because cost control is the force behind the medical effectiveness movement, the payers' preference, to link clinical guidelines to reimbursement, will likely prevail. We examine the utilization review programs of Medicare Part B carriers and the carriers' attempts to use clinical guidelines to determine medical necessity for the purpose of payment. We find that because the utilization review programs are driven by concerns about cost control, the carriers' actual review process relies on aggregate utilization and spending targets that have little to do with clinical guidelines. The carriers' medical review rhetoric--using the terms medically unnecessary and fraud and abuse as synonyms--also highlights their focus on cost control and the lack of concern about why services are used inappropriately.

A specialized mental health plan for persons with severe mental illness under managed competition.

Many concerns have been raised about the special problems of providing care for severely mentally ill persons in a reformed health care system based on managed competition. The authors describe what will likely be basic features of the reformed system and discuss potential problems in serving this population. The authors recommend the development of special mental health maintenance organizations (MHMOs) that would serve only persons with severe mental illness. The MHMO would emphasize case management in the community and would provide a fixed point of responsibility for clinical care of these patients. Two methods of reimbursing MHMOs are proposed. Each region's health insurance purchasing cooperative (HIPC) could reimburse the MHMO on a capitated risk-adjusted basis. Alternatively, HIPCs could require the general health plan to operate or contract for MHMOs. In each case, the HIPC would provide quality-of-care oversight and assign a team to act as a gatekeeper for referrals to the MHMO.

Deciding on access and levels of care: a comparison of Canada, Britain, Germany, and the United States.

Americans view universal coverage as a reality only if a minimum benefit package is explicitly defined, and discussions about expanding access take place under the slogan of minimum benefits. The policy environment is different in Canada, Britain, and Germany. There, health care costs are controlled and benefits provided under universal coverage plans. Yet the medical services provided in these countries result not from difficult decisions about rationing care at a "minimum" benefit level but from difficult political decisions about the structure of the health care system. Institutional factors rather than explicit policy influence the implicit health priorities in these countries. The United States, in contrast, develops policies that explicitly designate a minimum level of benefits.

The structure and characteristics of rural hospital consortia.

Rural hospital consortia are relatively new organizations that have been developed to help improve the viability of participating hospitals. This paper describes the characteristics of rural hospital consortia in the United States and develops and tests a measurement model of their underlying structure. The measurement model, which characterized consortia structure in terms of degree of member commitment, degree of complexity, scale of operations, and degree of formalization, provided a good fit to the sample data. Most consortia appear to have followed a relatively conservative course that involved the development of programs that had limited sensitivity and financial risk for individual hospitals. This suggests that rural hospital consortia may not become a model for major structural change in the rural health care system. Future research should examine the evolution of rural hospital consortia from an organizational life cycle perspective.

A highly sensitive, nonradioactive DNA labeling and detection system.

A highly sensitive method for detecting specific nucleotide sequences was recently developed. The method uses digoxigenin-labeled nucleic acid probes for hybridization to immobilized target nucleic acids. Probes can be labeled by the random-primed method, nick translation, oligonucleotide tailing, cDNA synthesis, photodigoxigenin or SP6/T7/T3 polymerase-mediated transcription. Hybrids are detected by an enzyme-linked immunoassay using an anti-digoxigenin antibody conjugate. Visualization of the bound antibody is accomplished by an enzymatic color reaction, enzymatic chemiluminescent reaction or immunofluorescence, depending on the antibody conjugate and enzymatic substrate used. Here we report the successful application of this technology in the detection of specific cloned DNA in colony and plaque hybridizations, specific detection of a single mRNA species in Northern blots and single-copy gene detection in genomic Southern blots.

Alternative models for the delivery of rural health services.

A number of alternatives to the solo, fee-for-service physician model have been pursued in an attempt to alleviate some of the specific problems associated with the delivery of primary care in rural areas. This article reviews and critiques the literature published in the 1980s for four alternative models: organized group practices, community health centers, community-oriented primary care and managed care systems. The review examines the strengths and shortcomings of the existing literature, and identifies high-priority research areas for each model. These four alternative models were introduced and promoted during the 1970s with little in the way of research evidence to support them. The subsequent literature pertaining to their performance was primarily descriptive. Of the quantitative studies, many utilized 1970s data. Therefore, the effects of the many environmental changes in rural areas during the 1980s on the viability of these models is unknown. In addition, little, if anything, is known about the life cycle of these models. While longitudinal, cross-organizational studies present several logistic difficulties and require considerable resources for data collection, they hold the promise of extending the existing knowledge concerning alternative models beyond its present states.

Changing physician behavior: does medical review of Part B Medicare claims make a difference?

This study attempts to determine whether the implementation of mandatory review of Medicare Part B claims for medical necessity has resulted in physicians submitting fewer medically unnecessary claims. After summarizing the literature on the effectiveness of various methods for changing physician practices, we compare the rate at which physicians submitted medically unnecessary claims before and after HCFA imposed mandatory review screens. We find that, consistent with expectations from the literature, the screens are only marginally effective (at best) in reducing the rate at which medically unnecessary claims are submitted. We make some suggestions for lowering this rate.

Impact of nutrition on postnatal development of serine-threonine dehydratase and branched-chain keto acid dehydrogenase in the rat.

The developmental patterns of two amino acid-catabolizing enzymes, serine-threonine dehydratase (STD) and branched-chain alpha-keto acid dehydrogenase (BCKAD), were investigated in growing rats. At 10, 15, 20, 30, and 60 days of age, STD and BCKAD activities were determined in pup tissues. Hepatic STD activity increased more than 3-fold between 10 and 20 days of age; after this peak, activity decreased by 30 days of age. Threonine dosing did not affect STD activity at any age. In the liver, kidney, brain and skeletal muscle, total BCKAD activity increased 2- to 4-fold between 10 and 30 days of age. Percentages of BCKAD active in all tissues decreased between 10 and 15 days of age, associated with a large increase in total activity. The percentages of BCKAD activity were unchanged between 20 and 30 days of age but increased from 30 to 60 days of age, associated with a decrease in total activity. Leucine dosing did not affect total BCKAD activity at any age. These results demonstrate that both STD and BCKAD develop late during the suckling period and total activities are unresponsive to excess dietary amino acids.

Effects of adrenalectomy on energy balance in obese (ob/ob) mice fed high carbohydrate or high fat diets.

We reported previously that adrenalectomy reduced the energy density of body weight gain (an indicator of proportional gain in lean and fat tissue) and the efficiency of energy retention in obese (ob/ob) mice to values approximating those in lean mice, but that adrenalectomy had much less influence on these parameters in ob/ob mice fed a purified high fat diet. To determine if fat was the exclusive factor in the purified high fat diet that negated effects of adrenalectomy, ob/ob mice were fed a purified high carbohydrate (glucose) diet identical in composition to the high fat diet, except for the fat/carbohydrate ratio. Responses of adrenalectomized ob/ob mice fed the purified high glucose diet from 4 to 7 wk of age mimicked those of mice fed the purified high fat diet, not those of mice fed the high carbohydrate nonpurified diet. Plasma glucose responses to a glucose load in adrenalectomized ob/ob mice paralleled the diet-dependent changes in energy balance. These results demonstrate that diet composition interacts with adrenal secretions to influence energy and glucose metabolism in ob/ob mice; consumption of either a purified high glucose or high fat diet negates the beneficial effects of adrenalectomy on energy and glucose metabolism observed when adrenalectomized ob/ob mice consume a nonpurified diet.