Sunlight Exposure and Vitamin D Levels in Older People- An Intervention Study in Swedish Nursing Homes.

OBJECTIVES: Older people are recommended to take oral vitamin D supplements, but the main source of vitamin D is sunlight. Our aim was to explore whether active encouragement to spend time outdoors could increase the levels of serum 25-hydroxyvitamin D (25(OH)D) and increase the mental well-being of nursing home residents. DESIGN: A cluster randomized intervention trial. SETTING: Nursing homes in southern Sweden. PARTICIPANTS: In total 40 people >65 years. INTERVENTION: The intervention group was encouraged to go outside for 20-30 minutes between 11 a.m. and 3 p.m. every day for two months during the summer of 2018. MEASUREMENTS: We analyzed serum 25(OH)D before and after the summer. Data from SF-36 questionnaires measuring vitality and mental health were used for the analyses. RESULTS: In the intervention group, the baseline median (interquartile range (IQR)) of serum 25(OH)D was 42.5 (23.0) nmol/l and in the control group it was 52.0 (36.0) nmol/l. In the intervention group, the 25(OH)D levels increased significantly during the summer (p=0.011). In the control group, there was no significant change. The intervention group reported better self-perceived mental health after the summer compared to before the summer (p=0.015). In the control group, there was no difference in mental health. CONCLUSION: Active encouragement to spend time outdoors during summertime improved the levels of serum 25(OH)D and self-perceived mental health significantly in older people in nursing homes and could complement or replace oral vitamin D supplementation in the summer.

The impact of using sagittal abdominal diameter to predict major cardiovascular events in European patients with type 2 diabetes.

BACKGROUND AND AIMS: Obesity is associated with diabetes type 2 and one of the most important risk factors for cardiovascular disease. We explored if sagittal abdominal diameter (SAD) is a better predictor of major cardiovascular events than waist circumference (WC) and body mass index (BMI) in type 2 diabetes. METHODS AND RESULTS: The CARDIPP study consists of a cohort of patients with type 2 diabetes. In this study we used data from 635 participants with no previous myocardial infarction or stroke, with a mean follow-up time of 7.1 years. SAD, WC and BMI were measured at baseline and the end-point was first cardiovascular event, measured as a composite of ICD-10 codes for acute myocardial infarction, stroke or cardiovascular mortality. SAD was significantly higher in the major cardiovascular event group compared to participants that did not suffer a major cardiovascular event during follow-up (p < 0.001). SAD >25 cm was the only anthropometric measurement that remained associated with major cardiovascular events when adjusted for modifiable and non-modifiable factors (hazard ratio 2.81, 95% confidence interval 1.37-5.76, p = 0.005). CONCLUSION: SAD with the cut off level of >25 cm, if confirmed in larger studies, may be used as a more independent risk-assessment tool compared with WC in clinical practice, to identify persons with type 2 diabetes at high cardiovascular risk. ClinicalTrials.gov: NCT01049737.

Abdominal obesity and low-grade systemic inflammation as markers of subclinical organ damage in type 2 diabetes.

AIM: This study aimed to explore the associations between abdominal obesity, inflammatory markers and subclinical organ damage in 740 middle-aged patients with type 2 diabetes. METHODS: Waist circumference (WC) and sagittal abdominal diameter (SAD) were measured, and blood samples were analyzed for C-reactive protein (CRP) and IL-6. Carotid intima-media thickness (IMT) was evaluated by ultrasonography, and aortic pulse wave velocity (PWV) measured with applanation tonometry. RESULTS: Abdominal obesity as determined by SAD and WC was significantly correlated with IL-6 (WC: r=0.27, P<0.001; SAD: r=031, P<0.001), CRP (WC: r=0.29, P<0.001; SAD: r=0.29, P<0.001), IMT (WC: r=0.09, P=0.013; SAD: r=0.11, P=0.003) and PWV (WC: r=0.18, P<0.001; SAD: r=0.21, P<0.001). In multiple linear regressions with IMT and PWV as dependent variables, and age, gender, statin use, systolic blood pressure (SBP), body mass index (BMI), CRP and HbA1c as independent variables, both SAD and WC remained associated with IMT and PWV. On stepwise linear regression and entering both SAD and WC, the association between SAD and PWV was stronger than the association between WC and PWV. CONCLUSION: Both SAD and WC are feasible measures of obesity, and both provide information on inflammation, atherosclerosis and arterial stiffness in type 2 diabetes, while SAD appears to be slightly more robustly associated with subclinical organ damage than WC.

Fibrin glue reduces the dissolution rate of sodium hyaluronate.

Sodium hyaluronate (HA) is known to modulate wound healing and interact with inflammatory reactions. High concentrations of extracellular HA are for example correlated to scarless wound healing. Topical treatment with HA has, however, limited effect due to the rapid clearance of HA in the tissue. In an effort to prolong the dissolution rate and enhance the effect of topically administered HA, HA was incorporated in a cross linked fibrin clot and placed in NaCl. The concentration of HA in the NaCl solution was analysed after 30', 60', 4h, 8h, and 24h. It was found that the dissolution rate of HA incorporated in cross linked fibrin was dramatically decreased in vitro, especially when the HA-fibrin mixture was put at rest and not exposed to a mechanical stress. The findings indicate a new possibility for slow release of HA after topical administration.

Elimination of exogenously injected sodium-hyaluronate from rabbit flexor tendon sheaths.

To obtain information about the elimination of Na hyaluronate (NaHe) deposited in flexor tendon sheaths in rabbits, two flexor tendon sheaths from each hind limb were operatively exposed in 15 animals and each filled with one of four different NaHe preparations, namely: 10 mg/ml, MW 2.6 x 10(6); 10 mg/ml, MW 6.3 x 10(6); 19 mg/ml, MW 3.0 x 10(6); and 19 mg/ml, MW 6.5 x 10(6). One, 3, and 7 days after surgery, the NaHe concentration was measured in frozen specimens from the tendon sheaths. One day after surgery, NaHe concentration in the tendon sheath fluid was close to 10 mg/ml in all groups. Three and 7 days after surgery it was higher in tendon sheaths in which NaHe preparations with a high (19 mg/ml) rather than with a low concentration (10 mg/ml) had been deposited. At 3 days, NaHe concentration was also higher in sheaths that had received a preparation with a high MW (6.5 x 10(6)) than those with a lower MW (2.6 or 3.0 x 10(6)). The findings suggest that within the first 24 h, a dilution of concentrated NaHe preparations takes place, probably as a result of their hyperoncotic properties. After 24 h, there was a slower decline in NaHe concentration when more concentrated solutions (19 mg/ml) and solutions with high MWs (6.5 x 10(6)) were administered. Development of pharmaceutical NaHe preparations with slow elimination from tendon sheaths should reasonably be focused on a solution with a high MW and high concentration.

Alveolar accumulation of hyaluronan and alveolar cellular response in bleomycin-induced alveolitis.

Hyaluronan (HA) accumulating in the alveolar interstitial tissue of rats injured by a single intratracheal instillation of bleomycin has been visualized histologically and assayed. HA was present already by Day 1 after bleomycin treatment, increased to a maximum value on Days 3 and 7 and then declined. A time-dependent relationship between this early connective tissue response and the invasion of inflammatory cells in the alveolar tissue was apparent. The dominating invading cells by Day 1 were granulocytes showing positive staining for the monoclonal antibody OX-42 reflecting the C3b receptor. The numbers of macrophages expressing class II antigens started to increase on Day 1, reaching a maximum on Days 3-7 and then declined. Macrophages were the dominating OX-42+ cells by Day 7. The appearance of W3/13+ cells ("pan-T-lymphocytes") showed a similar pattern to that for the class II expressing macrophages. The number of cells expressing CD4 antigen increased until Day 3 and levelled off on Day 30 whilst the largest number of cells expressing CD8 antigen was seen on Day 30. Few cells expressing B-cell phenotype outside lymph nodules were identified. Alveolar lining epithelial cells, probably epithelial type II cells, expressed class II antigens by Days 3-14. The time-related accumulation of HA and the appearance of T-cells, macrophages and granulocytes expressing signs of activation suggests that these cells may be involved in the early connective tissue response of the lung injured by bleomycin.

Hyaluronic acid in flexor tendon sheath fluid after sheath reconstructions in rabbits. A comparison between tendon sheath transplantation and conventional two stage procedures.

Because the synovial environment has been suggested to be important in limiting the number of postoperative adhesions after flexor tendon surgery, and a high concentration of hyaluronic acid is a characteristic feature of synovial fluid, we have examined the hyaluronic acid concentrations in fluid collected from different kinds of reconstructed flexor tendon sheaths in rabbits. The hyaluronic acid concentration in normal rabbit flexor tendon sheaths was similar to that in normal human sheath and joint fluid. Rabbit tendon sheaths restored by autologous sheath grafts and partial pseudosheaths contained similar or somewhat lower hyaluronic acid concentrations, whereas in complete pseudosheaths the concentrations were considerably lower. The data suggest that tendon sheath reconstructions should consist at least partly of synovial membrane so that they are as similar as possible to normal synovial membrane.

An improved technique for hyaluronan histochemistry using microwave irradiation.

A hyaluronan binding protein (HABP), extracted from cartilage, was biotin-labelled and used for histochemical localization of hyaluronan (HA) in tissue sections. Various tissues were fixed for a mixture of formaldehyde and glutaraldehyde during microwave irradiation. The microwave oven when set at 700 W and 45 degrees C yielded an intense and specific staining of HA. Under these conditions the relative proportion of the two aldehydes did not influence the staining intensity. Aldehyde fixation during microwave irradiation for HA histochemistry, (1) save time, (2) eliminates the use of cetylpyridinium chloride (CPC), and (3) improves the reproducibility.

Hyaluronan reflects the pre-fibrotic inflammation in irradiated rat lung: concomitant analysis of parenchymal tissues and bronchoalveolar lavage.

The pathogenesis of pulmonary fibrosis is a complicated chain of interactions between cells and molecules. During recent years bronchoalveolar lavage (BAL) in patients with various interstitial lung disorders has increased our knowledge of the fibrosis process and focused on new interesting interactions. Here we present an animal model which makes it possible to apply both morphological and immunohistochemical tissue staining to perform bronchoalveolar lavage in the same animal. Irradiation is an established method for experimentally evoking lung fibrosis in animals. Rats received irradiation (30 Gy) to the lower parts of both lungs. Bronchoalveolar lavage was performed in the right lung. The biochemical determination of lavage concentration of hyaluronan (HA) and cellular differential counts were compared with interstitial morphology. Animals were sacrificed and analysed 2, 4, 6, 8 and 10 weeks after irradiation. After 6 weeks a massive increase in connective tissue mast cells was seen in the peribronchial and alveolar-interstitial tissue. This mastocytosis was closely related to a marked increase in HA. It became obvious that, in this model, cellular analysis of BAL fluid did not correctly reflect the cellular changes in the lung interstitium. While BAL revealed a pronounced increase of neutrophils with no--or only very few--mast cells, a concomitant increase in mononuclear cells and mast cells was seen in the lung interstitium. In contrast an increase in HA in BAL correlated well with an increase in HA-deposition in the lung interstitium, indicating that measurement of a non-cellular component, such as HA, may better reflect the tissue inflammation.

The localization of hyaluronan in normal and rejected human kidneys.

Hyaluronan (HYA) is a large glycosaminoglycan with a high capacity to immobilize water. Increased levels of HYA have previously been observed in plasma as well as in affected tissues in various inflammatory conditions. The morphological localization of HYA has, however, not been described in normal or rejected human kidneys. Using a recently developed method for localization of HYA in tissue sections by means of a biotin-labeled hyaluronan binding protein used as a probe, we have now investigated the distribution of HYA in normal and irreversibly rejected human kidneys. In the normal kidney HYA was essentially confined to the medulla. In the rejected kidneys increased amounts of HYA were observed primarily in the cortex and in sclerotic vessels. Incubating tissue sections with hyaluronidase abolished the staining for HYA, showing the specificity of the staining procedure. The increased amounts of HYA of the rejected kidney may play a role in local edema formation, and thereby alter graft function.

Visualization of hyaluronic acid in the anterior segment of rabbit and monkey eyes.

Hyaluronic acid was visualized using a new histochemical technique: the hyaluronic acid-binding region of the cartilage proteoglycan was isolated, linked with biotin and used in histological sections as a ligand for hyaluronic acid. Staining was performed using the avidin-biotin-peroxidase technique. The presence of hyaluronic acid in the anterior segments of rabbit and monkey eyes was studied in fresh-frozen, as well as in fixed paraffin sections; addition of cetyl-pyridinium chloride to the fixative was essential. In both species intense staining was seen in the stroma of the conjunctiva, the connective tissue of the ciliary processes, and on the apical membranes of the corneal endothelium. Species differences were observed in both conventional and unconventional outflow pathways. In rabbits, the tissue located directly adjacent to the aqueous plexus and the connective tissue surrounding the poorly developed ciliary muscle were stained. In monkeys, only the anterior, non-filtering part of the trabecular meshwork and the outer wall of Schlemm's canal showed intense staining in paraffin sections. In frozen sections, the inner wall (cribriform region of the trabecular meshwork) was also stained. The spaces between the anterior part of the ciliary muscle bundles remained almost unstained.

Accumulation of hyaluronan in bronchoalveolar lavage fluid is independent of iron-, complement- and granulocyte-depletion in bleomycin-induced alveolitis in the rat.

Previous studies on bleomycin-induced alveolitis in rats have demonstrated a transient histological accumulation of hyaluronan (hyaluronate or hyaluronic acid) in the alveolar interstitium, corresponding to increases in hyaluronan (HA) levels in bronchoalveolar lavage (BAL) fluid and lung tissue extracts. The accumulation of HA was related to the influx of inflammatory cells, especially polymorphonucleated cells (PMNs) in BAL fluid and the increase in lung water. In this study we have investigated the influence of iron, complement and PMN dependent mechanisms on the early connective response of the lung in the bleomycin rat model. Iron depletion had no effect on HA or the cellular composition of lavage fluid recovered on day 4 post bleomycin. Treatment of bleomycin-injured rats with cobra venom factor (CVF), totally neutralized complement haemolytic activity but had no effect on lavage HA or the cell invasion in BAL. Treatment with anti-neutrophil serum (ANS), reduced blood and lavage PMN by 70-80%, but had no influence on HA levels in BAL. These results suggest that regulatory mechanisms other than those dependent on iron, complement activation or PMN recruitment are responsible for HA accumulation in this fibrosing alveolitis animal model.

High-dose corticosteroids during bleomycin-induced alveolitis in the rat do not suppress the accumulation of hyaluronan (hyaluronic acid) in lung tissue.

A single intratracheal injection of bleomycin in rats induced, 4 days later, a considerable accumulation of hyaluronan (hyaluronate, hyaluronic acid) in the lung tissue. This connective tissue reaction was quantified biochemically by analysing hyaluronan (HA) in bronchoalveolar lavage fluid (BAL) and lung tissue extracts. The molecular weight of the HA recovered during lavage was 0.2-0.3 X 10(6) daltons. The HA accumulation was related to an increase in lung water content and associated with an increased influx of eosinophils, neutrophils and lymphocytes into BAL fluid. High-dose corticosteroid treatment (prednisolone 15 mg.kg-1 bw per day) to bleomycin injured rats had no effect on the lung tissue content of HA, the recovery of HA during BAL or the molecular weight of HA accumulated in the alveolar space. Furthermore, steroids did not influence the increased lung water content, or the appearance of inflammatory cells in lavage fluid. These findings indicate that the early connective tissue response to the bleomycin lung injury is mediated by a mechanism which is unaffected by systemic high-dose corticosteroids.

Accumulation of hyaluronan (hyaluronic acid) in myocardial interstitial tissue parallels development of transplantation edema in heart allografts in rats.

By using biotin-labeled proteoglycan core protein, hyaluronan (hyaluronic acid; HA) was visualized in rat heart grafts at different times (2, 4, and 6 d) after transplantation. In normal, nontransplanted hearts HA was present in the adventitia of arteries and veins and in the myocardial interstitial tissue. An increased accumulation of HA was evident in the edematous interstitial tissue, infiltrated with lymphocytes, on day 4 after allogeneic transplantation, and was even more pronounced by day 6. No apparent increase in HA was seen in syngeneic grafts. Biochemical assay of HA in heart tissue demonstrated that the myocardial content of HA had increased 60% by day 2 after transplantation in allogeneic as well as syngeneic grafts, indicating that surgical trauma may induce some HA accumulation in heart grafts. The extractable amount of HA declined during the following days in the syngeneic grafts, but increased progressively during the development of rejection in the allogeneic grafts, and increased on average three times by day 6. The relative water content also increased progressively during rejection of allogeneic grafts and correlated with the HA accumulation. The interstitial accumulation of HA, a glycosaminoglycan with unique water-binding qualities, is presumably implicated in the development of interstitial edema during rejection of heart grafts.

Lung accumulation of hyaluronan parallels pulmonary edema in experimental alveolitis.

The glycosaminoglycan hyaluronan (HA, hyaluronate or hyaluronic acid) was quantified in rat lung during the development of bleomycin-induced lung injury. Extracted lung HA was measured by a radiometric assay. In control rats, the HA lung content was 95 +/- 5 (SE) micrograms/g of freeze-dried and homogenized lung tissue. After a single intratracheal instillation of bleomycin, the HA content of the lung increased significantly on day 1. Peak HA concentrations on days 3-7 averaged 70% higher than normal lung HA concentrations. From day 7 the HA concentrations progressively declined and had returned to normal by day 30. Qualitative assessment of lung HA has previously demonstrated that HA is accumulated in the edematous interstitial alveolar space during the alveolitis phase of bleomycin injury. Since high-molecular-weight HA has unique hydrophilic properties, another aim of the study was to elucidate the possible link between the increase in lung HA and the development of interstitial-alveolar edema postbleomycin. HA recovered by bronchoalveolar lavage from bleomycin-injured lungs increased with increasing total lung HA and had a mean molecular size of 220,000 +/- 50,000 (SE), indicating the minimum size of HA accumulated in the alveolar space. The relative lung water increased significantly on days 3-7 after bleomycin administration. A close relationship (P less than 0.001) and time dependence between the increase in relative lung water and the increase in lung HA were found.

Hyaluronate binding to intact corneas and cultured endothelial cells.

Sodium hyaluronate (HA) protects the corneal endothelium during cataract surgery. Recently, HA receptors have been found on liver endothelial cells that play an important role in HA catabolism. It is unknown if similar receptors are present on the corneal endothelium. In this study we have used two different methods to follow the interaction of HA with corneal endothelial cells: (1) binding of 3H-HA to cells or intact corneas was determined in the presence or absence of unlabelled glycosaminoglycans after solubilization with KOH, and (2) the HA-binding region of bovine cartilage proteoglycan was used as a histochemical probe and visualized by an avidin-biotin method. 3H-HA bound both to intact rat corneas pretreated with Streptomyces hyaluronidase and to cultured monkey corneal endothelial cells. The fraction-bound 3H-HA increased with time and was saturable. Cultured endothelial cells were estimated to have 1700-2100 binding sites per cell with a binding constant of 5.6-8.5 X 10(9) liters/mol. Furthermore, unlabelled HA displaced the tritiated in a dose-dependent manner and the displacing efficiency was dependent on molecular weight. The histochemical method disclosed that HA forms a continuous layer on the endothelium. If Healon was injected into the anterior chamber, the thickness and staining intensity of this layer increased conspicuously.

Hyaluronan in experimental serous and purulent otitis media.

The concentration of hyaluronan (hyaluronic acid; sodium hyaluronate; HA), a major component of connective tissue, was analyzed by a specific radioassay in tissue samples from normal rat middle ears and in tissue and effusions from middle ears affected by experimentally induced otitis media. In normal ears the pars flaccida of the tympanic membrane contained a considerably higher concentration of HA (60 to 200 micrograms/g) than the pars tensa and areas of the medial wall (2 to 7 micrograms/g). In purulent otitis media the pars flaccida contained less HA than in normal ears. The HA concentrations were lower in purulent (3 to 40 micrograms/mL) than in serous (20 to 190 micrograms/mL) effusions. The weight average molecular weight of HA in serous fluid was high (greater than 10(6] and comparable to that in lymph. It is inferred from the study that the subepithelial tissue and its matrix components, eg, HA, must be considered when attempting to understand the function of the middle ear normally and in otitis media.

Serum hyaluronate levels reflect disease activity in experimental arthritis models.

Serum hyaluronate (HA) levels were measured in rats subjected to adjuvant or type II collagen induced arthritis. As the arthritic lesions developed, both models showed an increase in serum HA levels of approximately 5 times, from a baseline level of 61-126 ng/ml (range). Furthermore a positive correlation was found between HA level and arthritic score. The increase in HA was not related to metabolic impairment, as the half life of serum HA in adjuvant arthritic rats was similar to that of normal rats. Serum HA may thus serve as a useful variable for evaluation of the severity of experimental arthritis.

Accumulation of hyaluronic acid in the alveolar interstitial tissue in bleomycin-induced alveolitis.

By using biotin-labeled proteoglycan core protein and an avidin-enzyme system, hyaluronic acid (HA) was visualized in the lungs of rats at different times (4, 10, and 20 days) after bleomycin injury. Four days after an intratracheal injection of bleomycin, HA was accumulated in the edematous alveolar septa of the focal areas with lung tissue injury. An interstitial cellular infiltrate of mainly lymphocytes was present. In normal rat lung, HA was not seen in the alveolar tissue but confined to peribronchial and perivascular spaces. Ten and twenty days after bleomycin administration, increasing numbers of macrophages were apparent in the alveolar space. Proliferating fibroblasts and deposition of collagen in the alveolar tissue were observed while the diffuse HA accumulation was becoming less prominent in the alveolar interstitial tissue. HA was more distinctly located in the surroundings of proliferating fibroblasts. A few scattered alveolar macrophages showed a positive staining for HA. An increased water content of the lung was most apparent 4 days after bleomycin administration. The accumulation of HA, a glycosaminoglycan with unique qualities to immobilize water, in the alveolar interstitium suggests a role for HA in the alveolar interstitial edema. The appearance of HA in alveolar macrophages might indicate that macrophage phagocytosis contributes to the elimination of HA from inflamed lung tissue.

Elevated levels of serum hyaluronate and correlation with disease activity in experimental models of arthritis.

The serum hyaluronate (HA) concentration was measured in groups of rats immunized for adjuvant or type II collagen arthritis. Serum HA increased as the arthritic lesions developed, correlating with the severity of the disease. This increase in HA was not related to metabolic impairment, because rats with adjuvant arthritis metabolized intravenously administered tritiated HA at a rate similar to that of normal rats. Serum HA levels may be useful as an indicator of synovitis in experimental and possibly in clinical arthritis. Further, this model could serve as an experimental approach for studies of HA metabolism in chronic joint inflammation.