Medicine and Health Sciences Commons^™

Chondroinduction From Naturally Derived Cartilage Matrix: A Comparison Between Devitalized And Decellularized Cartilage Encapsulated In Hydrogel Pastes., Emily C. Beck, Marilyn Barragan, Tony B. Libeer, Sarah L. Kieweg, Gabriel L. Converse, Richard A. Hopkins, Cory J. Berkland, Michael S. Detamore

Manuscripts, Articles, Book Chapters and Other Papers

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with …

Dog Walking Among Adolescents: Correlates And Contribution To Physical Activity., Jessa K. Engelberg, Jordan A. Carlson, Terry L. Conway, Kelli L. Cain, Brian E. Saelens, Karen Glanz, Lawrence D. Frank, James F. Sallis

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PURPOSE: To assess the association of dog walking with adolescents' moderate-to-vigorous physical activity (MVPA) and body mass index (BMI), and identify correlates of dog walking.

METHODS/DESIGN: Participants were 12-17year-olds (n=925) from the Baltimore, MD and Seattle, WA regions. Differences in accelerometer-assessed minutes/day of MVPA and self-reported BMI (percentile) were compared among adolescents (1) without a dog (n=441) and those with a dog who (2) did (≥1days/week, n=300) or (3) did not (n=184) walk it. Correlates of (1) dog walking (any vs. none) among adolescents with dogs (n=484), and (2) days/week of dog walking among dog walkers (n=300) were investigated. Potential …

Janus Kinase 2/Signal Transducer And Activator Of Transcription 3 Inhibitors Attenuate The Effect Of Cardiotrophin-Like Cytokine Factor 1 And Human Focal Segmental Glomerulosclerosis Serum On Glomerular Filtration Barrier., Mukut Sharma, Jianping Zhou, Jean-François Gauchat, Ram Sharma, Ellen T. Mccarthy, Tarak Srivastava, Virginia J. Savin

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Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) after renal transplantation is believed to be caused by a circulating factor(s). We detected cardiotrophin-like cytokine factor 1 (CLCF1), a member of the interleukin 6 family, in the plasma from patients with recurrent FSGS. We hypothesized that CLCF1 contributes to the effect of FSGS serum on the glomerular filtration barrier in vitro. Presently, we studied the effect of CLCF1 on isolated rat glomeruli using an in vitro assay of albumin permeability (P(alb)). CLCF1 (0.05-100 ng/mL) increased P(alb) and caused maximal effect at 5-10 ng/mL (P < 0.001). The increase in Palb was analogous to the effect of FSGS serum. Anti-CLCF1 monoclonal antibody blocked the CLCF1-induced increase in P(alb) and significantly attenuated the effect of FSGS serum (P < 0.001). The heterodimer composed of CLCF1 and cosecreted molecule cytokine receptor-like factor 1 (CRLF1) attenuated the increase in P(alb) caused by CLCF1 or FSGS serum. Western blot analysis showed that CLCF1 upregulated phosphorylation of signal transducer and activator of transcription 3 (STAT3) (Tyr705) in glomeruli. This effect was diminished by the heterodimer CLCF1-CRLF1. Janus kinase 2 (JAK2) inhibitor BMS-1119543 or STAT3 inhibitor Stattic significantly blocked the effect of CLCF1 or FSGS serum on P(alb) (P < 0.001). These novel findings suggest that when monomeric CLCF1 increases P(alb), the heterodimer CLCF1-CRLF1 may protect the glomerular filtration barrier. We speculate that albuminuria in FSGS is related to qualitative or quantitative changes in the CLCF1-CRLF1 complex, and that JAK2 or STAT3 inhibitors may be novel therapeutic agents to treat FSGS.

Scarnas Regulate Splicing And Vertebrate Heart Development., Prakash Patil, Nataliya Kibiryeva, Tamayo Uechi, Jennifer A. Marshall, James E. O'Brien, Michael Artman, Naoya Kenmochi, Douglas C. Bittel

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Alternative splicing (AS) plays an important role in regulating mammalian heart development, but a link between misregulated splicing and congenital heart defects (CHDs) has not been shown. We reported that more than 50% of genes associated with heart development were alternatively spliced in the right ventricle (RV) of infants with tetralogy of Fallot (TOF). Moreover, there was a significant decrease in the level of 12 small cajal body-specific RNAs (scaRNAs) that direct the biochemical modification of specific nucleotides in spliceosomal RNAs. We sought to determine if scaRNA levels influence patterns of AS and heart development. We used primary cells derived …

Ethanol At Low Concentrations Protects Glomerular Podocytes Through Alcohol Dehydrogenase And 20-Hete., Ellen T. Mccarthy, Jianping Zhou, Ryan Eckert, David Genochio, Rishi Sharma, Olurinde Oni, Alok De, Tarak Srivastava, Ram Sharma, Virginia J. Savin, Mukut Sharma

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Clinical studies suggest cardiovascular and renal benefits of ingesting small amounts of ethanol. Effects of ethanol, role of alcohol dehydrogenase (ADH) or of 20-hydroxyeicosatetraenoic acid (20-HETE) in podocytes of the glomerular filtration barrier have not been reported. We found that mouse podocytes at baseline generate 20-HETE and express ADH but not CYP2e1. Ethanol at high concentrations altered the actin cytoskeleton, induced CYP2e1, increased superoxide production and inhibited ADH gene expression. Ethanol at low concentrations upregulated the expression of ADH and CYP4a12a. 20-HETE, an arachidonic acid metabolite generated by CYP4a12a, blocked the ethanol-induced cytoskeletal derangement and superoxide generation. Ethanol at high …

Renal And Hematological Effects Of Clcf-1, A B-Cell-Stimulating Cytokine Of The Il-6 Family., Virginia J. Savin, Mukut Sharma, Jianping Zhou, David Gennochi, Timothy Fields, Ram Sharma, Ellen T. Mccarthy, Tarak Srivastava, Jos Domen, Aurélie Tormo, Jean-François Gauchat

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CLCF-1 is a cytokine known for B-cell stimulation and for neurotrophic properties. We have identified CLCF-1 as a potential injurious factor in the human renal disease focal segmental glomerulosclerosis (FSGS). We investigated its effects on renal cells and renal function in in vitro and in vivo studies. Methods include measurement of the effect of CLCF-1 on phosphorylation of target molecules of the JAK/STAT pathway, on cytoskeleton and cell morphology in cultured podocytes, on albumin permeability of isolated rat glomeruli, and on tissue phosphorylation and urine albumin after acute or chronic CLCF-1 injection. In addition, cell sorting was performed to determine …

Cyclooxygenase-2, Prostaglandin E2, And Prostanoid Receptor Ep2 In Fluid Flow Shear Stress-Mediated Injury In The Solitary Kidney., Tarak Srivastava, Uri S. Alon, Patricia A. Cudmore, Belal Tarakji, Alexander Kats, Robert E. Garola, R Scott Duncan, Ellen T. Mccarthy, Ram Sharma, Mark L. Johnson, Lynda F. Bonewald, Ashraf El-Meanawy, Virginia J. Savin, Mukut Sharma

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Hyperfiltration subjects podocytes to increased tensile stress and fluid flow shear stress (FFSS). We showed a 1.5- to 2.0-fold increase in FFSS in uninephrectomized animals and altered podocyte actin cytoskeleton and increased synthesis of prostaglandin E2 (PGE2) following in vitro application of FFSS. We hypothesized that increased FFSS mediates cellular changes through specific receptors of PGE2. Presently, we studied the effect of FFSS on cultured podocytes and decapsulated isolated glomeruli in vitro, and on solitary kidney in uninephrectomized sv129 mice. In cultured podocytes, FFSS resulted in increased gene and protein expression of cyclooxygenase (COX)-2 but not COX-1, prostanoid receptor EP2 …

Fluid Flow Shear Stress Over Podocytes Is Increased In The Solitary Kidney., Tarak Srivastava, Gianni E. Celsi, Mukut Sharma, Hongying Dai, Ellen T. Mccarthy, Melanie Ruiz, Patricia A. Cudmore, Uri S. Alon, Ram Sharma, Virginia A. Savin

Manuscripts, Articles, Book Chapters and Other Papers

BACKGROUND: Glomerular hyperfiltration is emerging as the key risk factor for progression of chronic kidney disease (CKD). Podocytes are exposed to fluid flow shear stress (FFSS) caused by the flow of ultrafiltrate within Bowman's space. The mechanism of hyperfiltration-induced podocyte injury is not clear. We postulated that glomerular hyperfiltration in solitary kidney increases FFSS over podocytes.

METHODS: Infant Sprague-Dawley rats at 5 days of age and C57BL/6J 14-week-old adult mice underwent unilateral nephrectomy. Micropuncture and morphological studies were then performed on 20- and 60-day-old rats. FFSS over podocytes in uninephrectomized rats and mice was calculated using the recently published equation …

Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets With Alopecia Resulting From A Novel Missense Mutation In The Dna-Binding Domain Of The Vitamin D Receptor., Peter J. Malloy, Jining Wang, Tarak Srivastava, David Feldman

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The rare genetic recessive disease, hereditary vitamin D resistant rickets (HVDRR), is caused by mutations in the vitamin D receptor (VDR) that result in resistance to the active hormone 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) or calcitriol). In this study, we examined the VDR from a young boy with clinical features of HVDRR including severe rickets, hypocalcemia, hypophosphatemia and partial alopecia. The pattern of alopecia was very unusual with areas of total baldness, adjacent to normal hair and regions of scant hair. The child failed to improve on oral calcium and vitamin D therapy but his abnormal chemistries and his bone X-rays normalized …

Bovine Immune Response To Shiga-Toxigenic Escherichia Coli O157:H7., Mark A Hoffman, Christian Menge, Thomas A Casey, William Laegreid, Brad T Bosworth, Evelyn A Dean-Nystrom

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Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the …

Effects Of Tacrolimus On Ischemia-Reperfusion Injury., Shawn D. St Peter, Adyr A. Moss, David C. Mulligan

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In addition to efficacious immunosuppression for the benefit of organ transplantation, tacrolimus has diverse actions that result in amelioration of ischemia-reperfusion injury. Knowledge is accumulating rapidly on the mechanisms through which tacrolimus exerts these cytoprotective effects, including alterations in microcirculation, free radical metabolism, calcium-activated pathways, inflammatory cascades, mitochondrial stability, apoptosis, stress-response proteins, and tissue recovery. Within the nucleus, actions mediating the effects of tacrolimus appear to be dominantly influenced by interactions with the transcription factor, nuclear factor-kappaB. Because tacrolimus is a cornerstone agent in immunosuppression regimens throughout the world and knowledge of its cellular mechanisms is evolving, it is important …

Optimisation Of Bile Production During Normothermic Preservation Of Porcine Livers., Charles J. Imber, Shawn D. St Peter, Inigo Lopez De Cenarruzabeitia, Hugh Lemonde, Mike Rees, Andrew Butler, Peter T. Clayton, Peter J. Friend

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Machine perfusion of livers may provide a mechanism for extended preservation of marginal donor organs before transplantation, as well as a method for viability assessment. It has proved possible in a series of experimental porcine liver perfusions to maintain liver viability for up to 72 h. However, a reduction in bile production with associated histological evidence of cholestasis was seen after 10 h of perfusion, damaging the biliary canaliculi during the preservation period and leaving these organs in an unacceptable condition for transplantation. It was proposed that reduction in bile production was the result of a relentless depletion of available …

Hepatic Steatosis And Its Relationship To Transplantation., Charles J. Imber, Shawn D. St Peter, Ashok Handa, Peter J. Friend

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Fatty infiltration of the liver is common in the brain-dead donor population and has a strong correlation with primary nonfunction after cold preservation, a condition that is catastrophic to liver transplant recipients. This literature review examines factors associated with the development, diagnosis, quantification, and clinical management of this difficult condition.

Extended Preservation Of Non-Heart-Beating Donor Livers With Normothermic Machine Perfusion., Shawn D. St Peter, C J. Imber, I Lopez, D Hughes, P J. Friend

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BACKGROUND: Non-heart-beating donor (NHBD) livers represent an important organ pool, but are seldom utilized clinically and require rapid retrieval and implantation. Experimental work with oxygenated perfusion during preservation has shown promising results by recovering function in these livers. This study compared sanguinous perfusion with cold storage for extended preservation of the NHBD liver in a porcine model.

METHODS: Porcine livers were subjected to 60 min of in vivo total warm ischaemia before flushing, after which they were preserved by one of two methods: group 1 (n = 4), University of Wisconsin (UW) solution by standard cold storage for 24 h; …

Beta-Galactosidase As A Marker Of Ischemic Injury And A Mechanism For Viability Assessment In Porcine Liver Transplantation., Shawn D. St Peter, Charles J. Imber, Inigo Lopez De Cenarruzabeitia, James Mcguire, Tim James, Richard Taylor, Peter J. Friend

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Glycohydrolases are a group of enzymes contained predominantly within lysosomes, which are released during Kupffer cell activation or death. One of these, beta-galactosidase, has been proposed as a marker of ischemia-reperfusion injury in the liver because Kupffer cell activation represents a primary event in the injurious reperfusion cascade. In this study, we compared B-galactosidase with more traditional indicators of liver injury and function in a porcine model of liver preservation. Porcine livers were allocated into two groups: group C (n = 5), preserved in University of Wisconsin solution by standard cold storage for 24 hours, and group W (n = …

Development Of A Blocking Enzyme-Linked Immunosorbent Assay For Detection Of Serum Antibodies To O157 Antigen Of Escherichia Coli., W Laegreid, M Hoffman, J Keen, R Elder, J Kwang

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The O157 antigen of Escherichia coli shares structural elements with lipopolysaccharide (LPS) antigens of other bacterial species, notably Brucella abortus and Yersinia enterocolitica 09, a fact that confounds the interpretation of assays for anti-O157 antibodies. To address this problem, a blocking enzyme-linked immunosorbent assay (bELISA) was designed with E. coli O157:H7 LPS as the antigen and a monoclonal antibody specific for E. coli O157, designated 13B3, as the competing antibody. The bELISA had equivalent sensitivity to, and significantly higher specificity than, the indirect ELISA (iELISA), detecting anti-O157 antibodies in sera from cattle experimentally inoculated with O157:H7. Only 13% of sera …

Biochemical And Mutational Analysis Of The Histidine Residues Of Staphylococcal Enterotoxin A., M Hoffman, M Tremaine, J Mansfield, M Betley

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The goal of this study was to examine the role of histidine residues in the biological activities of staphylococcal enterotoxin A (SEA). Carboxymethylated SEA was unable to stimulate murine T-cell proliferation but was resistant to monkey stomach lavage fluid degradation, suggesting that native conformation was intact. Site-directed mutagenesis of the histidine residues of SEA was subsequently performed. SEA-H44A (SEA with histidine 44 replaced with alanine), SEA-H44D, SEA-H50A, SEA-H50D, SEA-H114A, SEA-H114D, SEA-H187A, and SEA-H187D retained superantigen and emetic activities, whereas SEA-H225A and SEA-H225D were defective in the ability to stimulate T-cell proliferation. These mutants were unable to compete with SEA for …