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Biomedical subjects

L Philipson

Publications and source records attributed to L Philipson.

At least 19 recordsLinked to original sources
PubMed2004

The coxsackie-adenovirus receptor--a new receptor in the immunoglobulin family involved in cell adhesion.

The physiological and cell biological aspects of the Coxsackie-Adenovirus Receptor (CAR) is discussed in this review. The receptor obviously recognizes the group C adenoviruses in vivo, but also fibers from other groups except group B in vitro. The latter viruses seem to utilize a different receptor. The receptor accumulates at, or close to, the tight junction in polarized epithelial cells and probably functions as a cell-cell adhesion molecule. The cytoplasmic tail of the receptor is not required for virus attachment and uptake. Although there is a correlation between CAR and uptake of adenoviruses in several human tumor cells, evidence of an absolute requirement for integrins has not been forthcoming. The implication of these findings for adenovirus gene therapy is discussed.

Adenoviridae↗
PubMed2004

Islet isolation under cGMP conditions: replacing the coil.

INTRODUCTION: Clinical islet transplantation is increasingly regulated, with isolation standards defined under current good manufacturing practices (cGMPs). cGMP requires validation of equipment cleaning and sterilization. The automated process for islet isolation requires rapid thermal exchange during processing, manipulating a metal coil containing cellular product into and out of chilled/heated waterbaths. We recognize challenges of validating cleaning and sterilization of this coil and propose replacement with a disposable blood warming system. Our specific aims were to assess the system's ability to accommodate flow rates utilized during various phases of pancreatic digestion and to assess its efficiency of heat exchange and temperature control. METHODS: In a pancreas digestion circuit, heat exchange occurred via the coil in a digital water bath, or Warmflo blood warming bag in a digital warming unit. Temperature within the digestion chamber was assessed using an inserted thermocouple. Time to achieve 37 degrees C was measured for set heating element temperatures 38.5 degrees C to 41 degrees C. Circuit temperature maintenance characteristics were also recorded. RESULTS: The Warmflo bloodbag easily accommodated flows of 150 and 300 mL/min. At all set temperatures, the bag resulted in shorter or equivalent time to 37 degrees C than the coil. Maintenance of 37 degrees C was also equivalent. We have utilized this system for four human islet isolations with mean time to 37 degrees C of 5.5 minutes, without difference in digestion quality. CONCLUSIONS: Use of a disposable blood warming system in place of the coil during islet isolation provides adequate flow characteristics, heat exchange, and temperature control and may facilitate evolution of islet isolation toward cGMP compliance.

Cell Culture Techniques↗
PubMed2002

Human urinary bladder carcinomas express adenovirus attachment and internalization receptors.

The use of adenoviral vectors as potent gene delivery systems requires expression of the Coxsackievirus/adenovirus receptor (CVADR) on the target cell surface. This receptor is important for virus attachment to the cell surface. For effective internalization of the vector into the target cell the integrins alpha(v)beta(3) and/or alpha(v)beta(5) are needed. Since there have been reports of loss of CVADR in bladder cancer cell lines, we wanted to investigate the expression of this receptor in bladder carcinoma biopsies. Surgical biopsies, as well as five human bladder cancer cell lines, were analyzed for expression of CVADR, the integrins alpha(v)beta(3) and alpha(v)beta(5) and MHC class I. Further, we studied the ability to transduce these cell lines using adenoviral vectors. Immunohistochemistry revealed that all biopsies (27/27) were positive for CVADR. Some variation in expression was evident, and superficially growing tumors stained more strongly than invasive ones. Most human tumors expressed the integrin alpha(v)beta(5) (14/24), whereas integrin alpha(v)beta(3) was less frequently seen (3/20). The established cell lines were efficiently transduced with adenoviral vectors, and transduction could be reduced with anti-CVADR antibodies. The abundance of appropriate viral receptors on tumor biopsy cells is a further argument for using adenoviral vectors in gene therapy of bladder cancer.

Adenoviridae↗
PubMed2001

A mouse model for adenovirus gene delivery.

The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.

Adenoviridae↗
PubMed2001

Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism.

Mutations in hepatocyte nuclear factor 1alpha (HNF-1alpha) lead to maturity-onset diabetes of the young type 3 as a result of impaired insulin secretory response in pancreatic beta-cells. The expression of 50 genes essential for normal beta-cell function was studied to better define the molecular mechanism underlying the insulin secretion defect in Hnf-1alpha(-/-) mice. We found decreased steady-state mRNA levels of genes encoding glucose transporter 2 (Glut2), neutral and basic amino acid transporter, liver pyruvate kinase (L-Pk), and insulin in Hnf-1alpha(-/-) mice. In addition, we determined that the expression of several islet-enriched transcription factors, including Pdx-1, Hnf-4alpha, and Neuro-D1/Beta-2, was reduced in Hnf-1alpha(-/-) mice. These changes in pancreatic islet mRNA levels were already apparent in newborn animals, suggesting that loss of Hnf-1alpha function rather than chronic hyperglycemia is the primary cause of the altered gene expression. This expression profile was pancreatic islet-specific and distinct from hepatocytes, where we found normal expression of Glut2, L-Pk, and Hnf-4alpha in the liver of Hnf-1alpha(-/-) mice. The expression of small heterodimer partner (Shp-1), an orphan receptor that can heterodimerize with Hnf-4alpha and inhibit its transcriptional activity, was also reduced in Hnf-1alpha(-/-) islets. We characterized a 0.58-kb Shp-1 promoter and determined that the decreased expression of Shp-1 may be indirectly mediated by a downregulation of Hnf-4alpha. We further showed that Shp-1 can repress its own transcriptional activation by inhibiting Hnf-4alpha function, thereby establishing a feedback autoregulatory loop. Our results indicate that loss of Hnf-1alpha function leads to altered expression of genes involved in glucose-stimulated insulin secretion, insulin synthesis, and beta-cell differentiation.

Animals↗
PubMed2000

The gas 5 gene shows four alternative splicing patterns without coding for a protein.

The murine gas5 gene was originally isolated based on its preferential expression in the growth arrest phase of the cell cycle. This gene contains 12 exons from which two alternatively spliced transcripts have been initially identified. More recently, it has been reported that both human and murine gas5 genes contain in their introns sequences homologous to the small nucleolar RNAs involved in the processing of ribosomal RNA. Here we report on the identification and analysis of the expression pattern of two novel alternatively spliced mouse gas5 mRNAs which contain no obvious open reading frame (ORF). Using antibodies generated against the putative amino acid sequence deduced from the gas5 cDNAs, we were not able to detect any Gas5 protein in cultured cells or murine tissues extracts. Even more definitive evidence that the gas5 gene may not encode a protein was obtained by cloning and sequencing the rat gas5 gene which revealed that the putative ORF is interrupted by a stop codon after the first 13 amino acids.

3T3 Cells↗
PubMed2000

Expression of the adenovirus receptor and its interaction with the fiber knob.

The coxsackievirus group B (CVB) and adenovirus (Ad) receptor (HCVADR, formerly HCAR) is a cell surface protein with two immunoglobulin-like regions (IG1 and IG2) that serves as a receptor for two structurally unrelated viruses. We have established the tissue distribution of the receptor in the rodent by immunohistochemistry and show that the receptor is broadly expressed during embryonic development in the central and peripheral nervous systems and in several types of epithelial cells. The tissue distribution is more restricted in the adult but remains high mainly in epithelial cells. Using site-directed mutagenesis, based on computer modeling of the IG1 region, Ad5 binding could be inhibited but CVB attachment was unaffected. A double amino acid substitution in a three-stranded anti-parallel beta sheet that may form a face of the receptor completely inhibited Ad5 binding. Therefore, we conclude that the molecular interactions critical for Ad5 binding to HCVADR do not overlap with those of CVB3. In fact a specific antibody interfering with only CVB binding recognizes the IG2 domain in the receptor, suggesting that the CVB interacts with this region or an overlap between the IG1 and the IG2 regions.

3T3 Cells↗
PubMed1998

Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1alpha gene, insulin secretion and intracellular calcium ([Ca2+]i) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+ currents (KATP) in beta-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+]i, and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1alpha diabetes results from defective beta-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Adenosine Triphosphate↗
PubMed1998

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.

Animals↗
PubMed1997

Myc represses transcription of the growth arrest gene gas1.

Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. We show that c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen was sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in promotion of cell growth by Myc.

3T3 Cells↗
PubMed1997

HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses.

The subgroup C of the adenoviruses (Ad) and the group B coxsackieviruses (CVB) are structurally unrelated viruses that are known to compete for an unidentified cell surface receptor. We now describe the isolation of cDNAs from human and mouse that encode the human CVB and Ad2 and 5 receptor (HCAR) and the mouse CVB Ad2 and 5 receptor (MCAR). Both are 46-kDa glycoproteins whose primary amino acid sequences are highly homologous. Structurally, HCAR and MCAR appear to be transmembrane proteins that contain two extracellular immunoglobulin-like domains and therefore belong to this superfamily. Transfection of either of these cDNA molecules into receptor-negative NIH 3T3 cells conferred susceptibility to CVB infection and permitted the expression of beta-galactosidase from a recombinant Ad5 vector. In addition, HCAR and MCAR mRNAs could be detected on Northern blots of oligo(dT)-selected RNA from receptor-positive HeLa cells and TCMK-1 as well as several tissues of human and mouse origin that are known to be targets for Ad and CVB infections. Finally, Western blots using antibodies that inhibit virus binding to either the human or mouse CVB receptors detected 46-kDa proteins in HCAR- and MCAR-transfected cells, respectively. Taken together, these results confirm that the isolated cDNAs encode the receptors for the subgroup C Ad and CVB.

3T3 Cells↗
PubMed1997

The diagnosis of carpal tunnel syndrome. Sensitivity and specificity of some clinical and electrophysiological tests.

The study group consisted of 100 persons referred with suspected carpal tunnel syndrome. Clinical and neurophysiological examinations were performed blinded from each other. The gold standard for the carpal tunnel syndrome (CTS) diagnosis was based on the results of these examinations but relief of CTS symptoms after surgery was also required. The sensitivity and specificity for the combined results of the clinical examinations were 94% and 80% respectively, and for the neurophysiological examinations, 85% and 87%. Of the neurophysiological methods used, the quotient of sensory nerve conduction velocity between palm to wrist and wrist to elbow was best and the cut-off for this test was studied by means of an ROC-curve. According to our results clinical examination by an experienced doctor seems to be sufficient if there are typical symptoms of carpal tunnel syndrome, but if there is a history of pain, atypical symptoms or earlier fractures in the arm, wrist or hand, it is important to add a neurophysiological examination.

Adolescent↗
PubMed1996

The effect of continuous epidural infusion of ropivacaine (0.1%, 0.2% and 0.3%) on nerve conduction velocity and postural control in volunteers.

BACKGROUND: Continuous epidural infusions of local anaesthetics have become increasingly popular in postoperative pain treatment, especially as they permit early mobilisation. Ropivacaine is a promising new agent which induces more pronounced sensory than motor blockade. This study was focused on the influence of continuous epidural infusion of ropivacaine on impulse conduction in large nerves (by measurement of F and H latencies), and on the subjects' ability to maintain postural control during mobilisation. METHODS: Healthy male volunteers received 0.1%, 0.2% or 0.3% ropivacaine, and bupivacaine 0.25% was used as reference. A bolus epidural injection of 10 ml of the drug, at L2/3 level, was followed by continuous infusion at 10 ml/h for 21 h. Motor blockade was assessed by mechanical measurements of force during big toe flexion and by recording of F latency. Sensory blockade was monitored by pin-prick and Thermotest methods, and by H latency recording. The subjects' ability to perform a postural test was evaluated by posturography. RESULTS: The F and H latencies became prolonged/abolished dose-dependently. With ropivacaine, F latency recovered significantly later than motor function (P = 0.0002), and H latency recovered later than normal pin-prick perception (P = 0.0006). However, the duration of partial blockade of thermoperception was comparable to that of H latency prolongation. Posturographically, the subjects receiving 0.1% ropivacaine differed significantly from all others (P < 0.001) in that they were able to maintain postural control during the infusion. The recovery period after termination of infusion was significantly shorter with ropivacaine than with bupivacaine for all measured variables. CONCLUSION: Recovery of postural control with 0.2% and 0.3% ropivacaine is significantly faster than with bupivacaine 0.25%. H latency recording allows detection of epidural blockade intensity that does not prevent subjects from performing postural tests.

Adult↗
PubMed1996

Myoelectric activity in the stomach and duodenum after epidural administration of morphine or bupivacaine.

BACKGROUND: Gastric emptying is delayed in patients receiving postoperative pain relief with epidural morphine compared to patients receiving epidural bupivacaine. The electrophysiological basis for this effect is unknown. The aim of this study was to compare the effects of epidural morphine with epidural bupivacaine on gastroduodenal myoelectric activity (EMG) in patients after surgery. METHODS: Fourteen patients with epidural analgesia who underwent open cholecystectomy were randomly assigned to receive either epidural morphine (EM) or epidural bupivacaine (EB) for postoperative pain relief. During surgery EMG electrodes were placed in the subserosa in the antrum and duodenum and the EMG registration started 3.5 hours after the end of surgery. Gastric emptying measured with the acetaminophen test was studied in the morning the day after surgery. RESULTS: The spike activity in the antrum was significantly lower 160-340 minutes after the administration of morphine in the EM-group compared to the activity in the EB-group (P < 0.04). The incidence of regular slow wave rhythm in the antrum was significantly lower 160-520 minutes after the administration of morphine in the EM-group compared to the EB-group (P < 0.03). Duodenal Phase III activity measured with EMG was significantly more frequent during 160-520 minutes after the morphine administration in the EM-group compared to the EB-group (P < 0.02). The acetaminophen absorption was significantly delayed in the epidural morphine group compared to the epidural bupivacaine group. CONCLUSIONS: Gastroduodenal electromyographic activity was significantly changed during epidural morphine compared to epidural bupivacaine. The delayed gastric emptying during epidural morphine may be explained by decreased and uncoordinated contractile activity in the antrum, shown by decreased spike activity and irregular slow wave rhythm. The increased pressure activity in the duodenum, shown by increased Phase III activity, may also impair gastric emptying.

Adult↗
PubMed1995

The growth-inhibitory block of TGF-beta is located close to the G1/S border in the cell cycle.

Transforming growth factor-beta (TGF-beta) inhibits DNA synthesis in dense cultures of young human embryonic fibroblasts and antagonizes the mitogenic action of platelet-derived growth factor (PDGF). The inhibition of the PDGF-BB action by TGF-beta was independent of the induction of mRNAs for the PDGF-A chain and PDGF-beta receptor, the predominant types of PDGF receptor in human fibroblasts. The TGF-beta-mediated inhibition did not influence the expression of various genes that are involved in the transition from the arrested (GO) state to the S phase of the cell cycle. Indeed, TGF-beta upregulated the "early" genes c-myc, c-fos, and junB and downregulated the growth arrest-specific (gas) genes. These results suggest that the inhibition of DNA synthesis by TGF-beta in human fibroblasts is independent of modulation of expression of early and gas genes, placing the TGF-beta block comparatively late in the GO to S transition. In cultures of senescent human fibroblasts TGF-beta stimulated DNA synthesis but, nevertheless, had the same effect as in young cells on the expression of PDGF chains and receptor genes, as well as on early and gas genes, with the exception of a significantly lower induction of c-fos.

Cell Cycle Proteins↗