Effect of rejection on electrophysiologic function of canine intestinal grafts: Correlation with histopathology and na-k-ATPase activity

To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na-K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18-24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were snitched between a pair of dogs (allograft). Animals receiving intestinal autotransplantation were used as controls. AIIograji recipients were sacrificed 3, 4, 5, 7, or 9 days after transplantation, and autograft recipients were sacrificed 3, 7, or 14 days afier transplantation. Immunosuppression was not used. Electrophysiologic measurements were done with an Ussing chamber. Histological analysis was performed blindly using whole thickness sections. Na-K-ATPase activity in the mucosal tissue, which is said to regulate the potential difference, was also measured. Potential difference, resistance, and Na-K-ATPase activity of the allografi intestine decreased with time and were significantly lower 7 and 9 days after transplantation compared to host intestine, normul intestine, and graft intestine of controls (autograft). Potential difference, resistance, and Na-K-ATPase activity of the native intestinal tissue and the autografts did not decrease with time. Detection of histologically mild rejection of the intestine, which is important for appropriate immunosup-pressive treatment in clinical cases, could not be achieved based on electrophysiology or Na-K-ATPase activity. Deterioration of electrophysiologic function during rejection correlated with the histological rejection process and Na-K-ATPase activity; however, electrophysiology my not be a reliable tool for monitoring grafrs, since it cannot detect early intestinal rejection. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch

Development and stability of Th17 cells in ovarian cancer requires nitric oxide and endogenous NOS2 activity in cancer-associated CD4+ T cells

Th17 cells play reciprocal roles in different forms and at different stages of cancer. We report that the presence of Th17 cells in ovarian cancer ascites correlates with local expression of nitric oxide synthase-2 (NOS2). Furthermore, the development of RORγt+IL-23R+IL-17+ Th17 cells from human naive-, memory- or tumor-infiltrating CD4+ T cells critically depends on NO and endogenous NOS2 induced in CD4+ T cells by Th17-inducing cytokines (IL-1β/IL-6/IL-23) or by cancer-associated IL-1β/IL-6/IL-23/NO-producing MDSCs. Inhibition of NOS2 or its downstream cGMP/cGK signaling pathway abolishes de novo induction of Th17 cells. Moreover, even short-term blockade of NOS/cGMP suppresses the IL-17 production by established Th17 cells isolated from ovarian cancer patients, demonstrating the novel key role of NOS/cGMP in Th17 cell physiology and providing for new therapeutic targets to manipulate Th17- and NOS/cGMP-associated immunity in precancerous lesions and advanced cancer

Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor kappaB, degradation of inhibitor of kappaB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor kappaB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage

Combined In Silico, In Vivo, and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO2-/NO3- data from "middle-aged" (6-8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for "young" (2-3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging. © 2013 Namas et al

Effect of Strain Magnitude on the Tissue Properties of Engineered Cardiovascular Constructs

Mechanical loading is a powerful regulator of tissue properties in engineered cardiovascular tissues. To ultimately regulate the biochemical processes, it is essential to quantify the effect of mechanical loading on the properties of engineered cardiovascular constructs. In this study the Flexercell FX-4000T (Flexcell Int. Corp., USA) straining system was modified to simultaneously apply various strain magnitudes to individual samples during one experiment. In addition, porous polyglycolic acid (PGA) scaffolds, coated with poly-4-hydroxybutyrate (P4HB), were partially embedded in a silicone layer to allow long-term uniaxial cyclic mechanical straining of cardiovascular engineered constructs. The constructs were subjected to two different strain magnitudes and showed differences in biochemical properties, mechanical properties and organization of the microstructure compared to the unstrained constructs. The results suggest that when the tissues are exposed to prolonged mechanical stimulation, the production of collagen with a higher fraction of crosslinks is induced. However, straining with a large strain magnitude resulted in a negative effect on the mechanical properties of the tissue. In addition, dynamic straining induced a different alignment of cells and collagen in the superficial layers compared to the deeper layers of the construct. The presented model system can be used to systematically optimize culture protocols for engineered cardiovascular tissues

Multi-omic analysis in injured humans: Patterns align with outcomes and treatment responses

Trauma is a leading cause of death and morbidity worldwide. Here, we present the analysis of a longitudinal multi-omic dataset comprising clinical, cytokine, endotheliopathy biomarker, lipidome, metabolome, and proteome data from severely injured humans. A "systemic storm" pattern with release of 1,061 markers, together with a pattern suggestive of the "massive consumption" of 892 constitutive circulating markers, is identified in the acute phase post-trauma. Data integration reveals two human injury response endotypes, which align with clinical trajectory. Prehospital thawed plasma rescues only endotype 2 patients with traumatic brain injury (30-day mortality: 30.3 versus 75.0%; p = 0.0015). Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) was identified as the most predictive circulating biomarker to identify endotype 2-traumatic brain injury (TBI) patients. These response patterns refine the paradigm for human injury, while the datasets provide a resource for the study of critical illness, trauma, and human stress responses