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Retrospective observational study. Data derived from two multinational ICU studies (Ruby and Sapphire). Critically ill patients with early stage 2-3 AKI. None. We analyzed three consecutive uCCL14 measurements at 12-hour intervals after diagnosis of stage 2-3 AKI by Kidney Disease Improving Global Outcomes criteria. Primary outcome was persistent severe AKI, defined as 72 consecutive hours of stage 3 AKI, death, or receipt of dialysis prior to 72 hours. uCCL14 was measured using the NEPHROCLEAR uCCL14 Test on the Astute 140 Meter (Astute Medical, San Diego, CA). Based on predefined, validated cutoffs, we categorized uCCL14 as: low (≤ 1.3 ng/mL), medium (> 1.3 to ≤ 13 ng/mL), or high (> 13 ng/mL). Seventy-five of 417 patients with three consecutive uCCL14 measurements developed persistent severe AKI. Initial uCCL14 category strongly correlated with primary endpoint and, in most cases (66%), uCCL14 category was unchanged over the first 24 hours. Compared with no change and accounting for baseline category, decrease in category was associated with decreased odds of persistent severe AKI (odds ratio [OR], 0.20; 95% CI, 0.08-0.45; p < 0.001) and an increase in category with increased odds (OR, 4.04; 95% CI, 1.75-9.46; p = 0.001). In one-third of patients with moderate to severe AKI uCCL14 risk category altered over three serial measurements and such changes were associated with altered risk for persistent severe AKI. Serial CCL-14 measurement may detect progression or resolution of underlying kidney pathology and help refine AKI prognosis.

Bioactive films with a nanoplate structure were prepared on the surface of a biodegradable AZ31B magnesium (Mg) alloy via anodization in simulated body fluid (SBF) as an electrolyte to control Mg biodegradability and improve surface bioactivity. The effect of the electrolyte temperature and pH values on the formation of the biomimetic film were studied. The electrolyte was set at three different temperatures of 37, 50, and 80 °C, with pH values ranging from 7.4 to 8 for the lower electrolyte temperature and 11.5–12 for the two higher levels of temperature. The apatite films on the different samples were characterized using X-ray diffraction spectroscopy (XRD), field emission scanning electron microscopy (FE-SEM), EDS element mapping, X-ray photon spectroscopy (XPS), and FTIR spectroscopy. The water contact angles of the different surfaces were evaluated, moreover, the corrosion behaviors of the different samples were studied using electrochemical potentiodynamic DC, electrochemical impedance spectroscopy (EIS), and immersion tests. The human fetal-osteoblast cell line hFOB 1.19 was used in a cell culture test, and the biological response and cell function were evaluated in vitro using DNA and PCR. The decomposition of the apatite film was affected by the anodization electrolyte temperature, resulting in an amorphous structure. It is observed that the apatite structure has nanoplates at electrolyte temperature (37 to 50) °C and these have a tendency to disappear at 80 °C.

Magnesium (Mg), and its alloys have good potential for using in biomedical applications due to its good mechanical properties and biodegradability. However, the poor corrosion resistance and insufficient mechanical stability of Mg in biological fluids have limited its clinical applicability. As a result, surface modification techniques are being investigated to improve corrosion resistance for biomedical applications. In this study, a bioactive CaP film were deposited on the surface of the AZ31B magnesium alloy via anodization with SBF solution used as the electrolyte with and without ZrO 2 Nanoparticles (NPs). The samples were evaluated and characterized in vitro to assess their degradation and cytocompatability behaviour via FE-SEM, electrochemical corrosion test, immersion test, and cell culture tests. The results indicate that the AZ31B Mg alloy surface anodized in SBF with and without ZrO 2 NPs consists of CaP nanoplate leaf-like apatite structures and porous 3-D structures, respectively. The nanomorphology showed uniform growth on the anodized surface that had created the porous layer on the surface of the samples three days after immersion in the SBF solution. The formation of such nanostructures on the anodized sample could provide the implant materials with extra biocompatibility. Moreover, this unique deposited layer can improve the corrosion resistance and cytocompatability of the Mg alloys, which make it a promising material for use in biomedical applications.

Three-dimensional in vitro tumor models are needed to obtain more information about drug behavior in tumors. The aim of this study is to establish a new model for hepatocellular carcinoma (HCC) using decellularized rat livers. After generating the rat liver scaffolds, HepG2 liver cancer cells were perfused via the portal vein and placed in a bioreactor for 10 days. Histology was performed to analyze cell distribution within the scaffolds. Function and tumor-related gene expression were examined by polymerase chain reaction (PCR). We evaluated the function of HepG2 cells grown on scaffolds in the presence of a well-known anti-cancer drug to investigate the potential application of our system for drug screening. The scaffolds were devoid of cellular materials and preserved extracellular matrix components. HepG2 cells grew well on the scaffolds. The PCR results showed that the cells maintained function and invasion ability at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. These findings strengthen the potential use of rat liver scaffolds for screening HCC drugs.

Effects of placental insufficiency and preterm birth on neurodevelopment can be modeled in experimental settings of neonatal hypoxia in rodents. Here, rat pups were reared in reduced oxygen (9.5%) for eleven days, starting on postnatal day three (P3). This led to a significant reduction in brain and body weight gain in hypoxic pups when compared to age matched normoxia reared controls, plausibly reflecting inability to fulfill energetic needs of normal growth and development. Adaptive processes designed to augment energetic capacity in eukaryotes include stimulation of mitochondrial biogenesis. We show that after eleven days of sustained hypoxia, the levels of nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) are elevated and the content of mitochondrial DNA (mtDNA) is greater in the hypoxic P14 pup brain when compared to normoxic conditions. Corresponding immunohistochemical analyses reveal increased density of mtDNA in large cortical neurons. In contrast, no changes in mtDNA content are observed in the brain of pups reared for 24 h (P3–P4) under hypoxic conditions. Together, these data suggest that prolonged inadequate oxygenation may trigger a compensatory increase in neuronal mitochondrial DNA content to partially mitigate compromised energy homeostasis and reduced energetic capacity in the developing hypoxic brain.

Objective One of the major obstacles in applying decellularized organs for clinical use is the recellularization step, during which huge numbers of cells are required to develop whole livers. We established a simple protocol for constructing a bioartificial hepatic lobe and investigated its biocompatibility. Methods The right lateral lobe of porcine liver was decellularized using 0.1% sodium dodecyl sulfate through the right branch of the portal vein. Decellularized lobes were evaluated by histological and biochemical analyses. DNA content was quantified to validate the decellularization protocol. The presence of immunogenic and pathogenic antigens was checked to exclude potential rejection and thrombosis after xenotransplantation. Xeno-reactivity of decellularized tissue against human peripheral blood mononuclear cells was examined. Cytotoxicity was evaluated against hepatocarcinoma cells. Finally, scaffolds were incubated in collagenase for biodegradation testing. Results The decellularized lobe preserved the three-dimensional architecture, ultrastructure, extracellular matrix components, and vasculature. Scaffolds were almost depleted of DNA in addition to antigenic and pathogenic antigens, which are considered barriers to xenotransplantation. The human immune response against scaffolds was considered non-significant. Our matrices were biocompatible and biodegradable. Conclusions We successfully developed a non-cytotoxic, non-immunogenic, and biodegradable porcine hepatic lobe for future liver regeneration and bioengineering.

Purpose The aims of this study were to investigate the effects of sterilization with peracetic acid (PAA) and ethanol on the biological activity of porcine liver scaffolds and to develop a new technique for sterilization using slightly acidic electrolyzed water (SAEW). Methods Decellularization of liver slices was performed using 0.1% sodium-dodecyl-sulfate, then evaluated by histological and polymerase chain reaction analyses. Decellularized slices were treated with either PAA or ethanol or SAEW, and then DNA content was quantified. We determined sterilization efficiency by culturing scaffolds in culture medium and on blood agar. We next analyzed the glycosaminoglycan and collagen contents of the scaffolds. Finally, we tested the cytotoxicity of the scaffolds as well as the effects of sterilization on host cell attachment and proliferation. Results Complete cell and antigenic epitopes removal emphasized the decellularization efficiency. PAA and SAEW treatments achieved the highest efficiency of sterilization compared to that of the ethanol treated scaffolds, and were able to remove a considerable fraction of DNA from decellular-ized livers. The retained glycosaminoglycan content decreased in all treatments in the following order: SAEW, ethanol, and PAA. Ethanol caused a significant loss in collagen content compared to the other groups. A cytotoxicity evaluation revealed that all scaffolds were nontoxic. SAEW-treated scaffolds supported cell attachment and proliferation at a significantly higher rate than other groups. Conclusions These data suggest that SAEW is highly efficient for sterilizing scaffolds and allowed the scaffolds to retain their bioactivity in addition to its high efficiency for cell remnant removal.

Combustion smoke contains gases and particulates, which act via hypoxia and cytotoxicity producing mechanisms to injure cells and tissues. While carbon monoxide (CO) is the major toxicant in smoke, its toxicity is exacerbated in presence of other compounds. Here, we examined modulations of mitochondrial and cytosolic energy metabolism by inhalation of combustion smoke versus CO, in vivo, in the rat brain. Measurements revealed reduced activities of respiratory complexes, with greater inhibition by smoke than equivalent CO in ambient air. In the case of respiratory complex IV, inhibition by CO and smoke was similar; suggesting that complex IV inhibition is primarily by the action of CO. In contrast, inhibition of complexes I and III was greater by smoke. Increases in cytosolic lactate dehydrogenase and pyruvate kinase activities accompanied inhibition of respiratory complexes, likely reflecting compensatory increases in cytosolic energy production. Together, the data provide new insights into the mechanisms of smoke inhalation-induced perturbations of brain energetics, which impact neuronal function and contribute to the development of neuropathologies in survivors of exposures to CO and combustion smoke.

Tumor recurrence after curative resection is a major problem in the management of colon cancer therapy. Identifying molecular markers for tumor recurrence is critical for successfully selecting patients who are more likely to benefit from adjuvant chemotherapy. We analyzed the value of thymidylate synthase (TS) and methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms as a prognostic marker in stage II and stage III colon cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy.Between 1987 and 2007, blood samples were obtained from 197 patients with stage II or stage III colon cancer at medical facilities at the University of Southern California. DNA was extracted from peripheral blood, and the genotypes were analyzed using PCR-restriction fragment length polymorphism technique.Patients harboring the TS 3RG/+6-bp haplotype were at greatest risk to develop tumor recurrence [relative risk (RR): 2.25; 95% confidence interval (CI): 1.04-4.85; adjusted P value=0.032]. TS enhancer region 3RG alone (RR: 3.48 years; 95% CI: 1.61-7.54; adjusted P value=0.013) or in combination with TS 1494del6 bp (RR: 3.41 years; 95% CI: 1.33-8.75; adjusted P value=0.044) proved to be adverse prognostic markers in both univariate and multivariable analysis.'High-expression' variants of TS 2R/3R repeat, TS enhancer region 3R G/C, TS 1494del6 bp, and TS haplotype analysis might help to identify stage II and stage III colon cancer patients who are at great risk of developing tumor recurrence, and also those who are more likely to benefit from 5-fluorouracil-based adjuvant chemotherapy. Larger, independent, prospective studies are, however, needed to confirm and validate our preliminary findings.

Unrepaired or erroneously repaired DNA lesions drive genomic instability and contribute to cellular and organ decline. Since delayed neuropathologies are common in survivors of smoke inhalation injuries, we asked whether the integrity of brain DNA might be compromised by acute exposure to combustion smoke. Although many studies demonstrate that the brain is equipped to repair oxidatively damaged DNA, to date, the capacity for accurate DNA repair under conditions of disrupted oxygenation and oxidative stress has not been defined. We show that DNA adducts detectable by their ability to block PCR amplification form in the rat hippocampus after acute exposure to smoke. To identify the different types of adducts and to dissect their temporal formation and repair profiles in vivo in the brain, we used DNA-modifying enzymes to convert specific adducts into strand breaks prior to PCR amplification. Using this strategy, we detected formation of oxidative DNA adducts early on after smoke inhalation, while mismatched bases emerged at the later recovery times, potentially due to an erroneous DNA repair process. Erroneous repair can be mutagenic and because the initial smoke-induced oxidative damage to DNA is extensive, compromised fidelity of DNA repair may underlie neurotoxicity and contribute to delayed death of hippocampal neurons.

Acute smoke inhalation causes death and injury in victims of home and industrial fires as well as victims of combat situations. The lethal factors in combustion smoke inhalation are toxic gases and oxygen deficiency, with carbon monoxide (CO) as a primary cause of death. In survivors, inhalation of smoke can result in severe immediate and delayed neuropathologies. To gain insight into the progression of molecular events contributing to smoke inhalation sequelae in the brain, we developed a smoke inhalation rat model and conducted a genome-wide analysis of gene expression. Microarray analysis revealed a modified brain transcriptome with changes peaking at 24 h and subsiding within 7 days post-smoke. Overall, smoke inhalation down regulated genes associated with synaptic function, neurotransmission, and neurotrophic support, and upregulated genes associated with stress responses, including nitric oxide synthesis, antioxidant defenses, proteolysis, inflammatory response, and glial activation. Notably, among the affected genes, many have been previously implicated in other types of brain injury, demonstrating the usefulness of microarrays for analysis of changes in gene expression in complex insults. In accord with previously described modulations of nitric oxide homeostasis in CO poisoning, microarray analysis revealed increased brain expression of nitric oxide synthase (NOS) and NOS ligand after inhalation of smoke. Furthermore, immunostaining showed significant elevations in perivascular NOS and in protein nitration, corroborating the involvement of nitric oxide perturbations in post-smoke sequelae in the brain. Thus, the new rat model, in combination with microarray analyses, affords insight into the complex molecular pathophysiology of smoke inhalation in the brain.

Repair of most modified and mispaired bases in the genome is initiated by DNA glycosylases, which bind to their respective targets and cleave the N-glycosyl bond to initiate base excision repair (BER). The mammalian homolog of the Escherichia coli MutY DNA glycosylase (MYH) cleaves adenine residues paired with either oxidized or non-modified guanines. MYH is crucial for the avoidance of mutations resulting from oxidative DNA damage. Multiple N-terminal splice variants of MYH exist in mammalian cells and it is likely that different variants result in the production of enzymes with altered properties. To investigate whether modifications in the N-terminus are consequential to MYH function, we overexpressed intact and N-terminal-deletion rat MYH proteins and examined their activities. We found that deletion of 75 amino acids, which perturbs the catalytic core that is conserved with E.coli MutY, abolished excision activity. In contrast, deletions limited to the extended mammalian N-terminal domain, differentially influenced steady-state excision rates. Notably, deletion of 50 amino acids resulted in an enzyme with a significantly lower K(m) favoring formation of excision products with 3'-OH termini. Our findings suggest that MYH isoforms divergent in the N-terminus influence excision rates and processing of abasic sites.

Canine kidney preservation models have historically used autotransplants to avoid the complications of rejection, although clinically all transplants are allografts. This study investigated the effects of preservation time and method on early kidney function in a canine allograft vs. autograft model. Kidneys were harvested from beagles, preserved by cold storage (CS) in UW solution for 0, 24 or 72 h, or by machine perfusion (MP) with Belzer MPS for 72 h. In some experiments 45 min of warm ischemia (WI) was performed in situ before harvest. Allograft recipients received steroid immunosuppression. Kidney function was assessed by serum creatinine and survival for 7 days. Allografts preserved for 0 and 24 h performed as well as autografts. Allografts preserved for 72 h by either CS or MP had a higher incidence of primary nonfunction (PNF) compared with autografts, as determined by survival (50% vs. 100%, p < 0.003). Primary nonfunction kidneys had thrombotic microangiopathy, vascular and peritubular capillary binding of IgM and complement C4d, and evidence of circulating donor-specific antibodies; all consistent with humoral rejection. These responses were dependent on hypothermia time and were not attributable to ischemia, immunosuppression, preservation solution, or cellular rejection. In conclusion, prolonged hypothermia can cause PNF in allografts owing to acute humoral rejection.

The potentially detrimental influence of parenchymal cells on endothelial function during preservation in UW solution was examined by co-storage of rat abdominal aortic rings with isolated liver cells. Cold storage of rings in UW solution alone for up to 96 h had no effect on the response to acetylcholine, though constriction was progressively lost. Co-storage of rings with liver cells resulted in no loss of sodium nitroprusside response, but the relaxation response to acetylcholine was reduced. The loss of acetylcholine response could not be attributed to Kupffer cells, the lowering of pH, oxygen depletion, or the loss of constriction. A similar loss of endothelial function was observed in rings stored in pieces of liver, kidney or heart. We conclude that parenchymal cells exude factors during preservation by cold storage which reversibly inhibit vascular NO production. These factors could significantly impair whole organ function on reperfusion.

Background Donor brain death (BD) has been implicated as a risk factor for the poor performance of kidneys after transplantation in small but not large animal models. This study determined the effects of donor BD on renal function and lipid mediator metabolism in a large animal model of renal hypothermic preservation injury. Methods Adult beagle donors were subjected to explosive BD for 16 hr. After BD, the kidneys were removed, cold stored for 24 hr in cold University of Wisconsin solution, and allotransplanted into recipient dogs for either 4 hr (group 1) or 7 days (group 2). Controls for both groups consisted of kidneys obtained from living donors. Renal allograft function and tissue arachidonic acid (AA) metabolism were determined after reperfusion. Results Short-term renal function after transplantation was generally unaffected by BD. Renal blood flow decreased after reperfusion but was not altered during the 16-hr BD period. Neutrophil infiltration significantly increased in kidneys from brain-dead donors before storage and after 4 hr of reperfusion. Renal cortex and medulla AA metabolism were not significantly affected by BD after short-term reperfusion except when thiol-ether leukotrienes (LTC(4)/D(4)/E(4)) were increased with BD. Serum creatinine was elevated during 7 days, but, surprisingly, BD significantly attenuated this injury. Conclusion BD in large mammals does not significantly affect renal allograft function or AA metabolism after transplantation. The role of BD in human renal preservation injury and inflammation should be reevaluated.

To determine the effect of chronic corneal epithelial debridement on epithelial and stromal morphology and extracellular matrix components, and to compare those changes to those in spontaneous chronic corneal epithelial defects (SCCED) in dogs.Axial corneal epithelial wounds, 10 mm in diameter, were created weekly for 8 weeks in five normal adult laboratory beagles. Slit lamp biomicroscopy and corneal pachymetry were performed weekly before wounding. Three days after the last debridement the dogs were killed humanely, and corneas were processed for light and electron microscopy and immunohistochemistry for collagen IV, collagen VII, fibronectin, and laminin.No significant changes in corneal thickness were found. All samples demonstrated epithelial dysmaturation adjacent to the wound edge, and, in four of five, a narrow zone of nonadherent epithelium formed adjacent to the exposed stroma. All samples had a stromal acellular zone in the area of the defect and continuing for a short distance under the adjacent attached epithelium. Experimentally wounded dogs did not form the superficial hyaline acellular lamina found in 92% of dogs with SCCED. Laminin, collagen IV, and fibronectin were present on the stromal surface in all samples, and collagen VII was present in four of five samples. Transmission electron microscopy (TEM) demonstrated the presence of basement membrane on the surface of the exposed stroma.Epithelial changes are similar between experimentally wounded dogs and dogs with SCCED. The stromal acellular zone that forms in experimentally wounded dogs is distinct from the hyaline lamina observed in dogs with SCCED. The difference in the acellular stromal layers between chronically wounded dogs and dogs with SCCED may be of relevance to our understanding of the pathophysiology of persistent epithelial defects.