Vitamin D Status and Parkinson's Disease.

Parkinson's disease (PD) is a complex and progressive neurodegenerative disease, characterized by resting tremor, rigidity, slowness of movement, and postural instability. Furthermore, PD is associated with a wide spectrum of non-motor symptoms that add to overall disability. In recent years, some investigations, from basic science to clinical applications, have focused on the role of vitamin D in PD, often with controversial findings. Vitamin D has widespread effects on several biological processes in the central nervous system, including neurotransmission in dopaminergic neural circuits. Various studies have recorded lower levels of vitamin D in PD patients than in healthy controls. Low vitamin D status has also been correlated with the risk for PD and motor severity, whereas less is known about the effects vitamin D has on cognitive function and other non-motor symptoms. This review aims to better characterize the correlation between vitamin D and PD, clarify the role of vitamin D in PD prevention and treatment, and discuss avenues for future research in this field.

Optimization of Fibrin Scaffolds to Study Friction in Cultured Mesothelial Cells.

To study the friction of cell monolayers avoiding damage due to stress concentration, cells can be cultured on fibrin gels, which have a structure and viscoelasticity similar to that of the extracellular matrix. In the present research, we studied different gel compositions and surface coatings in order to identify the best conditions to measure friction in vitro. We examined the adhesion and growth behavior of mesothelial cell line MET-5A on fibrin gels with different fibrinogen concentrations (15, 20, and 25 mg/mL) and with different adhesion coatings (5 µg/mL fibronectin, 10 µg/mL fibronectin, or 10 µg/mL fibronectin + 10 µg/mL collagen). We also investigated whether different substrates influenced the coefficient of friction and the ability of cells to stick to the gel during sliding. Finally, we studied the degradation rates of gels with and without cells. All substrates tested provided a suitable environment for the adherence and proliferation of mesothelial cells, and friction measurements did not cause significant cell damage or detachment. However, in gels with a lower fibrinogen concentration, cell viability was higher and cell detachment after friction measurement was lower. Fibrinolysis was negligible in all the substrates tested.

Role of MUC1 in lubrication of pleural mesothelial cells cultured on fibrine gel.

To elucidate the role of sialomucin in friction reduction, we investigated the sliding friction of pleural mesothelial cells monolayers cultured on fibrine gel. These measurements were performed on normal (4/4 RM-4) and on tumor (CARM-L1 TG3) cell lines. The effect of treatment with neuraminidase, which removes sialic acid from sialomucin, and of dexamethasone, which has shown to increase sialomucin expression, were also assessed. Furthermore, the expression of the main form of cell-surface-associated mucin (MUC1) present in the mesothelium, was assessed by western blot and immunofluorescence, under different experimental conditions. Expression of MUC1 was not significantly different in the two cell lines. Moreover, dexamethasone did not increase the expression of MUC1. Coefficient of kinetic friction (µ) was significantly higher in tumor cells than in normal cells. Neuraminidase increased µ in both cell lines. These results suggest that sialomucin may play a role in reducing the friction of pleural mesothelial cells.

Effects of Creatine Treatment on Jejunal Phenotypes in a Rat Model of Acidosis.

We investigated the effects of creatine treatment on jejunal phenotypes in a rat model of oxidative stress induced by acidosis. In particular, the activities of some antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase), the level of lipid peroxidation, the expression of heat shock proteins (HSP70), and the expression of the major carriers of the cells (Na+/K+-ATPase, sodium-glucose Transporter 1-SGLT1, and glucose transporter 2-GLUT2) were measured under control and chronic acidosis conditions. Creatine did not affect the activity of antioxidant enzymes in either the control or acidosis groups, except for catalase, for which the activity was reduced in both conditions. Creatine did not change the lipid peroxidation level or HSP70 expression. Finally, creatine stimulated (Na+/K+)-ATPase expression under both control and chronic acidosis conditions. Chronic acidosis caused reductions in the expression levels of GLUT2 and SGLT1. GLUT2 reduction was abolished by creatine, while the presence of creatine did not induce any strengthening effect on the expression of SGLT1 in either the control or chronic acidosis groups. These results indicate that creatine has antioxidant properties that are realized through direct interaction of the molecule with reactive oxygen species. Moreover, the administration of creatine seems to determine a functional strengthening of the tissue, making it more resistant to acidosis.

Diaphragmatic Mobility Loss in Subjects With Moderate to Very Severe COPD May Improve After In-Patient Pulmonary Rehabilitation.

BACKGROUND: The diaphragm changes in COPD lead to functional inefficiency correlated to lung function loss. Muscle-fiber shortening follows lung hyperinflation, which results in a chronic mechanical disadvantage that impairs diaphragmatic mobility that worsens in COPD exacerbations. OBJECTIVES: To correlate the diaphragmatic mobility loss to COPD severity by using M-mode ultrasonography and to verify if the diaphragmatic mobility can improve after in-patient pulmonary rehabilitation. METHODS: We used M-mode ultrasonography to access diaphragmatic mobility during normal breathing or breathing at rest and deep inspiration in 52 subjects with moderate to very severe COPD who underwent pulmonary rehabilitation and 16 healthy subjects. Lung function test, arterial blood gas analysis, and a 6-min walk test were also performed. The measurements were performed at rehabilitation admission and discharge. RESULTS: We screened 30 subjects with severe to very severe COPD who had completed pulmonary rehabilitation. At discharge, inspiratory capacity improved, from 1.58 ± 0.5L to 1.7 ± 0.6 L (P = .04). Diaphragmatic mobility during deep inspirations increased from (mean ± SD) 4.58 ± 1.83 cm to 5.45 ± 1.56 cm (P = .05) after pulmonary rehabilitation. The diaphragmatic mobility during rest breathing was higher in the subjects with COPD (2.25 ± 0.83 cm) than in the healthy subjects (1.27 ± 0.3 cm) (P = .01). The diaphragmatic mobility for the rest breathing and deep inspirations were correlated to an FEV1 decrease (r = -0.74, P < .001; and r = 0.8, P < .001, respectively). CONCLUSIONS: Our findings demonstrated diaphragmatic mobility loss in the subjects with moderate to very severe COPD. These changes were correlated with COPD severity, and diaphragmatic mobility loss improved after in-patient pulmonary rehabilitation. (ClinicalTrial.gov registration NCT02838953.).

Pleural liquid and kinetic friction coefficient of mesothelium after mechanical ventilation.

Volume and protein concentration of pleural liquid in anesthetized rabbits after 1 or 3h of mechanical ventilation, with alveolar pressure equal to atmospheric at end expiration, were compared to those occurring after spontaneous breathing. Moreover, coefficient of kinetic friction between samples of visceral and parietal pleura, obtained after spontaneous or mechanical ventilation, sliding in vitro at physiological velocity under physiological load, was determined. Volume of pleural liquid after mechanical ventilation was similar to that previously found during spontaneous ventilation. This finding is contrary to expectation of Moriondo et al. (2005), based on measurement of lymphatic and interstitial pressure. Protein concentration of pleural liquid after mechanical ventilation was also similar to that occurring after spontaneous ventilation. Coefficient of kinetic friction after mechanical ventilation was 0.023±0.001, similar to that obtained after spontaneous breathing.

Error signals as powerful stimuli for the operant conditioning-like process of the fictive respiratory output in a brainstem-spinal cord preparation from rats.

Respiratory neuromuscular activity needs to adapt to physiologic and pathologic conditions. We studied the conditioning effects of sensory fiber (putative Ia and II type from neuromuscular spindles) stimulation on the fictive respiratory output to the diaphragm, recorded from C4 phrenic ventral root, of in-vitro brainstem-spinal cord preparations from rats. The respiratory burst frequency in these preparations decreased gradually (from 0.26±0.02 to 0.09±0.003 bursts(-1)±SEM) as the age of the donor rats increased from zero to 4 days. The frequency greatly increased when the pH of the bath was lowered, and was significantly reduced by amiloride. C4 low threshold, sensory fiber stimulation, mimicking a stretched muscle, induced a short-term facilitation of the phrenic output increasing burst amplitude and frequency. When the same stimulus was applied contingently on the motor bursts, in an operant conditioning paradigm (a 500ms pulse train with a delay of 700ms from the beginning of the burst) a strong and persistent (>1h) increase in burst frequency was observed (from 0.10±0.007 to 0.20±0.018 bursts(-1)). Conversely, with random stimulation burst frequency increased only slightly and declined again within minutes to control levels after stopping stimulation. A forward model is assumed to interpret the data, and the notion of error signal, i.e. the sensory fiber activation indicating an unexpected stretched muscle, is re-considered in terms of the reward/punishment value. The signal, gaining hedonic value, is reviewed as a powerful unconditioned stimulus suitable in establishing a long-term operant conditioning-like process.

Pleural mesothelium lubrication after phospholipase treatment.

Coefficient of kinetic friction (µ) of rabbit pleural mesothelium increased after short treatment of specimens with phospholipase C. This increase was removed by addition of a solution with hyaluronan or sialomucin, as previously shown in post-blotting Ringer or after short pronase treatment. After phospholipase µ decreased with increase in sliding velocity, but at highest velocity it was still greater than control; this difference was removed by addition of hyaluronan or sialomucin, as in post-blotting Ringer or after short pronase treatment. Hyaluronan placed on specimen before phospholipase treatment reduced increase in µ by protecting phospholipids from enzyme, as shown by others for alveolar and synovial phospholipids. Samples of parietal pleura stained with silver nitrate showed that mesothelial cells were not disrupted by short phospholipase treatment. Instead, they were disrupted if this treatment was preceded by a short pronase treatment; but even after this disruption addition of hyaluronan or sialomucin brought µ back to control.

Mixed lubrication after rewetting of blotted pleural mesothelium.

Coefficient of kinetic friction (µ) of pleural mesothelium blotted with filter paper, and rewetted with Ringer solution markedly increases; this increase is removed if a sufficient amount of sialomucin or hyaluronan is added to Ringer (Bodega et al., 2012. Respiratory Physiology and Neurobiology 180, 34-39). In this research we found that µ of pleural mesothelium blotted, rewetted, and sliding at physiological velocities and loads, decreased with increase of velocity, mainly at low velocities. Despite this decrease, µ at highest velocity was still double that before blotting. With small concentration of sialomucin or hyaluronan µ was markedly smaller at each velocity, decreased less with increase of velocity, and at highest velocity approached preblotting value. These findings indicate a regime of mixed lubrication in post-blotting Ringer, at variance with boundary lubrication occurring before blotting or postblotting with sufficient macromolecule addition. Greater roughness of mesothelial surface, caused by blotting, likely induces zones of elastohydrodynamic lubrication, which increase with velocity, while contact area decreases.

ß2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of ß2-adrenoreceptors (ß2AR). In this research we tried to provide molecular evidence for ß2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of ß2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed ß2AR and GRK2 specific bands.

Na+-glucose cotransporter is also expressed in mesothelium of species with thick visceral pleura.

Molecular evidence for Na+-glucose cotransporter (SGLT1) in rabbit pleural mesothelium has been recently provided, confirming earlier functional findings on solute-coupled liquid absorption from rabbit pleural space. In this research we checked whether SGLT1 is also expressed in pleural mesothelium of species with thick visceral pleura, which receives blood from systemic circulation, but drains it into pulmonary veins. To this end immunoblot assays were performed on total protein extract of scraped visceral and parietal mesothelium of lambs and adult sheep, and of a human mesothelial cell line. All of them showed SGLT1 specific bands. Moreover, confocal immunofluorescence images of lamb pleural mesothelium showed that SGLT1 is located in apical membrane. Therefore, a solute-coupled liquid absorption should also occur from pleural space of species with thick visceral pleura. Because of this protein-free liquid entering interstitium between visceral mesothelium and capillaries, inherent Starling forces should be different than hitherto considered, and visceral pleura capillaries could absorb liquid even in these species.

Pleural liquid and its exchanges.

After an account on morphological features of visceral and parietal pleura, mechanical coupling between lung and chest wall is outlined. Volume of pleural liquid is considered along with its thickness in various regions, and its composition. Pleural liquid pressure (P(liq)) and pressure exerted by lung recoil in various species and postures are then compared, and the vertical gradient of P(liq) considered. Implications of lower P(liq) in the lung zone than in the costo-phrenic sinus at iso-height are pointed out. Mesothelial permeability to H(2)O, Cl(-), Na(+), mannitol, sucrose, inulin, albumin, and various size dextrans is provided, along with paracellular "pore" radius of mesothelium. Pleural liquid is produced by filtration from parietal pleura capillaries according to Starling forces. It is removed by absorption in visceral pleura capillaries according to Starling forces (at least in some species), lymphatic drainage through stomata of parietal mesothelium (essential to remove cells, particles, and large macromolecules), solute-coupled liquid absorption, and transcytosis through mesothelium.

Expression of Na+-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura.

Indirect evidence for a solute-coupled liquid absorption from rabbit pleural space indicated that it should be caused by a Na(+)/H(+)-Cl(-)/HCO(3)(-) double exchanger and a Na(+)-glucose cotransporter [Agostoni, E., Zocchi, L., 1998. Mechanical coupling and liquid exchanges in the pleural space. In: Antony, V.B. (Ed.), Clinics in Chest Medicine: Diseases of the Pleura, vol. 19. Saunders, Philadelphia, pp. 241-260]. In this research we tried to obtain molecular evidence for Na(+)-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura. To this end we performed immunoblot assays on total protein extracts of scraped visceral or parietal mesothelium of rabbits. These showed two bands: one at 72kDa (m.w. of SGLT1), and one at 55kDa (which should also provide Na(+)-glucose cotransport). Both bands disappeared in assays in which SGLT1 antibody was preadsorbed with specific antigen. Molecular evidence for Na(+)/K(+) ATPase (alpha1 subunit) was also provided. Immunoblot assays for SGLT1 on cultured mesothelial cells of rabbit pleura showed a band at 72kDa, and in some cases also at 55kDa, irrespectively of treatment with a differentiating agent. Solute-coupled liquid absorption hinders liquid filtration through parietal mesothelium caused by Starling forces, and favours liquid absorption through visceral mesothelium caused by these forces.

Pleural liquid during hemorrhagic hypotension.

The effect of approximately 25% or 35% blood loss (b.l.) on volume, pressure, and protein concentration of pleural liquid has been determined in anesthetized rabbits in lateral or supine posture. Volume and pressure of pleural liquid did not change with 25% b.l. 30 and 60 min after beginning of hemorrhage, and with 35% b.l. at 30 min (bleeding time approximately 10 and 12 min, respectively). With 35% b.l. protein concentration of pleural liquid was 85% greater (P<0.01) than control; moreover, percent albumin was smaller (P<0.05), and percent globulin greater (P<0.05) than control. Decrease in arterial plasma protein concentration, hematocrit, and pH after hemorrhage fit literature data. Ventilation at 15 and 30 min increased (P<0.01) by 16% and 23%, respectively, with 25% b.l., but it did not change with 35% b.l., a condition borderline to survival in anesthetized rabbits without ad hoc treatment. Pleural liquid seems protected against derangements from hemorrhage up to 25% b.l. for periods shorter than 1 h.

Distribution and mixing of a liquid bolus in pleural space.

Distribution and mixing time of boluses with labeled albumin in pleural space of anesthetized, supine rabbits were determined by sampling pleural liquid at different times in various intercostal spaces (ics), and in cranial and caudal mediastinum. During sampling, lung and chest wall were kept apposed by lung inflation. This was not necessary in costo-phrenic sinus. Here, 10 min after injection, lung inflation increased concentration of labeled albumin by 50%. Lung inflation probably displaces some pleural liquid cranio-caudally, increasing labeled albumin concentration caudally to injection point (6th ics), and decreasing it cranially. Boluses of 0.1-1 ml did not preferentially reach mediastinal regions, as maintained by others. Time for an approximate mixing was approximately 1 h for 0.1 ml, and approximately 30 min for 1 ml. This relatively long mixing time does not substantially affect determination of contribution of lymphatic drainage through stomata to overall removal of labeled albumin from 0.3 ml hydrothoraces lasting 3 h [Bodega, F., Agostoni, E., 2004. Contribution of lymphatic drainage through stomata to albumin removal from pleural space. Respir. Physiol. Neurobiol. 142, 251-263].

Labeled albumin in plasma and removal paths from pleural space in control and increased ventilation.

Increased ventilation was shown to markedly increase lymphatic drainage and plasma content of labeled proteins injected into pleural space relative to control ventilation. These proteins reach plasma by lymphatic drainage: directly through parietal pleura stomata, and indirectly from pleural interstitium, reached by diffusion, convection and transcytosis. Increased drainage from interstitium should not involve a comparable increase in protein removal from pleural space by these transports, while increased drainage through stomata involves a comparable increase in protein removal. Hence, relative increase in labeled protein removal from pleural space caused by increased ventilation should be marked only if drainage through stomata contributed most of this removal, whereas relative increase of labeled proteins in plasma should be marked in either case. We injected 3 ml of albumin-Ringer with albumin-Texas red into the pleural space of three groups of anesthetized rabbits: control, CO2-, or muscle stimulation-increased ventilation. Increased ventilation for 3 h (78 and 61%, respectively) increased (P < 0.01) labeled albumin in plasma by 132 and 106%, respectively, but did not significantly increase its removal. Hence, lymphatic drainage through stomata should not contribute most of liquid and protein removal from pleural space.

Albumin transcytosis from the pleural space.

Occurrence of transcytosis in pleural mesothelium was verified by measuring removal of labeled macromolecules from pleural liquid in experiments without and with nocodazole. To this end, we injected 0.3 ml of Ringer-albumin with 750 microg of albumin-Texas red or with 600 microg of dextran 70-Texas red in the right pleural space of anesthetized rabbits, and after 3 h we measured pleural liquid volume, labeled macromolecule concentration, and, hence, labeled macromolecule quantity in the liquid of this space. Labeled albumin left was 318 +/- 28 microg in control and 419 +/- 17 microg in nocodazole experiments (means +/- SE); hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. Labeled dextran left was 283 +/- 10 microg in control and 381 +/- 21 microg in nocodazole experiments; hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. These findings indicate occurrence of transcytosis from the pleural space. Liquid removed by transcytosis was 0.05 ml/h. This amount times unlabeled albumin concentration under physiological conditions (10 mg/ml) times lumen-vesicle partition coefficient for albumin (0.78) provides fluid-phase albumin transcytosis: approximately 203 microg. h(-1) kg(-2/3). Transcytosis might contribute a relevant part of protein and liquid removal from the pleural space.

Albumin transcytosis in mesothelium.

Apparent permeability to albumin (P(alb)) was measured with (125)I-albumin in specimens of rabbit parietal pericardium from lumen to interstitium (L-I) and from interstitium to lumen (I-L). With albumin concentration (C(alb)) 0.5%, P(alb) (x 10(-5) cm/s) L-I at 37 degrees C was 0.172 +/- 0.019 SE; it decreased to 0.092 +/- 0.022 I-L at 37 degrees C, 0.089 +/- 0.021 L-I at 12 degrees C, and 0.084 +/- 0.018 I-L at 12 degrees C. These findings provide evidence for an active transport L-I, likely transcytosis. With C(alb) 2.5%, 0.05%, and 0.005%, P(alb) L-I at 37 degrees C was 0.188 +/- 0.023, 0.156 +/- 0.021, and 0.090 +/- 0.021, respectively; at 12 degrees C it was 0.089 +/- 0.017, 0.083 +/- 0.019, and 0.087 +/- 0.026, respectively. Hence, active albumin transport ceases with C(alb) 0.005%; P(alb) values I-L at 12 degrees C and with C(alb) 0.005% are similar and provide diffusional permeability. With physiological C(alb) (approximately 1%), active albumin flux was approximately 5 x 10(-4) micromol x h(-1) x cm(-2). Apparent permeability to FITC-dextran 70 (P(dx)) was also measured. P(dx) (x 10(-5) cm/s) L-I at 37 degrees C with C(alb) 0.5% was 0.095 +/- 0.018; it decreased to 0.026 +/- 0.004 I-L (37 degrees C, C(alb) 0.5%), 0.038 +/- 0.007 at 12 degrees C (L-I, C(alb) 0.5%), 0.030 +/- 0.009 with C(alb) 0.005% (L-I, 37 degrees C), and 0.032 +/- 0.011 with nocodazole (L-I, 37 degrees C, C(alb) 0.5%). These findings provide evidence for transcytosis and confirm conclusions drawn from P(alb). Vesicular liquid flow, computed from vesicular dextran flux (fluid-phase only), was approximately 3.5 microl x h(-1) x cm(-2). Transcytosis seems a relevant mechanism, removing protein and liquid from serous cavities.