The impact of organisational characteristics of staff and facility on infectious disease outbreaks in care homes: a systematic review.

BACKGROUND: Infectious disease outbreaks are common in care homes, often with substantial impact on the rates of infection and mortality of the residents, who primarily are older people vulnerable to infections. There is growing evidence that organisational characteristics of staff and facility might play a role in infectious disease outbreaks however such evidence have not previously been systematically reviewed. Therefore, this systematic review aims to examine the impact of facility and staff characteristics on the risk of infectious disease outbreaks in care homes. METHODS: Five databases (MEDLINE, EMBASE, ProQuest, Web of Science, CINAHL) were searched. Studies considered for inclusion were of any design reporting on an outbreak of any infectious disease in one or more care homes providing care for primarily older people with original data on: facility size, facility location (urban/rural), facility design, use of temporary hired staff, staff compartmentalizing, residence of staff, and/or nursing aides hours per resident. Retrieved studies were screened, assessed for quality using CASP, and analysed employing a narrative synthesis. RESULTS: Sixteen studies (8 cohort studies, 6 cross-sectional studies, 2 case-control) were included from the search which generated 10,424 unique records. COVID-19 was the most commonly reported cause of outbreak (n = 11). The other studies focused on influenza, respiratory and gastrointestinal outbreaks. Most studies reported on the impact of facility size (n = 11) followed by facility design (n = 4), use of temporary hired staff (n = 3), facility location (n = 2), staff compartmentalizing (n = 2), nurse aides hours (n = 2) and residence of staff (n = 1). Findings suggest that urban location and larger facility size may be associated with greater risks of an infectious disease outbreak. Additionally, the risk of a larger outbreak seems lower in larger facilities. Whilst staff compartmentalizing may be associated with lower risk of an outbreak, staff residing in highly infected areas may be associated with greater risk of outbreak. The influence of facility design, use of temporary staff, and nurse aides hours remains unclear. CONCLUSIONS: This systematic review suggests that larger facilities have greater risks of infectious disease outbreaks, yet the risk of a larger outbreak seems lower in larger facilities. Due to lack of robust findings the impact of facility and staff characteristics on infectious disease outbreaks remain largely unknown. PROSPERO: CRD42020213585 .

Automatic recording of individual oestrus vocalisation in group-housed dairy cattle: development of a cattle call monitor.

Oestrus detection remains a problem in the dairy cattle industry. Therefore, automatic detection systems have been developed to detect specific behavioural changes at oestrus. Vocal behaviour has not been considered in such automatic oestrus detection systems in cattle, though the vocalisation rate is known to increase during oestrus. The main challenge in using vocalisation to detect oestrus is correctly identifying the calling individual when animals are moving freely in large groups, as oestrus needs to be detected at an individual level. Therefore, we aimed to automate vocalisation recording and caller identification in group-housed dairy cows. This paper first presents the details of such a system and then presents the results of a pilot study validating its functionality, in which the automatic detection of calls from individual heifers was compared to video-based assessment of these calls by a trained human observer, a technique that has, until now, been considered the 'gold standard'. We developed a collar-based cattle call monitor (CCM) with structure-borne and airborne sound microphones and a recording unit and developed a postprocessing algorithm to identify the caller by matching the information from both microphones. Five group-housed heifers, each in the perioestrus or oestrus period, were equipped with a CCM prototype for 5 days. The recorded audio data were subsequently analysed and compared with audiovisual recordings. Overall, 1404 vocalisations from the focus heifers and 721 vocalisations from group mates were obtained. Vocalisations during collar changes or malfunctions of the CCM were omitted from the evaluation. The results showed that the CCM had a sensitivity of 87% and a specificity of 94%. The negative and positive predictive values were 80% and 96%, respectively. These results show that the detection of individual vocalisations and the correct identification of callers are possible, even in freely moving group-housed cattle. The results are promising for the future use of vocalisation in automatic oestrus detection systems.

Anticipation and frequency of feeding affect heart reactions in domestic pigs.

Measuring heart reactions has become a widely used method for the assessment of emotions. Heart rate and its variability, which can quite easily be noninvasively recorded, reflect the inputs of the sympathetic and parasympathetic branches of the autonomous nervous system. We tested the hypothesis that frequent anticipation of a positive event results in an increased state of welfare in pigs, expressed as positive arousal in anticipation of announced feeding as well as lowered heart rate and augmented heart rate variability during resting periods. We used a controlled paradigm with 3 groups of young domestic pigs (Sus scrofa domestica). We compared frequent acoustic announcement of feed delivery (group 1: 3 feedings between 0730 h and 1030 h plus 3 feedings between 1200 h and 1530 h) with the same number of feedings as in group 1 but without a temporal relation to the sound (group 2) and with a fixed-schedule feeding (group 3: 2 feedings at 0600 h and 1500 h). Specific cardiac and behavioral reactions indicated short-term (1 min) anticipation in the conditioned group. In this group, heart rate increased (P < 0.001) mainly through vagal withdraw and behavior became more active (P < 0.001). Only the conditioned group displayed changing heart rate characteristics during the sound. Pigs in the frequent unpredictable feed group reacted to feed delivery with increased heart rates (P < 0.001), whereas the heart-rate characteristics of pigs with the fixed schedule were unchanged during the sound and while the other 2 treatment groups were feeding. Clear evidence for long-term anticipation (over the course of hours) was not present in the data. Comparisons between the 3 treatment groups suggested that in housing conditions where pigs cannot obtain feed by their actions but must wait for feed delivery, feeding at 2 fixed times would be preferred. Animals in this treatment group presented lower resting heart rates at the end of the experiment than animals in the other 2 groups (P < 0.01). Therefore, merely announcing a positive stimulus without giving control to its access is apparently not suitable for increasing welfare.

The "puffed cheek method" to evaluate mucosal thickness: case series.

OBJECTIVES: Mucosal thickness should be considered in implant treatment planning. Needle probing to measure mucosal thickness is invasive and therefore not used in routine diagnosis. The "puffed cheek" method is an established technique to visualize the vestibule in computed tomography (CT). As CT assesses bone availability, a simultaneous mucosal thickness measurement would be useful. The aim of this study was to evaluate the reliability of mucosal thickness measurement in CT with distended cheeks. MATERIALS AND METHODS: Buccal maxillary mucosa thickness was evaluated at four measurement sites in the incisor and molar area of 11 patients. Each site was evaluated via CT with cheek distension and needle probing. Measurement area was identified with the aid of a thermoplastic splint to localize the exact position by a gutta-percha marker point. The comparison between the two methods was performed by Bland-Altman diagram. RESULTS: The mean clinical thickness was 1.17 mm (±0.31) compared to 1.11 mm (±0.31) in CT evaluation. The mean difference between the two methods was 0.07 mm (±0.40; CI-0.14;0.12, P = 0.88, Krippendorff α = 0.38). According to Bland-Altman diagram the mucosal thickness may diverge by up to 0.9 mm from the radiologic thickness. CONCLUSIONS: The two measurement methods may not be interchangeably used. As additional information to three-dimensional bone analyses, CT may be performed as a pre-operative soft tissue analysis at most implant sites with distended cheeks. Nevertheless, this method yields less valid and reliable results than the gold standard.

Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows.

In addition to plasma metabolites and hormones participating as humoral signals in the control of feed intake, oxidative metabolic processes in peripheral organs also generate signals to terminate feeding. Although the degree of oxidation over longer periods is relatively constant, recent work suggests that the periprandial pattern of fuel oxidation is involved in regulating feeding behavior in the bovine. However, the association between periprandial oxidative metabolism and feed intake of dairy cows has not yet been studied. Therefore, the aim of this study was to elucidate possible associations existing between single feed intake events and whole-body net fat and net carbohydrate oxidation as well as their relation to plasma metabolite concentrations. To this end, 4 late-lactating cows equipped with jugular catheters were kept in respiratory chambers with continuous and simultaneous recording of gas exchange and feed intake. Animals were fed ad libitum (AL) for 24h and then feed restricted (RE) to 50% of the previous AL intake for a further 24h. Blood samples were collected hourly to analyze ß-hydroxybutyrate (BHBA), glucose, nonesterified fatty acids (NEFA), insulin, and acylated ghrelin concentrations. Cross-correlation analysis revealed an offset ranging between 30 and 42 min between the maximum of a feed intake event and the lowest level of postprandial net fat oxidation (FOX(net)) and the maximum level of postprandial net carbohydrate oxidation (COX(net)), respectively. During the AL period, FOX(net) did not increase above -0.2g/min, whereas COX(net) did not decrease below 6g/min before the start of the next feed intake event. A strong inverse cross-correlation was obtained between COX(net) and plasma glucose concentration. Direct cross-correlations were observed between COXnet and insulin, between heat production and BHBA, between insulin and glucose, and between BHBA and ghrelin. We found no cross-correlation between FOX(net) and NEFA. During RE, FOX(net) increased with an exponential slope, exceeded the threshold of -0.2g/min as indicated by increasing plasma NEFA concentrations, and approached a maximum rate of 0.1g/min, whereas COX(net) decayed in an exponential manner, approaching a minimal COX(net) rate of about 2.5 g/min in all cows. Our novel findings suggest that, in late-lactating cows, postprandial increases in metabolic oxidative processes seem to signal suppression of feed intake, whereas preprandially an accelerated FOX(net) rate and a decelerated COX(net) rate initiate feed intake.

TRPC1 channels regulate directionality of migrating cells.

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca(2+) channels coordinate these steps by arranging intracellular Ca(2+) signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin-Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca(2+) gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca(2+) gradients. We propose that the Ca(2+) signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration.

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.

Altered vocalization rate during the estrous cycle in dairy cattle.

Correct detection of estrus is a problem in dairy herds. In practice, several procedures exist for detection of estrus besides conventional visual observation by humans. These procedures deliver very different results regarding detection of estrus. It is known that the calls of female mammals can contain information about reproductive status. It is also suspected that the vocalizations of cattle contain information about age, sex, dominance status, and stage in the estrous cycle. In the present study, a methodology for the continuous automatic recording of vocalization of heifers during the periestrous period is presented. It was shown in 10 tethered heifers that the estrous climax results in an increase in vocalization rate. Vocalization rate of heifers increased approximately 84% from d -2 to 0 (related to observed estrus) and approximately 59% from d -1 to d 0. After d 0, vocalization rate decreased about 79%. Increased vocalization was correlated with the visual observation of estrus by humans. We also found 2 different structures in the vocalization of heifers. The harmonic structure showed regular frequency bands, whereas the nonharmonic structure was noisy. The hypothesis that the disharmonic structure increases near the estrous climax was confirmed. Hence, it seems possible to get information about stage of the estrous cycle of dairy cattle by means of monitoring vocalization. The presented method of automatically detecting the rate of cattle vocalization (patent pending) could be used solely or in combination with other automated systems for detecting estrus and could considerably increase current estrus detection rates once its applicability can be demonstrated in nontethered cattle.

Linear prediction coding analysis and self-organizing feature map as tools to classify stress calls of domestic pigs (Sus scrofa).

It is assumed that calls may give information about the inner (emotional) state of an animal. Hence, in the last years sound analysis has become an increasingly important tool for the interpretation of the behavior, the health condition, and the well-being of animals. A procedure was developed that allows the characterization, classification, and visualization of the cluster structures of stress calls of domestic pigs (Sus scrofa). Based on the acoustic model of the sound production the extraction of features from calls was performed with linear prediction coding (LPC). A vector-based self-organizing neuronal network was trained with the determined LPC coefficients, resulting in a feature map. The cluster structure of the calls was then visualized with a unified matrix and the neurons were labeled for their input origin. The basic applicability of the procedure was tested by using two examples which were of special interest for a possible evaluation of the normal farming practice. The procedure worked well both in discriminating individual piglets by their scream characteristics and in classifying pig stress calls vs other calls and noise occurring under normal farming conditions.

Effects of dominance and familiarity on behaviour and plasma stress hormones in growing pigs during social confrontation.

Behavioural patterns and plasma adrenaline, noradrenaline and cortisol responses were studied in domestic pigs with different dominance status during 10-h social confrontation tests with a familiar and an unfamiliar group. Eight trials were carried out, where in each trial two groups of nine growing pigs (12 weeks old) were randomly formed. The pigs with the highest (HR) and lowest (LR) rank from each group were selected as test animals, provided with surgically implanted catheters and kept in single housing for 2 to 3 weeks. After this period, each test animal was introduced once into the familiar and once into the unfamiliar group for 10 h. Introduction of the test animals into the groups caused frequent agonistic interactions during the first 30 min and significantly more agonistic interactions during the confrontation with the unfamiliar group. The agonistic behaviour was accompanied by a rapid increase of plasma catecholamines and cortisol. HR pigs showed significantly higher plasma catecholamine concentrations and more agonistic interactions during the first 30 min compared with the LR pigs. During confrontation with the unfamiliar group, HR pigs experienced more defeats and showed a higher increase of plasma cortisol levels than during the confrontation with the familiar group. No influences of rank or familiarity were found on the other behavioural patterns examined. The results show that agonistic behaviour and activation of the sympatho-adrenomedullary and the hypothalamic-pituitary-adrenal system in pigs during a social confrontation test are determined by the former dominance rank of the animals and the familiarity of the group.

Monitoring of piglets' open field activity and choice behaviour during the replay of maternal vocalization: a comparison between Observer and PID technique.

The paper presents a new system for the automatic monitoring of open field activity and choice behaviour of medium-sized animals. Passive infrared motion detectors (PID) were linked on-line via a digital I/O interface to a personal computer provided with self-developed analysis software based on LabVIEW (PID technique). The set up was used for testing 18 one-week-old piglets (Sus scrofa) for their approach to their mother's nursing vocalization replayed through loudspeakers. The results were validated by comparison with a conventional Observer technique, a computer-aided direct observation. In most of the cases, no differences were seen between the Observer and PID technique regarding the percentage of stay in previously defined open field segments, the locomotor open field activity, and the choice behaviour. The results revealed that piglets are clearly attracted by their mother's nursing vocalization. The monitoring system presented in this study is thus suitable for detailed behavioural investigations of individual acoustic recognition. In general, the PID technique is a useful tool for research into the behaviour of individual animals in a restricted open field which does not rely on subjective analysis by a human observer.

Perlecan is responsible for thrombospondin 1 binding on the cell surface of cultured porcine endothelial cells.

Thrombospondin 1 (TSP1), a high molecular weight glycoprotein of the extracellular matrix, interacts with glycosaminoglycan at the cell surface of porcine endothelial cells (Schön et al., Eur.J. Cell Biol. 59, 329-339 (1992)). In this study we identified and characterized the heparan sulfate proteoglycan (HSPG) responsible for TSP1 binding and uptake in endothelial cells and investigated some properties of the TSP1-proteoglycan interaction. Porcine endothelial cells synthesize proteoglycans containing heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS). CS/DS-containing compounds are present predominantly in the culture medium. On Sepharose CL-4B the cellular proteoglycan fraction yielded two HS-containing compounds with a Kav = 0.18 and Kav = 0.55. Only the larger HS-containing component was sensitive to alkaline treatment and was also found in the medium fraction. Trypsin treatment of endothelial cells revealed that the large HS-containing component represents a cell surface-associated proteoglycan, whereas the smaller fraction represents a pool of intracellular HS-chains. The cellular HSPG is partially localized at the apical cell surface but also incorporated and tightly bound to the subendothelial matrix. Deglycosylation of the high molecular weight HSPG resulted in the identification of a core protein of about 400 kDa. Using specific antibodies, in ELISA assays and in immunoblot analysis we observed that the large HSPG is identical to the extracellular matrix proteoglycan, perlecan. Immunohistochemical studies confirmed the location of perlecan on the apical cell surface and additionally as a dense fibrillar network surrounding the cells. Purified perlecan bound to TSP1 in a dose-dependent manner and the binding was mediated by its glycosaminoglycan side chains. In competition assays using various sulfated polysaccharides, heparin potently inhibited binding of perlecan to TSP1 immobilized on nitrocellulose. Dermatan sulfate was a less effective inhibitor. Calcium bound to TSP1 was found to influence its capacity for binding perlecan. The present data provide evidence that perlecan is required for binding and concentrating TSP1 at the apical surface of vascular endothelial cells during receptor-mediated endocytosis.

Proline 21, a residue within the alpha-helical domain of phiX174 lysis protein E, is required for its function in Escherichia coli.

PhiX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis-trans isomerization of proline residues within alpha-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded alpha-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli. Oligomerization of protein P21G-StrpA was not disturbed.

Simple determination of polysaccharide specific antibodies by means of chemically modified ELISA plates.

A new ELISA technique using Nunc CovaLink NH microtiter plates has been developed to measure anticapsular polysaccharide specific antibodies. Capsular polysaccharide (PS) of Haemophilus influenzae type b (PRP) and pneumococcal antigens types 3, 6, 8, 14, 19, 23 were immobilized on CovaLink NH. These are modified plates with secondary amino groups bound to their surface which, in the presence of a water-soluble carbodiimide as coupling reagent, facilitate the direct binding of polysaccharides. We compared the binding characteristics of PS antigens to CovaLink NH and a conventional polystyrene ELISA plate. Checkerboard titration of PS antigens between 0.04-30 micrograms/ml clearly demonstrated that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 microgram/ml, while binding of PS to the conventional plate was rather poor even at concentrations of 30 micrograms/ml. The CVs for the ELISA ranged from 1.1 to 2.8% for intra-assay comparisons and from 3.6 to 7.3% for inter-assay comparisons. In addition, when PRP-IgG antibodies were determined with the CovaLink NH ELISA and compared with the Farr assay an acceptable correlation ( r = 0.89, p < 0.0001) was obtained. The technique described provides a simple and sensitive tool for evaluating specific immunity to PS antigens.

Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli.

As a tool for determining the topology of the small, 91-amino acid phi X174 lysis protein E within the envelope complex of Escherichia coli, a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.

Cell-associated proteoheparan sulfate mediates binding and uptake of thrombospondin in cultured porcine vascular endothelial cells.

[125I]Thrombospondin (TSP) binds to porcine endothelial cells in a specific, saturable and time-dependent fashion and is endocytosed by a receptor-mediated process. The N-terminal heparin-binding domain is necessary for the interaction with the cell surface. Binding and uptake is inhibited by heparin and to a much smaller extent by other vascular glycosaminoglycans. Chemical modification of lysine and arginine residues of TSP, but not treatment of the molecule with neuraminidase, resulted in a pronounced loss of binding at the cell surface. Treatment of cells with heparitinase but not with chondroitin ABC lyase caused inhibition of binding and uptake of TSP. Inhibition of sulfation of proteoglycans on the cell surface by chlorate leads to a dose and time-dependent inhibition of binding and degradation of TSP. In the presence of chlorate, newly synthesized TSP is not incorporated into the cell matrix but mainly released into the culture medium, whereas localization and incorporation of newly synthesized fibronectin is not altered. A cell surface proteoheparan sulfate was identified as TSP binding macromolecule by affinity chromatography. The data emphasize the role of heparan sulfate proteoglycan as a receptor-like molecule for the specific interaction with thrombospondin.

Interactions between thrombospondin and the small proteoglycan decorin: interference with cell attachment.

Decorin, a ubiquitous small interstitial dermatan sulfate proteoglycan, interacts with several extracellular matrix components, e.g., with type I collagen and fibronectin. Using a solid phase assay it is shown that the intact proteoglycan as well as its glycosaminoglycan-free core protein exhibits with KD values of about 5 nM and 2 nM, respectively, high affinity binding also to thrombospondin. However, the polysaccharide chain was required for an interaction with Sepharose-bound thrombospondin and served itself as ligand. In light of the results of binding studies with an N-terminal heparin-binding fragment of thrombospondin it is concluded that several structural features of thrombospondin and of decorin contribute to the mutual interaction of the two macromolecules. Thrombospondin substrata allowed attachment but prevented spreading of human skin fibroblasts. The addition of decorin or of its glycosaminoglycan-free core protein led to a considerable delay of cell attachment on a thrombospondin substrate. The strength of cell attachment appeared to be reduced. These data support the antiadhesive role of decorin regardless of whether subsequent cell spreading is supported or not.

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.