The design, errors and costs of a local adult oral health survey.

OBJECTIVE: To evaluate the errors and costs of a sampling and survey design for producing reliable estimates of oral health in a demographically diverse local population with a potentially high non-response rate. BASIC RESEARCH DESIGN: A two-stage cluster sample of 45-54 year old adults was drawn from a sampling frame of patients registered with local GPs and cross checked against the electoral register. The modes of data collection were interview/examination and questionnaire. Statistical properties of the estimates were evaluated and costs of the survey described. MAIN OUTCOME MEASURES: The frequency of categories of non-response were described. Sample characteristics were compared with (1) the local population's (1991) census distribution with respect to gender, ethnic composition and socio-economic group (SEG); (2) local utilisation data. The bias effects on oral health values of gender, SEG and non-response, were estimated. The design effect was described for mean number of teeth and proportion with 21 teeth or more. Optimal primary and secondary sample sizes, and non-response follow-up fractions, were estimated. CONCLUSION: This paper describes and costs a sampling and survey design which efficiently produced statistically acceptable estimates of oral health, using small samples from a selected age group in a district, posing demographic and non-response problems in ensuring reliable epidemiological results. The findings indicate that national surveys may give reasonable projected estimates for even richly diverse local populations.

alpha IIb beta 3 redistribution triggered by receptor cross-linking.

Fibrinogen binding to alpha IIb beta 3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/alpha IIb beta 3 complexes on the platelet surface. Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and individual fibrinogen molecules have been demonstrated to trigger receptor redistribution. Here we examine the respective roles of receptor cross-linking and ligand occupancy of receptors in initiating this movement. Monovalent, alpha IIb beta 3-binding fibrinogen fragments RGDS and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting that ligand binding to a single receptor is an insufficient stimulus. Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did not trigger receptor movement. However, cross-linking these receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by conjugation of the anti-receptor antibody to large colloidal gold particles triggered receptor redistribution identical in rate, pattern, and final distribution to that previously seen with fibrinogen binding. We conclude that receptor cross-linking provides the signal for initiation of fibrinogen/alpha IIb beta 3 complex redistribution on platelet surfaces.

Self-association of bound fibrinogen on platelet surfaces.

Binding of fibrinogen to receptors on the surfaces of activated platelets triggers movement of the ligand-bound receptors. In this study this process was followed by using native and colloidal gold-labeled fibrinogen. Both labeled and unlabeled proteins on platelet surfaces could be visualized by low-voltage, high-resolution scanning electron microscopy. Fibrinogen and gold-conjugated fibrinogen were observed to bind to platelet surfaces and to trigger identical patterns of receptor-ligand complex redistribution. In addition to previously described long- and short-range translocation patterns, fibrinogen, either unlabeled or conjugated to gold particles, formed small, specific, nonfibrillar aggregates after binding to platelet surface receptors. Similar triggering and movement resulted from binding of gold-conjugated antibody to the fibrinogen receptor, but no subsequent self-association of the antibody-gold was observed.

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.

Evidence for two mechanisms of ligand-receptor movement on surface-activated platelets.

The movement of platelet membrane glycoproteins was studied by correlative light and electron microscopy. Two distinct classes of movement were seen. In the first type, binding of colloidal gold-labeled ligand or antibody to the fibrinogen receptor (GPIIb/IIIa) triggered a long-range centripetal movement of the receptor-ligand complexes, in the plane of the membrane, on the surfaces of fully spread platelets. This movement was dependent on the presence of the intact cytoplasmic actin cytoskeleton, as evidenced by its inhibition by treatment of the platelets with cytochalasins D and E to disrupt actin filaments. A second surface receptor, the platelet von Willebrand's factor receptor (GPIb), did not undergo this long range, actin-dependent movement in response to antibody binding. However, in a second class of movement, both GPIb and GPIIb/IIIa were translocated on the platelet membrane. Both receptors, GPIb and GPIIb/IIIa, in response to binding of gold-labeled antibody or ligand, accumulated in the channels of the open canalicular system when these were present, and between platelets, under the platelet margins at the sites of platelet-platelet contact. This type of movement occurred only over a much shorter distance than the centripetal movement seen with ligand-bound GPIIb/IIIa and was not affected by the disruption of filamentous actin by the cytochalasins. These results suggest that there are two active mechanisms driving ligand-receptor movement on surface-activated platelets, and that one receptor type may be driven by either mechanism. However, each mechanism predominates at a different stage in the sequence of platelet activation, adherence, and spreading, consistent with differences in function related to receptor-ligand complex formation at the different stages.

Cytochalasin D and E: effects on fibrinogen receptor movement and cytoskeletal reorganization in fully spread, surface-activated platelets: a correlative light and electron microscopic investigation.

This study investigates the involvement of actin microfilaments in fibrinogen receptor redistribution and cytoskeletal reorganization that takes place in fully spread, surface-activated platelets. Colloidal gold-labeled fibrinogen (Fgn-Au label) in conjunction with video-enhanced differential interference contrast light microscopy (VDIC) was used to identify fibrinogen binding sites, glycoprotein IIb/IIIb (GPIIb/IIIa), on fully spread platelets. Platelets were treated with cytochalasins D and E (5 x 10(-5) mol/L to 5 x 10(-8) mol/L) for 10 minutes, before or after incubation with Fgn-Au label. Results observed with VDIC were subsequently confirmed by high-voltage transmission and low voltage-high resolution scanning electron microscopic examination of the specimens. Preincubation of activated platelets with cytochalasin D or E (5 x 10(-5) and 5 x 10(-6) mol/L) inhibited fibrinogen receptor redistribution and abolished cytoskeletal reorganization in fully spread platelets. After surface-activated platelets were incubated with Fgn-Au label, treatment with the above concentrations of cytochalasin D or E disrupted cytoskeletal reorganization and caused random movement of previously redistributed receptor-ligand complexes. Incubation of platelets with cytochalasin E 5 x 10(-6) mol/L prevented platelet activation and spreading. Thus, actin filaments appear necessary for platelet spreading from the discoid to the fully spread stage. The ligand-triggered, cytoskeletally directed movement of fibrinogen receptors in fully spread platelets appears to be dependent on the presence of intact, polymerized actin. This movement is distinct from the cytochalasin-insensitive accumulation of GPIIb/IIIa-ligand in the channels of the open canalicular system.

Optimizing parameters for correlative immunogold localization by video-enhanced light microscopy, high-voltage transmission electron microscopy, and field emission scanning electron microscopy.

Correlative video-enhanced light microscopy, high-voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy were used to examine the binding of colloidal gold-labeled fibrinogen to platelet surfaces. Optimal conditions for the detection of large (18 nm) and small (3 nm) gold particles are described.

Exploratory analyses of caries data.

Some recent developments in statistical technique are applied to exploring published data of caries experience in 5-year old children. The methods are: robust regression and identification of outliers; analysis of compositional data derived from the dmf index; plotting compositional data using a ternary (or Ferrers) diagram, with a 96 per cent contour and principal axes; data-smoothing applied to 3-dimensional graphs of prevalence, severity and treatment response to caries. A strong relationship was found between prevalence and severity of caries, and 16 outlying observations were identified. The smoothed data demonstrated an insufficient treatment response to caries. The role of such analyses is discussed and recommendations made.

Distribution and movement of membrane-associated platelet glycoproteins: use of colloidal gold with correlative video-enhanced light microscopy, low-voltage high-resolution scanning electron microscopy, and high-voltage transmission electron microscopy.

Scanning electron microscopy (SEM), especially low-voltage (1 KeV) high-resolution SEM, can be used in conjunction with stereo pair high-voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface-related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to "prepare" cells for electron microscopy precludes observation in the living state. The use of several types of video-enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy. We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold-labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD-containing adhesive proteins, with gold-fibrinogen or gold-anti-IIb/IIIa. The initial binding and subsequent movement of gold-fibrinogen-IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low-voltage (1 KeV) high-resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position-in-transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).

Observations of colloidal gold labelled platelet microtubules: high voltage electron microscopy and low voltage-high resolution scanning electron microscopy.

18 nm colloidal gold-antitubulin and 4 nm colloidal gold-antitubulin were used to label microtubules in adherent, fully spread platelets. Both sizes of marker effectively labelled microtubules in the partially extracted platelets. However only the 4 nm gold penetrated the dense microfilament matrix of the inner filamentous zone so that portions of microtubules within this cytoskeletal zone could be tracked. The gold marker could be visualized well with 1 MeV high voltage transmission EM and with 5 kV or greater secondary imaging or 20 kV backscattered imaging of carbon only coated samples. 1 kV secondary imaging permitted high resolution imaging of the surface of tubules and the microfilaments with their respective associated material. Individual gold-antibody complexes were difficult to identify by shape alone due to the tendency of the antibody coats to blend together when in very close approximation and due to the presence of other molecules or molecular aggregates similar in size to the gold-antibody labels. Microtubules were seen to wind in and out of the inner and outer filamentous zones as they encircled the granulomere. Some tubules were seen to "dead end" at the peripheral web. Numerous smaller microtubule loops were present principally in the outer filamentous zone and tubules could be followed as they went from the outer filamentous zone through the inner filamentous zone and into the granulomere.

Probe size and bound label conformation in colloidal gold-ligand labels and gold-immunolabels.

Colloidal gold can be produced in sizes ranging from 1.0nm to 150nm. All sizes of gold can be conjugated, principally by hydrophobic bonding, to a variety of molecules including ligands, enzymes and antibodies, as well as lectins and polysaccharides. The activity of most of these biological molecules is retained on conjugation with gold particles irregardless of size range, although the ratio of protein surface area to gold particle surface area varies widely depending on particle and protein size. We have employed low voltage high resolution scanning electron microscopy to compare, microscopically, the shapes of biological molecules unbound, bound to very small (3nm) gold particles, and bound to larger (18nm-30nm) gold particles. When very small gold particles are conjugated to large protein molecules, several particles bind along the length of each molecule, while smaller protein molecules often wrap around a single small gold particle. With larger gold particles, several biological molecules bind to a single gold particle. In addition, the shape of protein molecules bound to larger gold particles differs from that of molecules bound to small gold particles.

Ex vivo platelet deposition on fibronectin-preadsorbed surfaces.

Temporal platelet deposition profiles of canine plasma fibronectin (CPFN) adsorbed to different polymers ex vivo and the in vitro characteristics of CPFN adsorption were studied in an attempt to correlate the two. The maximum platelet deposition (gamma pltmax) obtained at a protein preadsorption time of 30 min was greater than that obtained using an adsorption time of 120 min for all surfaces studied. At 30 min of preadsorption, gamma pltmax was 520,560 and 1230 platelets/1000 micron2 on Biomer, polyethylene (PE) and oxidized PE (OXPE), respectively. In contrast, the platelet deposition at 120 min. of fibronectin preadsorption was about 60 approximately 90 platelets/1000 micron2 on all polymers studied. The surface concentrations of adsorbed CPFN measured using 125I-CPFN, were in the order PE greater than OXPE greater than Biomer. The adsorbed protein concentration increased with increasing adsorption time. The surface distribution of adsorbed CPFN was visualized with antibody-labelled colloidal gold and scanning electron microscopy. The extent of staining was lowest on PE, greater on Biomer, and highest on OXPE, roughly similar to the order of platelet deposition. Platelet deposition ex vivo appears to correlate with the immunogold-stainable-adsorbed protein rather than with the total amount of adsorbed protein.

Utilization of immunogold labeling to compare the adsorption behavior of fibrinogen, fibronectin and albumin on polymers.

Immunogold labeling followed by scanning electron microscopy (SEM) was used to examine the surface distribution of adsorbed plasma proteins. Adsorption was performed under various conditions on six different polymers; [low density polyethylene (PE), chromic acid-oxidized PE (OXPE), solution grade Biomer (SB), Teflon-(FEP), a laboratory synthesized polyurethane containing some zwitterions (ZW) and a polydimethylsiloxane based polyurethane (ZS) also containing zwitterions]. The proteins used were purified human and canine fibrinogen, fibronectin, and serum albumin. The immunogold staining technique was successful in the labeling of the adsorbed proteins. The adsorbed proteins were distributed differently on the polymers selected. Human and canine fibrinogen were found to cover all surfaces in a dense, uniform fashion. Albumin covered most surfaces in a less uniform fashion and on the zwitterionomers covered only a portion of the surface, leaving large bare patches. Fibronectin appeared to deposit unevenly, forming a network on part of the surface and uniformly coating other parts.

Surface characterization of biomaterials by immunogold staining--quantitative analysis.

The labeling of target proteins by immunogold particles has been analyzed based on Einstein's law of Brownian motion. The theory was confirmed from the experiments which employed antifibrinogen gold markers to label fibrinogen molecules adsorbed on the polyethylene surface. The theory predicts that the degree of labeling depends on the concentration of gold markers, temperature, medium viscosity, size of gold markers, and staining time. Of these factors most important is the concentration of immunogold particles. Small change in the marker concentration results in a significant variation in the staining efficiency when other variables are kept constant. The effect of temperature is always accompanied with that of the medium viscosity. There is a linear relationship between the degree of labeling and the temperature when the viscosity effect is combined. The staining of fibrinogen molecules adsorbed on the polyethylene surface at three different temperatures shows a temperature dependence which is in close agreement with the theory. The degree of labeling is inversely related to a square root of the size of gold markers. This analysis makes it possible to maximize the staining sensitivity and to improve the reproducibility of the labeling. Thus, the immunogold staining under a well defined condition allows quantification as well as positive identification and localization of target proteins. This technique has been used to study protein adsorption on biomaterials.

Labeling of sweet taste binding sites using a colloidal gold-labeled sweet protein, thaumatin.

Thaumatin, an intensely sweet tasting protein, was bound to colloidal gold and applied to the taste bud-bearing foliate papillae of Rhesus monkeys. Examination of thin sections of taste pores showed that gold particles were bound to merocrine secretions of Type I taste bud cells, to some cell remnants of lysed cells, and, most importantly, to small, membrane bounded blebs of cytoplasm. These blebs are thought to be shed into the pore from the tips of taste bud cell microvilli, particularly those arising from Type II cells. The binding of gold particles to microvillus tips and to the blebs suggest that this may be an important means by which taste bud cells rid themselves of taste stimulus-receptor complexes.

Iodinating ability of various leukocytes and their bactericidal activity.

A rapid method that employs monolayers of different phagocytic cells, primarily from guinea pigs and mice, has allowed a kinetic determination of (a) ingestion by these cells of labeled particles, (b) fixation of (131)I and (c) microbicidal activity in the cells after periods as short as 5' of exposure of bacteria to phagocytes. Phagocytes so examined included polymorphonuclear leukocytes (PMN) elicited into the peritoneal cavity, elicited peritoneal mononuclear cells (monocytes) (MN), and peritoneal macrophages (MAC) obtained simply by lavage. Circulating PMN from normal human subjects and from children afflicted with chronic granulomatous disease were also studied. The potential for generation of H(2)O(2) (a key component of the iodinating system) of all the normal cells studied, gauged by their content of cyanide-insensitive NADH oxidase, seemed comparable. Peroxidase levels varied widely, and were highest in PMN and almost undetectable in MAC. Catalase was at negligible levels in all the cell types obtained from mice. The fixation of (131)I by phagocytes ingesting (14)C-labeled dead tubercle bacilli appeared to be primarily a function of the cellular peroxidase content. Thus, mouse macrophages, with virtually no peroxidase, displayed no fixation of iodide. PMN proved far more able to fix (131)I during phagocytosis than did MN. In experiments comparing PMN from normal human subjects and from children with chronic granulomatous disease (CGD), a sex-linked condition characterized by a deficiency of H(2)O(2) production during phagocytosis and low microbicidal activity, the iodination ratio of CGD cells was dramatically less than that of normal PMN (by about two orders of magnitude). Capacity for iodination was correlated with bactericidal activity toward E. coli. At low bacterial loads (ca. 5:1), phagocytes killed efficiently, and little discrepancy in ability among cell types was apparent. Under the stress of higher loads of (14)C-labeled E. coli (ca. 100:1), differences in bactericidal activity were exaggerated, and a substantial disparity between MN and PMN was observed in favor of the latter. The hierarchy for killing efficiencies therefore agreed with that for iodination, with one notable exception: mouse MAC were consistently competent in their killing activity, more so than MN, even though they virtually lack peroxidase and the ability to iodinate ingested bacteria.