Effects of Local Budesonide Treatment on the Cell-Mediated Immune Response in Acute and Relapsing Colitis in Rats

Glucocorticosteroids (GCS) are effective intreatment of inflammatory bowel disease (IBD), but alsohave unwanted systemic side effects. Here, we describethe effects of budesonide and dexamethasone on acute experimental colitis and on T cells inthymus and spleen, as well as the effect of budesonidetreatment on relapsing colitis. Acute colitis wasinduced by intracolonic administration of2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol, and a relapsewas induced by an intraperitoneal booster of TNBS. GCSwere administered intrarectally on days 1, 4, and 6after induction of acute colitis or a relapse.Inflammatory cells in the colon were studied on day 7, andin acute colitis also on days 13 and 16. Budesonidetreatment in acute and relapsing colitis resulted inreduction of macroscopic damage and decreased thenumbers of macrophages and neutrophils in the colon.Dexamethasone was less effective. Dexamethasone, but notbudesonide, reduced the number of T cells in the thymus.It is concluded that local budesonide is more effective in treatment of acute experimentalcolitis than dexamethasone and, in contrast todexamethasone, did not cause a general suppression of Tcells. Although budesonide was very effective in thetreatment of relapsing colitis, this effect was notaccomplished by affecting the number of T cells in thecolon.     

The Proximal Gastric Pouch Invariably Contains Acid-Producing Parietal Cells in Roux-en-Y Gastric Bypass

Background: Roux-en-Y gastric bypass (RYGBP) is well tolerated and effective in ameliorating diseases common to morbidly obese patients. A potential drawback, however, is the risk for stomal ulcers, probably due to acid and peptic digestion of the mucosa in the proximal Roux limb. Methods: In 23 RYGBP patients (mean BMI 45 kg/m², age 39 years), the gastro-jejunostomy was performed by circular stapler and the gastric suture ring retrieved for histological examination. 13 consecutive patients received our standard totally transected 4 × 3 cm proximal gastric pouch. The anvil was passed transgastricly and reference biopsies were taken from the gastrotomy in the corpus of the stomach. In the last 10 patients, the pouch size was reduced to 2 × 3 cm by a modified surgical technique. Results: All suture rings from the standard pouches consisted of corpus-fundus mucosa with a large amount of parietal cells, histologically identical to the reference biopsies from the gastrotomy. Also, the 10 suture rings from the modified small pouches contained corpus-fundus mucosa. In 5 of these samples, cardiac mucosa was found, but only in a small segment (6 mm). In addition, 3 patients had esophageal epithelium in the suture ring. Conclusion: The proximal pouch invariably contains acid-producing parietal cells. In order to reduce acid production and, hence, the risk of stomal ulcers, the pouch has to be made as small as possible.     

Cytotoxicity of ventricular cerebrospinal fluid from Parkinson patients: Correlation with clinical profiles and neurochemistry

Abstract

Other investigators have reported that the cerebrospinal fluid (CSF) from patients with Parkinson's disease (PD) might contain endogenous dystrophic factors. Using CSF samples drawn from individual PD patients during surgery, we investigated the toxic effect of ventricular CSF (vCSF) on the growth of PC12 cells and the correlation between the clinical profiles of the patients and CSF neurochemistry. Ventricular CSF samples from 28 patients with PD or essential tremor (ET) were collected during ventriculography for stereotactic pallidotomy or thalamotomy. PC12 cells were incubated with 20% vCSF from both clinical groups for up to 72 h. Microdialysis was used to analyze four neurochemical parameters (glucose, lactate, pyruvate, and glutamate) in each vCSF sample. We observed that vCSF drawn from PD patients exerted nonspecific growth inhibition on PC12 cells in a time-dependent manner. The growth inhibitory action of PD–vCSF decreased significantly after heat treatment. Microdialysis demonstrated no statistical differences between PD and ET samples among the four parameters studied. In addition, PC12 cell survival after 72 h incubation with PD-vCSF correlated with no neurochemical parameter or individual clinical profile (age, onset age, duration of disease, Hoehn & Yahr stage, disease progression rate), except for a slight correlation between vCSF and disease progression rate in heat treated samples from female patients. One or more endogenous cytotoxic factors in PD-vCSF inhibit PC12 cell growth. This factor or factors are partially sensitive to heat which suggests proteins or peptides as possible agents. The cytotoxic effect of PD-vCSF did not directly correlate with any clinical profiles studied or energy metabolism of PD brain.     

Histological Effect of (R)-α-Methylhistamine on Ethanol Damage in Rat Gastric Mucosa (Influence on Mucus Production)

(R)--Methylhistamine, a selective agonistof histamine H₃ receptors, preventsmacroscopically visible gastric lesions by absoluteethanol in the rat. A further insight into its activitywas the aim of our study. Rats were given saline or(R)--methylhistamine (100 mg/kg)intragastrically. After 30 min, absolute ethanol wasgiven and gastric mucosa was sampled 60 min later.Histologic damage and intracellular and adherent mucus werequantified. Luminal surface and mucous cells wereexamined by scanning and transmission electronmicroscopy. (R)--Methylhistamine reduced theextent of lesions by ethanol from 96 to 18%. Surface mucous cellsand mucous neck cells were increased in volume andnumber, packaging of intracellular mucus was modified,and the secretory processes were promoted by(R)-methylhistamine itself, although these modifications weremostly evident in stomachs subsequently exposed toethanol. Adherent mucus layer thickness was increased by(R)-methylhistamine only after ethanol exposure. It is concluded that(R)--methylhistamine predisposes mucous cells toreact to ethanol.     

Quantification of Inflammatory Mediators in Stool Samples of Patients with Inflammatory Bowel Disorders and Controls

Previous studies indicated that eosinophils andmast cells accumulate and become activated ininflammatory bowel disorders. The aim of the study wasto examine the presence of eosinophil and mast cellmediators in human stool samples of patients withinflammatory bowel disorders. We measured eosinophilcationic protein (ECP), eosinophil protein ×(EPX), methylhistamine, and₁-antitrypsin in fecal samples of 136 patients (62 Crohn's 1 disease, 24ulcerative colitis, 15 intestinal food allergy, 35 othergastrointestinal diseases) and 8 healthy controls. Wefound strongly elevated levels of ECP (median: 29,range: 0.4-1783 ng/g feces) and EPX (803, 10-33,225ng/g) in all patient groups compared to controls (ECP:1.5, 0.5-55 ng/g; EPX: 235, 12-746 ng/g). Similarresults, albeit less pronounced, were obtained formethylhistamine and alpha1-antitrypsin. Particularly highconcentrations of ECP and EPX were found in patientswith active mucosal inflammation. In conclusion, thestudy presents an easy and reliable method for thedetection of fecal ECP, EPX, and methylhistamine and mayprovide a tool to gain insight into the pathogenesis ofinflammatory bowel diseases and have potential asdiagnostic test.     

Interleukin-2 Fusion Protein (DAB389IL- 2) Selectively Targets Activated Human Peripheral Blood and Lamina Propria Lymphocytes

Activated mucosal T lymphocytes correlate withthe intestinal inflammation of inflammatory boweldisease. Activated T cells elaborate interferon-(IFN-) and express high-affinity interleukin-2(IL-2) receptors. The IL-2/diphtheria toxin fusionprotein (DAB₃₈₉IL-2) has been shown tospecifically kill high affinity IL-2 receptor-bearingcells. We tested whether DAB₃₈₉IL-2 couldspecifically target activated lamina propria lymphocytes. Lymphocytes 389were activated in vitro with phytohemagglutinin and IL-2for 24 - 48 hr. Toxin efficacy was determined by the[¹⁴C]leucine incorporation, IFN-ELISA, and flow cytometry. DAB₃₈₉IL-2 (10^-11 M) inhibited protein synthesis by 80% inactivated lamina propria 389 lymphocytes. Thisinhibition was blocked by coculture of either excessIL-2 or a nonfunctional IL-2 diphtheria toxin mutant protein.DAB₃₈₉IL-2 (10^-12 M) alsosignificantly reduced the numbers of activated helper Tcells and IFN- levels in 24-hr cultures.DAB₃₈₉IL-2 specifically targets activatedIL-2 receptor-positive lamina propria lymphocytesand is a potential new therapeutic agent for patientswith active inflammatory bowel disease.     

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1⁺CD45^–CD11b^–MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of ¹²⁵I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1⁺CD45^–CD11b^–MSCs into the bone marrow cavity of Rag2^−/− immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling. © 2012 American Society for Bone and Mineral Research.     

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH-releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by regulating a distinct set of downstream mediators. In this study, we find that GH is one of the most significantly upregulated genes by BMP9 in mouse MMCs through expression-profiling analysis. We confirm that GH is a direct early target of and upregulated by BMP9 signaling. Exogenous GH synergizes with BMP9 on inducing early and late osteogenic markers in MMCs. Furthermore, BMP9 and GH costimulation leads to a significant expansion of growth plate in cultured limb explants. Although GH alone does not induce de novo bone formation in an ectopic bone formation model, BMP9 and GH costimulated MMCs form more mature bone, which can be inhibited by silencing GH expression. The synergistic osteogenic activity between BMP9 and GH can be significantly blunted by JAK/STAT inhibitors, leading to a decrease in GH-regulated insulin-like growth factor 1 (IGF1) expression in MMCs. Our results strongly suggest that BMP9 may effectively regulate extrapituitary GH expression in MMCs. Thus, it is conceivable that the BMP9-GH-IGF axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.     

Osteogenic Differentiation of Bone-Marrow-Derived Stem Cells Cultured with Mixed Gelatin and Chitooligosaccharide Scaffolds

This study investigates the effect of scaffolds prepared from gelatin (G) and chitooligosaccharide (COS) on the osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (MSC). The sponge scaffolds at G/COS mixing ratios of 100:0, 70:30 and 50:50 were fabricated by freeze-drying, followed by glutaraldehyde cross-linking. The pore size of the G/COS scaffolds ranged from 70 to 105 μm. MSC cultured in the scaffolds in the osteogenic medium were differentiated into osteogenic cells for all G/COS scaffolds. Calcium nodules were homogeneously formed on the surface of scaffolds cultured with MSC. A Fourier transform infrared (FT-IR) analysis demonstrated the formation of hydroxyapatite spectroscopically. Among all G/COS scaffolds, the highest ALP activity and calcium content were observed for MSC cultured in the G/COS 70:30 scaffolds. The G/COS 70:30 scaffolds were then pre-cultured with MSC in the osteogenic medium for 28 days and subcutaneously implanted into nude mice to evaluate ectopic bone formation. Enhanced vascularization, cell infiltration, collagen formation and calcium deposition around the scaffolds implanted were histologically observed at 2 and 8 weeks after implantation. It was concluded that the G/COS scaffold with the mixing ratio of 70:30 was a promising organic material to induce osteogenic differentiation of MSC.     

In Vitro Biocompatibility of Sheath–Core Cellulose-Acetate-Based Electrospun Scaffolds Towards Endothelial Cells and Platelets

Typically, tissue-engineered scaffolds mimic the topographical properties of the native extracellular matrix. However, other physical properties, such as the scaffold mechanical stiffness, are not imitated. The purpose of this study was to fabricate scaffolds with improved mechanical properties and investigate their biocompatibility towards endothelial cells and platelets. To enhance mechanical properties, an electrospinning apparatus was developed that fabricates fibers with sheath–core morphologies. Different combinations of cellulose acetate and chitosan were chosen to modulate the mechanical properties of the formed fibers. We hypothesized that mechanically stiffer scaffolds would improve endothelial cell growth and that all scaffolds would be compatible towards endothelial cells and platelets. Endothelial cell-culture conditions were quantified up to 5 days. Migration onto scaffolds was monitored for 10 days. Platelet aggregation, antagonized by thrombin receptor agonist peptide 6, was measured after scaffold incubation. A platelet activation time-course was assessed with the modified prothrombinase assay. As scaffold mechanical stiffness increased, endothelial cell growth within and adhesion to and migration throughout the scaffolds was promoted. Also, scaffolds did not induce platelet aggregation or activation. These results indicate that the mechanical stiffness of engineered scaffolds regulates endothelial cell-culture parameters and that these sheath–core electrospun scaffolds are compatible towards endothelial cells and platelets.