Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation.

G protein-coupled receptors (GPCRs) constitute an abundant family of membrane receptors of high pharmacological interest. Cell-based assays are the predominant means of assessing GPCR activation, but are limited by their inherent complexity. Functional molecular assays that directly and specifically report G protein activation by receptors could offer substantial advantages. We present an approach to immobilize receptors stably and with defined orientation to substrates. By surface plasmon resonance (SPR), we were able to follow ligand binding, G protein activation, and receptor deactivation of a representative GPCR, bovine rhodopsin. Microcontact printing was used to produce micrometer-sized patterns with high contrast in receptor activity. These patterns can be used for local referencing to enhance the sensitivity of chip-based assays. The immobilized receptor was stable both for hours and during several activation cycles. A ligand dose-response curve with the photoactivatable agonist 11-cis-retinal showed a half-maximal signal at 120 nM. Our findings may be useful to develop novel assay formats for GPCRs based on receptor immobilization to solid supports, particularly to sensor surfaces.

Partial reactions of the Na,K-ATPase: determination of rate constants.

Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of patients with spondyloarthropathy seen in specialty clinics with those identified in a communitywide epidemiologic study. Has the classic case misled us?

BACKGROUND: Undiagnosed cases of seronegative spondyloarthropathy (Spa) are often observed during epidemiologic studies. OBJECTIVE: To determine the extent of and the reasons for the underdiagnosis of Spa. METHODS: We studied 2 groups of Alaskan native patients with Spa using a standardized protocol that included an interview, physical examination, medical record review, and radiographic and laboratory examinations. One group consisted of patients identified in a communitywide epidemiologic study; the other group consisted of patients from related but geographically separate populations who had been diagnosed by a specialist in the hospital or a specialty clinic. All cases met the current classification criteria for Spa. The clinical and demographic features of the cases in the 2 groups were compared. RESULTS: Fifty-five (72%) of the 76 community cases that we identified in the epidemiologic study had not been diagnosed previously as Spa. Among the undiagnosed patients were 34 (94%) of the 36 women, 11 (65%) of the 17 patients with ankylosing spondylitis, 12 (36%) of the 33 patients with reactive arthritis, and 24 (100%) of those with undifferentiated Spa. The community and specialty clinic patient groups were similar in age of onset of joint and back pain and in overall symptoms. The specialty clinic group had a higher proportion of men, more severe disease, and a higher frequency of iritis. CONCLUSIONS: The diagnosis of Spa was missed more often than not in the primary care setting, probably because most of the cases were of mild or moderate severity and did not fit the classic descriptions of spondyloarthropathic disorders. The higher proportion of men among the specialty clinic cases probably reflects provider expectation as well as a slightly milder disease course in women.

Discrepancies between patient recall and the medical record. Potential impact on diagnosis and clinical assessment of chronic disease.

BACKGROUND: During a case-control study, data necessary for fulfilling diagnostic and classification criteria for spondyloarthropathy were collected from 121 patients. OBJECTIVE: To study the potential impact of differences between patient recall and the medical record on diagnosis and clinical characterization of spondyloarthropathy as a model of chronic disease. METHODS: The study was conducted among four Alaskan Eskimo populations served by the Alaska Native Health Service. Two sets of historical data were compiled for each subject, one acquired during the interview and the other derived from the medical record. Paired items from the interview and the medical record were analyzed to determine discrepancies and consequent effects on diagnosis, classification, and disease characterization. RESULTS: Significant differences were observed in the reporting of genitourinary or diarrheal illnesses preceding or associated with arthritis, the occurrence of eye inflammation in association with joint pain, the occurrence of joint pain and back pain together, and the age at onset of back pain all of which are important to the diagnosis and classification of spondyloarthropathy. In contrast, for information needed to establish the probable inflammatory nature of back pain, patient interview was more helpful than the medical records, which did not provide adequate details to differentiate inflammatory from mechanical back pain. CONCLUSIONS: Patient recall bias can substantially affect diagnosis and clinical assessment of chronic disease, as exemplified by spondyloarthropathy. Reliance on records alone, however, may lead to underestimation of features that require subjective appraisal by the patient.

Prevalence of low femoral bone density in older U.S. women from NHANES III.

Data on the number of U.S. women with low femoral bone mineral density (BMD) are currently available only from indirect estimates. We used dual-energy X-ray absorptiometry (DXA) measurements of femoral BMD from phase 1 of the third National Health and Nutrition Examination Survey (NHANES III, 1988-1991) to estimate prevalences of low femoral BMD in women ages 50 years and older using an approach proposed recently by an expert panel of the World Health Organization (WHO). Cutpoints for low BMD were derived from BMD data of 194 non-Hispanic white (NHW) women aged 20-29 years from the NHANES III dataset. The prevalence of older U.S. women with femoral osteopenia (BMD between 1 standard deviation [SD] and 2.5 SD below the mean of young NHW women) ranged from 34-50% in four different femur regions, which corresponds to approximately 12-17 million women. The prevalence with osteoporosis (BMD > 2.5 SD below the mean of young NHW women) ranged from 17-20%, or approximately 6-7 million women. Prevalences were 1.3-2.4 times higher in NHW women than non-Hispanic black women (NHB), and 0.8-1.2 times higher in NHW versus Mexican American (MA) women. The estimated numbers of NHW, NHB, and MA women with osteopenia were 10-15 million, 800,000-1.2 million, and 300,000-400,000, respectively; corresponding figures for osteoporosis were 5-6 million, 200,000-300,000, and 100,000 respectively. Thus, the first data on BMD from a nationally representative sample of older women show a substantial number with low femoral BMD.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of low femoral bone density in older U.S. adults from NHANES III.

Most estimates of osteoporosis in older U.S. adults have been based on its occurrence in white women, even though it is known to affect men and minority women. In the present study, we used dual-energy X-ray absorptiometry measurements of femoral bone mineral density (BMD) from the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) to estimate the overall scope of the disease in the older U.S. population. Specifically, we estimate prevalences of low femoral BMD in women 50 years and older and explore different approaches for defining low BMD in older men in that age range. Low BMD levels were defined in accordance with an approach proposed by an expert panel of the World Health Organization and used BMD data from 382 non-Hispanic white (NHW) men or 409 NHW women ages 20-29 years from the NHANES III dataset. For women, estimates indicate 13-18%, or 4-6 million, have osteoporosis (i.e., BMD > 2.5 standard deviations [SD] below the mean of young NHW women) and 37-50%, or 13-17 million, have osteopenia (BMD between 1 and 2.5 SD below the mean of young NHW women). For men, these numbers depend on the gender of the reference group used to define cutoff values. When based on male cutoffs, 3-6% (1-2-million) of men have osteoporosis and 28-47% (8-13 million) have osteopenia; when based on female cutoffs, 1-4% (280,000-1 million) have osteoporosis and 15-33% (4-9 million) have osteopenia. Most of the older U.S. adults with low femur BMD are women, but, regardless of which cutoffs are used, the number of men is substantial.

Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes.

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thioethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50-60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 A, which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopepitde concentration in the outer monolayer.

Design and conduct of collaborative international epidemiologic studies of rheumatic diseases.

Collaborative international epidemiologic studies are important endeavors for improving our understanding of health and disease. They can help answer questions relating to genetic and environmental factors that may plan a role in the etiology and pathogenesis of the disease under study. These studies are challenging but can be very rewarding. There are many opportunities for collaborative international epidemiologic studies in rheumatology which can be explored.

Arthritis and other rheumatic conditions: who is affected now, who will be affected later? National Arthritis Data Workgroup.

OBJECTIVE: We present national and state estimates for 1990 and projections for the year 2020 of the prevalence of self-reported arthritis and other rheumatic diseases and related activity limitations. We further suggest a research and policy agenda to address this important and growing public health problem. METHODS: Estimates and projections were derived from household interviews conducted for the 1989-1991 National Health Interview Survey, and were applied to United States census population estimates for 1990 and projections for 2020. RESULTS: The prevalence rate of self-reported arthritis and other rheumatic conditions in the United States is projected to increase from 15.0% (37.9 million) of the 1990 population to 18.2% (59.4 million) of the estimated 2020 population. Activity limitation attributed to these conditions is projected to increase from 2.8% (7.0 million) of the 1990 population to 3.6% (11.6 million) of the 2020 population. Prevalence rates were higher for older persons, women, residents of nonmetropolitan areas, and those with less education or lower income. CONCLUSIONS: Arthritis and other rheumatic conditions are frequent and disabling public health problems now, and are projected to become even more so by 2020. Implementing the suggested research and public health agenda could reduce the occurrence and impact of these conditions.

Estimating the incidence and prevalence of rare rheumatologic diseases: a review of methodology and available data sources.

Where the need for descriptive epidemiology is great, population based registries can be established, at considerable cost, to provide the desired data. In many instances, however, there may be existing data and information systems that provide morbidity information on sufficiently large, well defined populations to allow reasonable estimates of the incidence and prevalence of rare rheumatologic diseases.

National Center for Health Care Technology assessment of therapeutic apheresis for rheumatoid arthritis, 1981.

The National Center for Health Care Technology coordinated the Public Health Service's assessment of therapeutic apheresis for rheumatoid arthritis in response to the Health Care Financing Administration's request for advice regarding Medicare coverage of this rapidly emerging technology. The information considered included reports available in the medical literature (as of early 1981), advice from the National Institutes of Health and Food and Drug Administration, advice from the appropriate medical societies, as well as information submitted by interested parties in response to a notice published in the Federal Register announcing the assessment. Synthesis of this information provided the basis for concluding that therapeutic apheresis for rheumatoid arthritis was considered experimental with the possible exception of treatment for life-threatening complications such as vasculitis, cryoglobulinemia, or hyperviscosity syndrome.

Incidence of hip fractures in the elderly: a cross-national analysis.

This paper reviews international data on incidence rates of hip fracture in persons 50 years of age and older, based on a bibliographic search of articles published since 1960. Incidence rates are higher in white populations than in black, Asian, and Hispanic populations. In both sexes and in all ethnic groups and geographic areas, incidence rates increase markedly with age. The steep increase with age, however, occurs later in black, Asiatic and Hispanic populations than in whites. The ratio of female to male incidence rates is higher than 1.0 in whites, while in blacks and Asians it has often been the reverse, with higher rates among men. In recent years in Hong Kong incidence rates in females have increased more rapidly than incidence rates in males, so that now the incidence rates in females are higher than those in males. In addition to the study in Hong Kong, most studies in Northern Europe and North America show an increase in age-adjusted hip fracture incidence rates over time over the past few decades. Methodological differences among the various studies (including differences in the definition of hip fracture, in case ascertainment, and in the selection and sample size of the study population) necessitate cautious interpretation of the findings of this report.

Incorporation of rhodopsin in laterally structured supported membranes: observation of transducin activation with spatially and time-resolved surface plasmon resonance.

Rhodopsin-transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with a receptor and its G protein. It served as a model system to show the feasibility to immobilize G protein-coupled receptors on solid supports and investigate receptor activation and interaction with G proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed by the self-assembly of lipids and rhodopsin from detergent solution onto functionalized gold surfaces. They formed micrometer-sized alternating regions of pure fluid phospholipid bilayers separated by bilayers composed of an outer phospholipid leaflet on a gold-attached inner thiolipid. Rhodopsin was found to incorporate preferentially into the phospholipid bilayer regions, whereas transducin was uniformly distributed over the entire outer surface of the supported patterned membrane. The influence of rhodopsin on the dark binding of transducin to lipid membranes was described quantitatively and compared with previously published data. Coupling reactions with transducin resembled closely the native system, indicating that the native functionality of rhodopsin was preserved in the supported membranes. The spatially varying properties of the membranes resulted in a pattern of rhodopsin activity on the surface. This combination of techniques is very promising for the investigation of the lateral diffusion of transducin, can be extended to include signalling proteins downstream of the G protein, and may be applied to functional screening of other G protein-coupled receptors. In the future, it may also serve as a basis for constructing biosensors based on receptor proteins.

Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies.

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

Global statistical tests for comparing multiple outcomes in rheumatoid arthritis trials. MIRA Trial Group.

OBJECTIVE: To evaluate global statistical tests (GSTs) of treatment effectiveness for rheumatoid arthritis (RA) trials measuring multiple outcomes. METHODS: Using outcome measures from American College of Rheumatology (ACR) core set variables available in 3 RA trials, GSTs were calculated using the O'Brien ranking procedure and a procedure for binary data. GSTs take correlations among outcomes into account. Power calculations using 1 trial data set provide comparisons of GSTs and ACR criteria for improvement. RESULTS: Spearman correlations among outcomes ranged from 0.21 to 0.73. Erythrocyte sedimentation rate had the lowest correlation with other outcomes in all 3 trials. Within a trial, joint swelling and joint tenderness or patient and physician assessment had the highest correlations, depending on the trial. Results were consistent with results using the ACR criteria, although the GST was more powerful. CONCLUSION: GSTs are a useful tool for comparing treatment effects across multiple clinically meaningful outcome measures. The GST allows easy inclusion of validated, reliable new measures that are not a part of ACR criteria, such as quality of life, and can be computed with or without selecting a cutoff point defining patient improvement. GSTs should be considered for rheumatic disease treatment trials.

Epidemiology of osteoporosis in women in southern Italy.

Studies on the distribution of bone mineral density (BMD) values in different age groups and in different populations are valuable for understanding the causes of the appreciable geographical variability in fracture incidence. We studied a population of southern Italy in an area where the incidence of hip fracture had been previously estimated. With a completion rate of 85%, we recruited a group of 264 women between 45 and 79 years of age, representative of non-institutionalized and active women in the population, and measured bone density both at the lumbar spine (L1-L4) and at the right femoral neck using a dual X-ray absorptiometry (DEXA) system. We report the age group distribution of BMD in this population. The elderly showed higher mineralization, as compared to an international pooled sample. The prevalence of osteoporosis among women of 50-79 years of age was 40%; the rate changed according to the measurement site. Our results show that a large proportion of women would not have been diagnosed as having osteoporosis if we had relied on a single measurement site. A very low percentage of cases (as low as 4% in the 50-59 years age group) was diagnosed at both sites. The lack of concordance in BMD estimate between measurement sites is significant at younger ages, with an almost dichotomous distribution of cases diagnosed either at the lumbar or femoral site, suggesting the hypothesis that distinct patterns of bone involvement and bone mass lowering exist and all eventually lead to systemic involvement. Longitudinal follow-up of this population should help address some of the questions raised by these results.

Prevalence of spondyloarthropathies in Alaskan Eskimos.

OBJECTIVE: To estimate the prevalence of spondyloarthropathies (SpA) in 2 Alaskan Eskimo populations, using improved methodology for case ascertainment and new, more inclusive classification criteria. METHODS: Through existing rheumatic disease registries, health care providers and the Alaska Area Native Health Service (AANHS) computerized patient information system, we identified all native residents of the 2 study regions with a diagnosis of any inflammatory arthritis or problems characteristic of SpA, such as iritis or persistent back pain. Individuals with such diagnoses or problems were evaluated in clinic, according to a standardized protocol (interview, examination), and by medical record review, pelvic radiography and laboratory tests. Each case was evaluated according to standard diagnostic criteria for the individual disease entities and by the Amor and European Spondylarthropathy Study Group (ESSG) preliminary classification criteria for SpA. RESULTS: We identified 104 cases of SpA in the combined Eskimo populations, an overall prevalence of 2.5% in adults aged 20 and over. Both undifferentiated (USpA) and reactive SpA were more common than ankylosing spondylitis (AS). CONCLUSION: Using the new criteria and a more effective approach to case ascertainment we found the prevalence of SpA to be about twice that found in our earlier studies of adult Eskimo populations. The prevalence of SpA was very similar in men and women. Despite the known high prevalence (25-40%) of HLA-B27 in the study populations we did not find the prevalence of any form of SpA to be as strikingly high as that of AS (6-10%) for the Canadian Haida.

Class I HLA antigens in spondyloarthropathy: observations in Alaskan Eskimo patients and controls.

OBJECTIVE: To assess the role of HLA-B27 and other class I histocompatibility antigens in overall risk and clinical manifestations of spondyloarthropathy (SpA) in Alaskan Eskimos. METHODS: Class I antigens were studied in 104 patients with SpA and in 111 controls. The frequencies of HLA-A, B, and Cw antigens were determined in patients with SpA with various clinical manifestations and compared to frequencies observed in controls. RESULTS: Only HLA-B27 differed significantly in cases and controls. Except for B27, no association of particular antigens with specific syndromes or disease features was found. Patients with B27 had more extraarticular manifestations than patients who lacked B27 antigen. Patients putatively homozygous for B27 did not appear to have more severe disease than those who were heterozygotic. B27 was most closely associated with ankylosing spondylitis [odds ratio (OR) = 210], less so with reactive arthritis (OR = 12.9) and undifferentiated SpA (OR = 4.6). CONCLUSION: Observations in other population groups that implicated B27 cross reactive group (CREG) and other A, B, and Cw antigens as risk factors for developing SpA were not confirmed in Alaskan Eskimos. Nor were CREG or other B antigens either alone or in combination with B27 associated with specific clinical syndromes. Only HLA-B27 was strongly associated with disease and with extraarticular manifestations.

Spondyloarthropathy in the community: differences in severity and disease expression in Alaskan Eskimo men and women.

OBJECTIVES: To determine the relative severity and compare the clinical expression of spondyloarthropathy (SpA) in men and women. METHODS: A clinical study was conducted in 43 women and 40 men who made up 80% of all individuals identified as having SpA in a community-wide epidemiologic study of Alaskan Eskimos. The study included interviews, physical, laboratory, radiographic and electrocardiographic examinations, record reviews, and functional assessments. A measure of relative severity was developed to evaluate disease impact in individual patients. The results in men and women were compared. RESULTS: No significant differences between men and women were found in many features, including the age of onset, frequency of inflammatory joint swelling or inflammatory back pain, physical signs of sacroiliitis, presence of skin changes, or positive family history of SpA. Women were less likely to have sacroiliac joint fusion, advanced spinal changes, uveitis, severe cardiac conduction and valvular abnormalities, and elevated erythrocyte sedimentation rates. According to our relative severity measure, a smaller proportion of women had severe disease than men. CONCLUSION: Although as many women as men were affected by SpA in the communities studied, severe disease was seen more often in men and a number of disease manifestations were more frequent or more marked in men. These discrepancies in disease severity and expression may contribute to the underdiagnosis of SpA in women and the long standing impression that SpA is a disease predominantly of men.