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Biomedical subjects

Brian A Babbin

Publications and source records attributed to Brian A Babbin.

9 recordsLinked to original sources
PubMed2006

Malabsorption work-up: utility of small bowel biopsy.

A wide variety of small intestinal mucosal diseases lead to malabsorption. Although stool studies, especially stool for excess fat, and functional tests for deficiency states are important clues to malabsorption, small intestinal biopsies are probably the most crucial part of the diagnostic process. Many mucosal disorders have distinctive histologic features that allow for precise diagnosis. However, these histologic changes might be subtle. The role of the gastroenterologist is to provide the pathologist with adequate clinical information and tissue material to ensure a complete examination pathologically. Celiac disease is the most common mucosal cause of chronic malabsorption in the western world. Celiac disease can present classically as large volume fatty diarrhea, but it more commonly presents with subtle clinical symptoms or iron deficiency anemia. Although the histologic hallmark of celiac disease is increased intraepithelial lymphocytosis along with villous atrophy, increased intraepithelial lymphocytosis alone in an appropriate clinical context might suggest the diagnosis of celiac disease. The aim of this review is to highlight the importance of close cooperation and communication between the gastroenterologist and the pathologist to optimize the diagnosis of mucosal diseases that result in malabsorption.

Adult↗
PubMed2006

Annexin I regulates SKCO-15 cell invasion by signaling through formyl peptide receptors.

Annexin 1 (AnxA1) is a multifunctional phospholipid-binding protein associated with the development of metastasis in some invasive epithelial malignancies. However, the role of AnxA1 in the migration/invasion of epithelial cells is not known. In this study, experiments were performed to investigate the role of AnxA1 in the invasion of a model epithelial cell line, SKCO-15, derived from colorectal adenocarcinoma. Small interfering RNA-mediated knockdown of AnxA1 expression resulted in a significant reduction in invasion through Matrigel-coated filters. Localization studies revealed a translocation of AnxA1 to the cell surface upon the induction of cell migration, and functional inhibition of cell surface AnxA1 using antiserum (LCO1) significantly reduced cell invasion. Conversely, SKCO-15 cell invasion was increased by approximately 2-fold in the presence of recombinant full-length AnxA1 and the AnxA1 N-terminal-derived peptide mimetic, Ac2-26. Because extracellular AnxA1 has been shown to regulate leukocyte migratory events through interactions with n-formyl peptide receptors (nFPRs), we examined the expression of FPR-1, FPRL-1, and FPRL-2 in SKCO-15 cells by reverse transcriptase-PCR and identified expression of all three receptors in this cell line. Treatment of SKCO-15 cells with AnxA1, Ac2-26, and the classical nFPR agonist, formylmethionylleucylphenylalanine, induced intracellular calcium release consistent with nFPR activation. Furthermore, the nFPR antagonist, Boc2, abrogated the AnxA1 and Ac2-26-induced intracellular calcium release and increase in SKCO-15 cell invasion. Together, these results support an autocrine/paracrine role for membrane AnxA1 in stimulating SKCO-15 cell migration through nFPR activation. The findings in this study suggest that activation of nFPRs stimulates epithelial cell motility important in the development of metastasis as well as wound healing.

Annexin A1↗
PubMed2006

Plasmid DNA and siRNA transfection of intestinal epithelial monolayers by electroporation.

This study was conducted to evaluate the ability of electroporation to efficiently transfect differentiated intestinal epithelial monolayers with plasmid DNA and to determine whether electroporation can transfect these monolayers with short-interfering RNA (siRNA) to cause gene silencing. Confluent T84 monolayers were transfected with reporter plasmids expressing luciferase or green-fluorescent protein or with siRNA directed against the nuclear envelope proteins lamin A/C using electroporation. Optimized electroporation conditions resulted in luciferase and GFP expression. Both intracellular uptake of fluorescently labeled plasmid and expression of the reporter genes increased with increasing electroporation strength and DNA concentration. When monolayers were transfected by lipofection with the reporter plasmids, expression and DNA uptake were less than for electroporation. Electroporation was also found to transfect monolayers with siRNA, which resulted in up to 90% inhibition of targeted protein production. Silencing occurred within 24h of transfection and increased with increasing siRNA concentration. These results suggest that electroporation can provide a valuable research tool for transfection of intestinal epithelial monolayers and other differentiated cell systems, and may ultimately be useful for clinical gene therapy applications.

Cell Line↗
PubMed2005

Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils.

Neutrophil (polymorphonuclear leukocytes [PMN]) transepithelial migration during inflammatory episodes involves a complex series of adhesive interactions and signaling events. Previous studies have shown that key adhesive interactions between leukocyte CD11b/CD18 and basally expressed fucosylated glycoproteins followed by binding to desmosomal-associated JAM-C are key elements of the transmigration response. Here we provide the first evidence that PMN-expressed junctional adhesion molecule-like protein (JAML) regulates transmigration via binding interactions with epithelial coxsackie and adenovirus receptor (CAR). Experiments with a JAML fusion protein revealed specific binding of JAML to epithelial CAR expressed at tight junctions in T84 cell monolayers and normal human colonic mucosa. Furthermore, JAML-CAR binding is mediated via the membrane distal immunoglobulin (Ig) loop of CAR and the membrane proximal Ig loop of JAML. PMN bound to immobilized CAR but not JAML in a divalent cation-independent manner. Lastly, in assays of PMN transepithelial migration, JAML/CAR fusion proteins and their antibodies significantly inhibited transmigration in a specific manner. Taken together, these results indicate that JAML and CAR are a novel pair of adhesion molecules that play an important role in modulating PMN migration cross epithelial tight junctions. These findings add a new element to a multistep model of PMN transepithelial migration and may provide new targets for anti-inflammatory therapies.

Antibodies, Monoclonal↗
PubMed2005

Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity.

Epithelial tight junctions form a selectively permeable barrier to ions and small molecules. Junctional adhesion molecule 1 (JAM1/JAM-A/F11R) is a tight junction-associated transmembrane protein that has been shown to participate in the regulation of epithelial barrier function. In a recent study, we presented evidence suggesting that JAM1 homodimer formation is critical for epithelial barrier function (Mandell, K. J., McCall, I. C., and Parkos, C. A. (2004) J. Biol. Chem. 279, 16254-16262). Here we have used small interfering RNA to investigate the effect of the loss of JAM1 expression on epithelial cell function. Consistent with our previous study, knockdown of JAM1 was observed to increase paracellular permeability in epithelial monolayers. Interestingly, knockdown of JAM1 also produced dramatic changes in cell morphology, and a similar effect was observed with expression of a JAM1 mutant lacking the putative homodimer interface. Further studies revealed that JAM1 knockdown decreased cell-matrix adhesion and spreading on matrix proteins that are ligands of beta1 integrins. These changes were characterized by a decrease in beta1 integrin protein levels and loss of beta1 integrin staining at the cell surface. Immunolabeling of cells for the small GTPase Rap1, a known activator of beta1 integrins, revealed colocalization of Rap1 with JAM1 at intercellular junctions, and knockdown of JAM1 resulted in decreased Rap1 activity. Lastly, knockdown of Rap1b resulted in diminished beta1 integrin expression and altered cell morphology analogous to that observed with knockdown of JAM1. Together, these results suggest that JAM1 regulates epithelial cell morphology and beta1 integrin expression by modulating activity of the small GTPase Rap1.

Cell Adhesion↗
PubMed2004

JAM-C is a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration.

Neutrophil (PMN) transepithelial migration is dependent on the leukocyte beta(2) integrin CD11b/CD18, yet the identity of epithelial counterreceptors remain elusive. Recently, a JAM protein family member termed JAM-C was implicated in leukocyte adhesive interactions; however, its expression in epithelia and role in PMN-epithelial interactions are unknown. Here, we demonstrate that JAM-C is abundantly expressed basolaterally in intestinal epithelia and localizes to desmosomes but not tight junctions. Desmosomal localization of JAM-C was further confirmed by experiments aimed at selective disruption of tight junctions and desmosomes. In assays of PMN transepithelial migration, both JAM-C mAbs and JAM-C/Fc chimeras significantly inhibited the rate of PMN transmigration. Additional experiments revealed specific binding of JAM-C to CD11b/CD18 and provided evidence of other epithelial ligands for CD11b/CD18. These findings represent the first demonstration of direct adhesive interactions between PMN and epithelial intercellular junctions (desmosomes) that regulate PMN transepithelial migration and also suggest that JAM-C may play a role in desmosomal structure/function.

Acrylamide↗
PubMed2004

Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification.

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, beta1-integrin. This was independent of cell-matrix adhesion through beta1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with beta1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.

Actins↗
PubMed2004

Identification of Krüppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer.

Krüppel-like factor 4 (KLF4 or GKLF) is an inhibitor of the cell cycle. The gene encoding KLF4 is localized on chromosome 9q, previously shown to exhibit allelic loss in colorectal cancer (CRC). In this study, we show that the mean level of KLF4 mRNA in a panel of 30 CRC was 52% that of paired normal colonic tissues. Similarly, the levels of KLF4 mRNA and protein in a panel of six established CRC cell lines were significantly lower than those of an untransformed colonic epithelial cell line. Using highly polymorphic DNA markers that flank the KLF4 locus, we found evidence for loss of heterozygosity (LOH) in two of eight surgically resected CRC specimens. In addition, LOH was observed in five of six CRC cell lines with one additional cell line exhibiting hemizygous deletion in the KLF4 gene. We also found that the 5'-untranslated region of KLF4 was hypermethylated in a subset of resected CRC specimens and cell lines. Lastly, the open-reading frame of KLF4 in two of three CRC cell lines examined contained several point mutations that resulted in a diminished ability to activate the p21(WAF1/Cip1) promoter. These findings indicate that KLF4 is a potential tumor suppressor gene in CRC.

5' Untranslated Regions↗