The effect of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> on TSLP,IL-33 and IL-25 expression in respiratory epithelium

Background

Airway epithelium is an active and important component of the immunological response in the pathophysiology of obstructive lung diseases. Recent studies suggest an important role for vitamin D₃ in asthma severity and treatment response.

Objective

Our study evaluated the influence of an active form of vitamin D₃ on the expression of selected mediators of allergic inflammation in the respiratory epithelium.

Material and Methods

Primary nasal and bronchial epithelial cells were exposed to1,25D₃ for 1 hour and were then stimulated or not with IL-4, TNF-α, LPS, and poly I:C. After 24 hours TSLP, IL-33, and IL-25 protein levels were measured in culture supernatants usingELISAandmRNAlevels in cells by real time PCR.

Results

1,25D₃ increased TSLP concentration in unstimulated nasal epithelial cells, but did not influence IL-33 and IL-25 expression. In IL-4-stimulated epithelial cell cultures 1,25D₃ mostly inhibited TSLP and IL-33 expression. In LPS-treated cultures 1,25D₃ decreased IL-33 expression. Simultaneously 1,25D₃ augmented IL-25 production in the same model of stimulation.

Conclusion

Our study revealed the dual nature of vitamin D₃ manifested in both pro- and anti-inflammatory properties observed in airway epithelial cells.

     

The subcommissural organ expresses D<Subscript>2</Subscript>, D<Subscript>3</Subscript>, D<Subscript>4</Subscript>, and D<Subscript>5</Subscript> dopamine receptors

Dopamine receptors have been found in certain populations of non-neuronal cells in the brain, viz., discrete areas of ciliated ependyma and the ependymal cells of the choroid plexus. We have studied the presence of both tyrosine-hydroxylase-immunoreactive nerve fibers and dopamine receptors in the subcommissural organ (SCO), an ependymal brain gland that is located in the roof of the third ventricle and that secretes, into the cerebrospinal fluid, glycoproteins that aggregate to form Reissners fiber (RF). Antibodies against D₂, D₃, D₄, and D₅ dopamine receptors were used in immunoblots of bovine striatum, fresh SCO, and organ-cultured SCO, and in immunocytochemistry of the bovine, rat, and mouse SCO. Only a few tyrosine-hydroxylase fibers appeared to reach the SCO. However, virtually all the secretory ependymal and hypendymal cells of the SCO immunoreacted with antibodies against D₂, D₄, and D₅ receptors, with the last-mentioned rendering the strongest reaction, especially at the ventricular cell pole of the secretory ependymocytes, suggesting that dopamine might reach the SCO via the cerebrospinal fluid. The antibodies against the four subtypes of receptors revealed corresponding bands in immunoblots of striatum and fresh SCO. Although the cultured SCO displayed dopamine receptors, dopamine had no apparent effect on the expression of the SCO-spondin gene/protein or on the release of RF-glycoproteins (SCO-spondin included) by SCO explants, suggesting that dopamine affects the function(s) of the SCO differently from the secretion of RF-glycoproteins.Financial support was provided by grants PI 030756 and Red CIEN, Instituto de Salud Carlos III, Spain (to J.M.P.F.), and 1030265 from Fondecyt, Chile (to E.M.R.)     

Ghrelin and insulin gene expression changes in streptozotocin-induced diabetic rats after rosiglitazone pretreatment

The aim of the study was to evaluate the effect of rosiglitazone treatment on islet ghrelin and insulin gene expressions in streptozotocin (STZ)-induced diabetic rats. Animals were divided into four groups. 1. Intact controls. 2. Rosiglitazone-treated controls. 3. STZ-induced diabetes. 4. Rosiglitazone-treated diabetes. Rosiglitazone was given for 7 days at a dose of 20 mg/kg body weight. Ghrelin and insulin gene expressions were investigated by immunohistochemistry and in situ hybridization. There was no statistically significant difference in body weight between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats during the experimental period. Furthermore, there were no significant differences in blood glucose levels and insulin immunoreactive cell numbers between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats. There was a tendency towards a reduction of ghrelin gene expression in diabetic animals compared with intact controls. We found, in addition, that ghrelin immunoreactive and ghrelin mRNA expressing cells were frequent in the epithelial lining of the ducts suggesting ductal epithelium might be the source of the regenerating islet ghrelin cells, as is known for other islet cells. The results show that short-term rosiglitazone pretreatment had no significant effect on ghrelin and insulin gene expressions.     

N-acetyl-beta-D-glucosaminidase activity in serum, kidney and liver of streptozotocin-induced diabetic rat

Serum N-acetyl-beta-D-glucosaminidase (NAG) activity in streptozotocin-induced diabetic rats was significantly increased. There was neither a difference in total NAG activity in kidney and liver, nor in optimal pH of NAG in serum, kidney and liver between diabetic and control rats. The ratio of the thermounstable fraction of NAG increased in diabetic kidney and liver, while there was no difference in thermostability of between diabetic and control rats. Isoelectricfocusing of diabetic serum NAG indicated an increase in the neutral form. That of kidney and liver NAG indicated an increase in the acid form. These results may suggest that NAG clearance from the serum is decreased diabetic state.     

Comparison of biologically effective spectra for erythema and pre-vitamin D<Subscript>3</Subscript> synthesis

The short wavelength cut-off (λ_c), the wavelength of the maximum spectral UV (λ_Max) of spectral pre-vitamin D₃ effective solar UV irradiance (UV_D3), and the spectral erythemal UV (UV_Ery) were compared at 5-min intervals over a 6-month period at solar zenith angles (SZA) ranging from 4.7° to 80°. Averaged over the entire period, λ_c for UV_D3 is higher by 1.05 nm than that for UV_Ery. The λ_Max is higher for UV_D3 compared to UV_Ery for SZA < ~50°. For higher SZA (>55°), the ratio of λ_Max for UV_D3 to that for UV_Ery is less than 1. As the erythemal action spectrum extends into the UVA, the ratio of UV_D3 to UV_Ery irradiances decreases with increasing SZA, along with a decrease in the ratio of λ_Max for UV_D3 compared to UV_Ery. The changes in λ_c and λ_Max influence both personal UV_D3 and UV_Ery exposure and, to take this into account, a dual calibration technique for polysulphone dosimeters has been developed to simultaneously provide measurements of both types of exposure.     

Heterogeneity of human liver, muscle, and adipose tissue insulin receptor

We have studied the structure and function of the human insulin receptor in liver, skeletal muscle and adipose tissue. The alpha-subunit of the insulin receptor for liver, muscle and adipose tissue migrated on SDS-PAGE with Mrs 137632 +/- 216, 134034 +/- 1080, and 133575 +/- 165, respectively (p less than 0.05). Treatment of these receptors with neuraminidase decreased their molecule sizes and eliminated the relative size differences between the receptors. Three monoclonal antibodies (5A1, 10D9, and 20H3), directed towards different epitopes of the human insulin receptor alpha-subunit were used to probe immunological differences among the receptors. Antibodies 5A1 and 20H3 recognized all the receptors, whereas 10D9 recognized muscle and adipose tissue receptors but not liver receptors. The mobility of insulin receptor beta-subunit in the absence of insulin was the same in all tissues with a similar phosphorylation-induced decrease in mobility in SDS-PAGE in the presence of insulin. However, insulin stimulated autophosphorylation per receptor was different being greatest (p less than 0.05) in muscle (334 +/- 104 32P cpm) and similar in adipose tissue (114 +/- 10) and liver (183 +/- 68). These studies indicate, therefore, that the human insulin receptor is heterogeneous among the major target tissues for insulin, and raise the possibility that this heterogeneity may account for tissues' specific differences in insulin's biological messages.     

Hormone replacement therapy,calcium and vitamin D<Subscript>3</Subscript> versus calcium and vitamin D<Subscript>3</Subscript>alone decreases markers of cartilage and bone metabolism in rheumatoid arthritis: a randomized controlled trial [ISRCTN46523456]

This study aimed to evaluate the effects of hormone replacement therapy (HRT), known to prevent osteoporosis and fractures, on markers of bone and cartilage metabolism. Furthermore, we assessed whether changes in these markers corresponded to alterations in bone mineral density and radiographic joint destructions in postmenopausal women with rheumatoid arthritis. Eighty-eight women were randomized to receive HRT, calcium, and vitamin D3, or calcium and vitamin D3 alone, for 2 years. Bone turnover was studied by analyzing serum levels of C-terminal telopeptide fragments of type I collagen (CTX-I), C-terminal telopeptide of type I collagen (ICTP), bone sialoprotein, and C-terminal propeptide of type I procollagen (PICP) and cartilage turnover by urinary levels of collagen type II C-telopeptide degradation fragments (CTX-II) and cartilage oligomeric matrix protein (COMP) in serum. Treatment with HRT resulted in decrease in CTX-I (P < 0.001), ICTP (P < 0.001), PICP (P < 0.05), COMP (P < 0.01), and CTX-II (P < 0.05) at 2 years. Reductions in CTX-I, ICTP, and PICP were associated with improved bone mineral density. Of the markers tested, CTX-I reflected bone turnover most sensitively; it was reduced by 53 +/- 6% in the patients receiving HRT. Baseline ICTP (P < 0.001), CTX-II (P < 0.01), and COMP (P < 0.05) correlated with the Larsen score. We suggest that biochemical markers of bone and cartilage turnover may provide a useful tool for assessing novel treatment modalities in arthritis, concerning both joint protection and prevention of osteoporosis.     

3,5-Diiodo-L-Thyronine increases F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats

Short-term effects of 3,5-L-diiodothyronine (T₂) administration to hypothyroid rats on F_oF₁-ATP synthase activity were investigated in liver mitochondria. One hour after T₂ injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F_oF₁-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T₂ administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F_oF₁-ATP synthase was found, ruling out a T₂ genomic effect. In T₂-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T₂ administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T₂-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F_oF₁-ATP synthase activity observed following injection of T₂ to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

P2X<Subscript>7</Subscript> receptor and klotho expressions in diabetic nephropathy progression

Diabetes mellitus is characterized by increased levels of reactive oxygen species (ROS), leading to high levels of adenosine triphosphate (ATP) and the activation of purinergic receptors (P2X₇), which results in cell death. Klotho was recently described as a modulator of oxidative stress and as having anti-apoptotic properties, among others. However, the roles of P2X₇ and klotho in the progression of diabetic nephropathy are still unclear. In this context, the aim of the present study was to characterize P2X₇ and klotho in several stages of diabetes in rats. Diabetes was induced in Wistar rats by streptozotocin, while the control group rats received the drug vehicle. From the 1st to 8th weeks after the diabetes induction, the animals were placed in metabolic cages on the 1st day of each week for 24 h to analyze metabolic parameters and for the urine collection. Then, blood samples and the kidneys were collected for biochemical analysis, including Western blotting and qPCR for P2X₇ and klotho. Diabetic rats presented a progressive loss of renal function, with reduced nitric oxide and increased lipid peroxidation. The P2X₇ and klotho expressions were similar up to the 4th week; then, P2X₇ expression increased in diabetes mellitus (DM), but klotho expression presented an opposite behavior, until the 8th week. Our data show an inverse correlation between P2X₇ and klotho expressions through the development of DM, which suggests that the management of these molecules could be useful for controlling the progression of this disease and diabetic nephropathy.     

Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in Vmax (i.e., maximal enzyme activity) without a change in Km (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.     

Low dose pramipexole is neuroprotective in the MPTP mouse model of Parkinson's disease,and downregulates the dopamine transporter via the D<Subscript>3</Subscript> receptor

Background  

Our aim was to determine if pramipexole, a D₃ preferring agonist, effectively reduced dopamine neuron and fiber loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model when given at intraperitoneal doses corresponding to clinical doses. We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+).     

A<Subscript>2A</Subscript> and A<Subscript>3</Subscript> adenosine receptor expression in rheumatoid arthritis: upregulation,inverse correlation with disease activity score and suppression of inflammatory cytokine and metalloproteinase release

Introduction  

The reduction of the inflammatory status represents one of the most important targets in rheumatoid arthritis (RA). A central role of A_2A and A₃ adenosine receptors (ARs) in mechanisms of inflammation has been reported in different pathologies. The primary aim of this study was to investigate the A_2A and A₃ARs and their involvement in RA progression measured by Disease Activity Score in 28 or 44 joints (DAS28 or DAS).     

The polythiophene molecular segment as a sensor model for H<Subscript>2</Subscript>O,HCN, NH<Subscript>3</Subscript>, SO<Subscript>3</Subscript>, and H<Subscript>2</Subscript>S: a density functional theory study

Studying the interaction of some atmospheric gases (H₂O, HCN, NH₃, SO₃ and H₂S) with 3PT oligomers is important in the development of polymeric sensors for gas detection. In the present study, we studied the relaxed geometries, interaction energies, charge analysis, HOMO–LUMO orbital analysis, and UV–vis spectra of all interacted systems using first-principles density functional theory (DFT). All these analyses indicated the potential of polythiophene as an inexpensive polymeric sensor for the analytes mentioned. Interaction energy values of ?19.90, ?19.66, ?14.01, ?8.70, and ?4.76 kJ mol^?1 were achieved for adsorption of SO₃, H₂O, NH₃, HCN, and H₂S on 3PT, respectively. Consequently, clarification of their physical parameters became the major focus of this study.     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.