Inhibition of the pro-inflammatory NF-κB pathway by a grape seed and grape marc meal extract in intestinal epithelial cells.

In pigs and other monogastric animal, the weaning phase is commonly accompanied by an increased susceptibility to gut disorders such as diarrhoea owing to the induction of an inflammatory process in the intestine during weaning. Given the unfavourable effects of intestinal inflammation on feed consumption, digestive capacity of the intestine and growth of animals, controlling intestinal inflammation is a reasonable approach for the maintenance of performance characteristics of livestock animals. Therefore, this study aimed to study the anti-inflammatory potential of a commercial polyphenol-rich grape seed (GS) and grape marc (GM) meal-based feed additive in a well-established in vitro intestinal epithelium model (polarized Caco-2 cells). The anti-inflammatory potential was evaluated by studying the effect of an ethanolic extract obtained from the GS and GM meal-based feed additive (GSGME) on the pro-inflammatory transcription factor NF-κB, which is considered to play a key role in the induction of weaning-associated intestinal inflammation. The highest non-cytotoxic concentrations of the ethanolic GSGME dose dependently reduced TNFα-induced NF-κB transactivation and decreased TNFα-induced mRNA levels of the NF-κB target genes IL-1ß, IL-8, MCP-1 and CXCL1 in Caco-2 intestinal cells (p < 0.05). No effect of the ethanolic GSGME was observed on the cytoprotective Nrf2 pathway in Caco-2 cells as evidenced by an unaltered Nrf2 transactivation and unchanged mRNA levels of Nrf2 target genes, such as GPX-2, NQO1, CYP1A1 and UGT1A1. In conclusion, this study shows that an ethanolic GSGME exerts anti-inflammatory effects in intestinal cells under in vitro conditions. Thus, polyphenol-rich GSGM meal-based feed additives may be useful for the inhibition or prevention of inflammatory processes in the intestine of livestock animals, in particular during states with inappropriate NF-κB activation in the intestinal tissue, such as the weaning phase. Future studies are warranted to prove the in vivo anti-inflammatory potential of GSGM meal-based feed additives.

The behavior of osteoblast-like cells on various substrates with functional blocking of integrin-beta1 and integrin-beta3.

This study was designed to examine the influence of integrin subunit-beta1 and subunit-beta3 on the behavior of primary osteoblast-like cells, cultured on calcium phosphate (CaP)-coated and non coated titanium (Ti). Osteoblast-like cells were incubated with specific monoclonal antibodies against integrin-beta1 and integrin-beta3 to block the integrin function. Subsequently, cells were seeded on Ti discs, either non coated or provided with a 2 microm carbonated hydroxyapatite coating using Electrostatic Spray Deposition. Results showed that on CaP coatings, cellular attachment was decreased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. On Ti, cell adhesion was only slightly affected after a pre-treatment with anti-integrin-beta3 antibodies. Scanning electron microscopy showed that on both types of substrate, cellular morphology was not changed after a pre-treatment with either antibody. With quantitative PCR, it was shown for both substrates that mRNA expression of integrin-beta1 was increased after a pre-treatment with either anti-integrin-beta1 or anti-integrin-beta3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-beta3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-beta1 and beta3 seem to play different roles in attachment, proliferation, and differentiation of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation.

The influence of the crystallinity of electrostatic spray deposition-derived coatings on osteoblast-like cell behavior, in vitro.

This article describes the influence of the crystallinity of carbonate apatite (CA) coatings on osteoblast-like cell behavior. Porous CA coatings were produced with electrostatic spray deposition (ESD), and subsequently, received heat treatments of 400, 500, or 700 degrees C to induce various coating crystallinities. As a result, an amorphous calcium phosphate (ACP), a crystalline CA (CCA), and a crystalline carbonated hydroxyapatite (CHA) structure were formed, respectively. Uncoated titanium substrates served as the control group. After seeding rat osteoblast-like cells, the initial cell attachment was similar between the groups, and approached 100% after 6 h. Between the various coatings, no differences were observed for proliferation, differentiation, or mineralization. However, proliferation of the osteoblast-like cells was lower on all coated substrates after longer culture periods, compared to the uncoated substrates, while at the same time differentiation was stimulated. Furthermore, after 8 and 16 days of incubation, scanning electron microscopy showed more signs of mineralization on coated substrates, compared to the uncoated substrates. In conclusion, porous ESD-derived CA coatings have a positive effect on the in vitro differentiation of osteoblast-like cells, compared to uncoated, as-machined titanium. However, this effect is not further enhanced by the degree of crystallinity of the ESD-derived CA coatings.

Osteoclastic resorption of calcium phosphate coatings applied with electrostatic spray deposition (ESD), in vitro.

Calcium phosphate (CaP) coatings have been applied on titanium implants to improve the bioactivity in order to favor the initial bone healing response. Recently, a new technique has been developed to apply CaP coatings: electrostatic spray deposition (ESD). Although ESD-derived coatings have several benefits, it is not known whether they are degradable. This study was designed to examine the cell-mediated degradation of two ESD-derived coatings with different chemical compositions, that is, beta-tricalcium phosphate (beta-TCP) and carbonate apatite (CA). First, coatings were deposited and analyzed physiochemically. Subsequently, rat bone marrow-derived osteoclastlike cells were seeded on the coatings, and analyzed with osteoclast-specific markers, scanning electron microscopy, and transmission electron microscopy. Results showed that both coatings exhibited porous morphologies, with an average pore size of less than 1 microm (beta-TCP), or larger than 1 microm (CA). After heat treatment, both coatings were crystalline in structure. The Ca/P ratios were 1.4 to 1.5 for the beta-TCP coating, and 1.8 to 2.0 for the CA coating. After 8 and 12 days of culture, multinucleated osteoclastlike cells were observed on both coatings. The osteoclast phenotype was confirmed by tartrate resistant acid phosphatase (TRAP) staining, and immunostaining against the calcitonin receptor. Using scanning electron microscopy, numerous resorption lacunae were observed in both coatings. Finally, transmission electron microscopy of TRAP-positive cells confirmed the osteoclastlike aspect of the cells revealing multiple nuclei and a ruffled border. In conclusion, CaP coatings produced with the ESD process can be degraded by osteoclasts.

Integrins as linker proteins between osteoblasts and bone replacing materials. A critical review.

The adhesion of osteoblasts to substrates is mediated through proteins that have adsorbed to the substrate, providing integrins on the cell membrane with ligands to connect to. The integrins regulate cell behavior through bi-directional signaling pathways. This critical review has the purpose to consider the research that has been performed with osteoblasts, integrins, and bone replacing materials. Until now, most research has been done to investigate the integrin expression of osteoblasts in culture during cellular adhesion. However, it remains difficult to draw general conclusions from this research. Nevertheless, it can be concluded that the used substrates and protein or peptide coatings can influence the integrin expression and cellular behavior. Additional research has to be done to fully understand all the parameters involved in integrin expression, the adhesion of cells to substrates, and the subsequent cellular behavior. For this purpose, model substrates are under development. The signaling pathway is receiving more and more attention, but for biomaterial purposes, too little consideration is paid to the translation of the in vitro results to the in vivo situation, and to practical applications.

Electrostatic spray deposition (ESD) of calcium phosphate coatings, an in vitro study with osteoblast-like cells.

Electrostatic spray deposition (ESD) is a recently developed technique to deposit a calcium phosphate (CaP) coating upon substrates. With this technique, an organic solvent containing calcium and phosphate is pumped through a nozzle. Between the nozzle and substrate a high voltage is applied. As a consequence, droplets coming out the nozzle disperse into a spray, and this spray is deposited upon the substrate. When the solvent has evaporated, a coating is formed on the substrate. ESD allows for a variation in coating composition and morphology. Titanium alloy (TiAl6V4) substrates were coated with a CaP layer using two different methods; radio frequency magnetron sputtering, and ESD. These surfaces were characterized with X-ray diffraction, Fourier transform infrared spectroscopy, an universal surface tester, scanning electron microscopy, and energy dispersive spectrometry. Subsequently, bone marrow cells were isolated from rat femora and cultured 1, 4, 8, 14 and 16 days. Cell proliferation, alkaline phosphatase activity, and osteocalcin concentration were assayed. RT-PCR was done for collagen type I and osteocalcin. SEM was also performed to observe cellular behaviour during culture. Two separate runs of the experiment were performed. In the first run, osteoblast-like cells on both CaP coatings showed similar results in all assays. In the second run, proliferation and osteogenic expression had increased on ESD coatings. On basis of these results, we conclude that the novel ESD coating behaved similar to, or even better than the known RF magnetron sputter coating. Thus, ESD could be a valid addition to already existing CaP coating processes.

Poor positive predictive value of low sensitive thyrotropin assay levels for hyperthyroidism in nursing home residents.

We assessed the positive predictive value of a low thyrotropin (TSH) level on sensitive TSH (STSH) assay as an indicator of hyperthyroidism. In 742 determinations on nursing home residents who were not taking thyroid hormone, we identified 15 with low TSH levels. None of the residents had a completely suppressed (undetectable) TSH level upon initial testing or an elevated total triiodothyronine (T3) or thyroxine (T4) level. Half the patients in whom total T3 was measured had low levels. Of 11 surviving residents, four subsequently had a normal TSH level and six others had a normal free T4 level. Only one patient had a slight elevation of the free T4 level. None of the residents were diagnosed as having hyperthyroidism.

Hyperthyroidism as a cause of atrial fibrillation in long-term care.

BACKGROUND: We measured sensitive thyrotropin-stimulating hormone (sTSH) in 50 consecutive nursing home patients (39 men) with atrial fibrillation to determine the frequency of hyperthyroidism. METHODS: Patients were identified in a long-term care facility by an electrocardiogram demonstrating atrial fibrillation. The sensitive thyrotropin-stimulating hormone assay had a detection limit of less than or equal to 0.02 mU/L (normal range, 0.46 to 3.60 mU/L). RESULTS: No subject had a suppressed sensitive thyrotropin-stimulating hormone. CONCLUSIONS: Hyperthyroidism is not a common cause of atrial fibrillation in male nursing home residents.

Analysis of angiotensin II binding to human platelets: differences in young and old subjects.

We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125I-AII alone, with 125I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects.

Platelet angiotensin receptors in young and old humans.

Platelets from healthy human volunteers were studied for angiotensin II (AII) binding sites. Platelets were isolated from whole blood by differential centrifugation, and binding sites were analyzed by Scatchard plots of radioactive ligand binding data. The number of platelet AII receptor sites was significantly higher in human beings of advanced age compared with younger persons. The affinity of receptor sites was not different in young and old participants. The increased number of binding sites bore no relationship to salt intake as documented by history, plasma renin activity, or blood pressure. A significant portion of the increase in platelet AII receptor sites in older adults in this study is related directly to the age of the individuals.

Increasing the pneumococcal vaccination rate of elderly patients in a general internal medicine clinic.

To improve the pneumococcal vaccination status of an elderly patients group, those older than 64 years of age were identified from a computer file of all continuing care patients in a general internal medicine clinic. In a randomly chosen study group (N = 163), 91 elderly patients (56 per cent) had received the pneumococcal vaccine. Factors associated with a higher rate of pneumococcal vaccination included receiving the previous year's influenza vaccine, a medical problem list attached to the patient's chart, active clinic status (i.e., seen in the year before the study began), and more than two problems listed in the computer record. Letters encouraging pneumococcal vaccination were then sent to patients who had not been vaccinated. Twenty of 72 patients (28 per cent) who received the letter were vaccinated during the next year; 8 per cent of control patients (three of 39) who did not receive the letter were vaccinated. The 95 per cent confidence limit for the relative difference between the study and control group is 6 to 53 per cent. The relative difference was also significant for influenza vaccination between the intervention group and the portion of the control group that had not been vaccinated at the first chart review. Factors associated with pneumococcal vaccination rate following the mailing of the reminder letter were active clinic status and being up to date for either influenza or tetanus vaccination.(ABSTRACT TRUNCATED AT 250 WORDS)