Biomedical subjects

A L Beaudet

Publications and source records attributed to A L Beaudet.

At least 19 recordsLinked to original sources

PubMed1995

Novel isoforms of murine intercellular adhesion molecule-1 generated by alternative RNA splicing.

Intercellular adhesion molecule-1 (ICAM-1)-deficient mice, produced by homologous recombination and previously recognized to be devoid of the common form of ICAM-1, are shown to express residual amounts of ICAM-1 Ag in thymus and lung. We demonstrate that this expression of ICAM-1 is possible because the mutated exon 5 in these animals has been skipped by alternative splicing of RNA. Three different alternative isoforms of ICAM-1 are expressed in mutant mice. Moreover, two of these isoforms are expressed in wild-type mice together with two additional alternative isoforms that cannot be produced in mutant mice. All alternatively spliced isoforms of ICAM-1 detected are missing complete extracellular Ig domains. In both mutant and wild-type mice, expression of alternatively spliced isoforms is up-regulated following stimulation of animals with LPS. Furthermore, all alternative isoforms of ICAM-1, except one, retain the ability to bind to the well-recognized ICAM-1 counter-receptor LFA-1. These findings, along with the restricted tissue distribution in mutant mice, indicate that alternative isoforms of ICAM-1 are significant physiologic adhesion structures which could play a distinct role in the functioning of the immune system of intact animals.

Alternative Splicing↗

PubMed1995

Cloning and mapping of the mouse alpha 7-neuronal nicotinic acetylcholine receptor.

We report the isolation of cDNA clones for the mouse alpha 7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind alpha-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse alpha 7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The alpha 7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 +/- 1.7 cM-Gas2-0.9 cM +/- 0.9 cM-D7Mit70-1.8 +/- 1.2 cM-Acra7-4.4 +/- 1.0 cM-Hras1-ps1/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14.

Amino Acid Sequence↗

PubMed1995

Sequential contribution of L- and P-selectin to leukocyte rolling in vivo.

Leukocyte recruitment into inflammatory sites is initiated by a reversible transient adhesive contact with the endothelium called leukocyte rolling, which is thought to be mediated by the selectin family of adhesion molecules. Selectin-mediated rolling precedes inflammatory cell emigration, which is significantly impaired in both P- and L-selectin gene-deficient mice. We report here that approximately 13% of all leukocytes passing venules of the cremaster muscle of wild-type mice roll along the endothelium at < 20 min after surgical dissection. Rolling leukocyte flux fraction reaches a maximum of 28% at 40-60 min and returns to 13% at 80-120 min. In P-selectin-deficient mice, rolling is absent initially and reaches 5% at 80-120 min. Rolling flux fraction in L-selectin-deficient mice is similar to wild type initially and declines to 5% at 80-120 min. In both wild-type and L-selectin-deficient mice, initial leukocyte rolling (0-60 min) is completely blocked by the P-selectin monoclonal antibody (mAb) RB40.34, but unaffected by L-selectin mAb MEL-14. Conversely, rolling at later time points (60-120 min) is inhibited by mAb MEL-14 but not by mAb RB40.34. After treatment with tumor necrosis factor (TNF)-alpha for 2 h, approximately 24% of all passing leukocytes roll in cremaster venules of wild-type and P-selectin gene-deficient mice. Rolling in TNF-alpha-treated mice is unaffected by P-selectin mAb or E-selectin mAb 10E9.6. By contrast, rolling in TNF-alpha-treated P-selectin-deficient mice is completely blocked by L-selectin mAb. These data show that P-selectin is important during the initial induction of leukocyte rolling after tissue trauma. At later time points and in TNF-alpha-treated preparations, rolling is largely L-selectin dependent. Under the conditions tested, we are unable to find evidence for involvement of E-selectin in leukocyte rolling in mice.

Antibodies, Monoclonal↗

PubMed1995

Simultaneous analysis of mutant and normal alleles for multiple cystic fibrosis mutations by the ligase chain reaction.

The ligase chain reaction (LCR) involves repetitive cycles of ligation of two adjacent pairs of oligonucleotides to form longer ligated products in a template-dependent manner. This study demonstrates the application of LCR for analysis of multiple small mutations. We adapted the technology for the simultaneous determination of the normal and mutant alleles in a competition format, as well as multiple mutations in a multiplex format. For these purposes, we used mutations causing cystic fibrosis, namely the delta F508, W1282X, and G551D mutations. Blunt ligation was compared to a strategy with a single base gap on one or both strands to be filled by thermostable polymerase prior to ligation. Blunt or gap strategies worked well for detection of the delta F508 mutation. Detection of the W1282X mutation worked well with a blunt strategy when high K+ concentration (180-220 mM) was used to reduce template-independent ligation. For reliable detection of the G551D mutation, we used mismatches in the oligonucleotides 2-5 bp away from the ligation site and hot start of the reaction to achieve allele specificity. Excellent discrimination of mutations was achieved using competitive LCR with six oligonucleotides (two common on one side of the mutation plus two wild type and two mutant on the opposite side with the mutation site at the end adjacent to the common oligonucleotides) and with multiplex-competitive LCR using 12 oligonucleotides to detect both alleles for two mutations in a single tube.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

PubMed1995

Gene targeting for inflammatory cell adhesion molecules.

Using gene targeting in mouse embryonic stem cells, it is possible to introduce diverse mutations into specific genes. Using these methods, various laboratories have reported mutations for a variety of inflammatory cell adhesion molecules including CD18, alpha 5 integrin, ICAM-1, P-selectin, and L-selectin; preliminary reports of other mutations are also available. Mutations in CD18 and ICAM-1 cause impaired inflammatory and immune responses, mutations in P-selectin and L-selectin cause decreased leukocyte rolling and emigration, and a mutation in alpha 5 integrin causes embryonic lethality. Gene targeting complements other approaches for analyzing the function of inflammatory cell adhesion molecules.

Animals↗

PubMed1995

Severe phenotype in mice with termination mutation in exon 2 of cystic fibrosis gene.

Mice with a termination codon mutation in exon 2 of the cystic fibrosis (CF) gene were generated using homologous recombination in embryonic stem cells. Animals homozygous for the mutant allele display a severe intestinal phenotype similar to that previously reported for CF mutant mice. The null nature of this allele was demonstrated by the absence of detectable wild-type mRNA, by the absence of detectable CFTR in the serous gland collecting ducts of salivary tissues, and by the lack of cAMP-mediated short-circuit current responses in colonic epithelium of mutant animals.

Animals↗

PubMed1995

Intercellular adhesion molecule 1 gene associations with immunologic subsets of inflammatory bowel disease.

BACKGROUND & AIMS: Inflammatory bowel disease is characterized by a failure to down-regulate the usual self-limited gut inflammatory response, suggesting that one or more of the predisposing genes could be those that determine the level of the immune response along the inflammatory pathway. The aim of this study was to examine potential associations of intercellular adhesion molecule 1 (ICAM-1) gene polymorphisms with inflammatory bowel disease or subsets of inflammatory bowel disease. METHODS: One hundred eighteen patients with ulcerative colitis (UC) and 130 patients with Crohn's disease (CD) as well as 77 ethnically matched controls were tested for antineutrophil cytoplasmic antibodies (ANCAs) and genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for ICAM-1 polymorphisms at codon 241 (exon 4) and codon 469 (exon 6). RESULTS: There was no significant difference between all patients with UC, patients with CD, and controls in either polymorphism. However, when stratified by ANCA status, ANCA-negative UC showed a significantly increased frequency of allele R241 compared with ANCA-positive UC (16.0% vs. 6.6%; P = 0.047, Fisher's Exact Test). In contrast, it was ANCA-positive CD that had an increased allele frequency compared with ANCA-negative CD (19.6% vs. 8.4%; P = 0.027, Fisher's Exact Test). CONCLUSIONS: Because the codon 241 polymorphism is in a functionally important domain III of ICAM-1, we may have identified an actual responsible genetic variation for genetically heterogeneous subsets of both UC and CD.

Alleles↗

PubMed1995

Aerosol delivery of a beta-galactosidase adenoviral vector to the lungs of rodents.

Aerosol delivery of adenoviral vectors is of particular interest in regard to gene therapy for cystic fibrosis (CF), with potential advantages of more uniform respiratory delivery, a less invasive approach, and ease of repetition. The AdHCMVsp1LacZ (AdLacZ) adenoviral vector was used to evaluate the feasibility of aerosol delivery to the respiratory epithelium in rodents. The adenoviral vector tolerated aerosol generation as measured by recovery in an all-glass impinger. Using an Andersen sampler to mimic the human respiratory tract, aerosol particles were found to have an average mass mean aerodynamic diameter of 1.6 microns and a geometric standard deviation of 1.7 microns. Cotton rats and mice exposed to viral aerosols demonstrated beta-galactosidase expression in up to 10-30% of the epithelial surface of the small and large airways, whereas expression in Sprague Dawley rats was largely limited to the alveolar epithelium. Transgene expression was distributed uniformly through both lungs in animals treated by aerosol. The variables for aerosol delivery are complex and include viral titer, aerosol device, duration of exposure, species of recipient, and respiratory behavior among other factors. Species differences in expression in airways as compared to alveolar epithelium have important implications for clinical application.

Adenoviridae↗

PubMed1995

P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli.

Neutrophil emigration during an inflammatory response is mediated through interactions between adhesion molecules on endothelial cells and neutrophils. P-Selectin mediates rolling or slowing of neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Removing the function of either molecule partially prevents neutrophil emigration. To analyze further the role of P-selectin and ICAM-1, we have generated a line of mice with mutations in both of these molecules. While mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum during Streptococcus pneumoniae-induced peritonitis, double mutant mice show a complete loss of neutrophil emigration. In contrast, neutrophil emigration into the alveolar spaces during acute S. pneumoniae-induced pneumonia is normal in double mutant mice. These data demonstrate organ-specific differences, since emigration into the peritoneum requires both adhesion molecules while emigration into the lung requires neither. In the peritoneum, P-selectin-independent and ICAM-1-independent adhesive mechanisms permit reduced emigration when one of these molecules is deficient, but P-selectin-independent mechanisms cannot lead to ICAM-1-independent firm adhesion and emigration.

Animals↗

PubMed1994

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis.

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G+C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci.

Base Sequence↗

PubMed1994

Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster.

Genetic polymorphisms in leukocyte and endothelial cell adhesion molecules may be important variables with regard to susceptibility to multifactorial disease processes that include an inflammatory component. For this reason, polymorphisms were sought for intercellular adhesion molecule-1 (ICAM-1; gene symbol ICAM1) and for the three genes in the selectin cluster, P-selectin, L-selectin, and E-selectin (gene symbols SELP, SELL, and SELE, respectively). Two amino acid polymorphisms were identified for ICAM-1; Gly or Arg at codon 241 and Lys or Glu at codon 469. Dinucleotide repeat polymorphisms were identified in the 3'-untranslated region for ICAM-1 and in intron 9 for P-selectin. Restriction fragment length polymorphisms were found using cDNAs for each of the three selectin genes as probes; E-selectin with BglII, P-selectin with ScaI, and L-selectin with HincII. Linkage analysis was performed for the selectin gene cluster and for ICAM-1 using the CEPH families; ICAM-1 is very tightly linked to the LDL receptor on chromosome 19, and the selectin cluster is linked to markers at chromosome 1q23.

Base Sequence↗

PubMed1994

Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region.

To determine the molecular basis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS), we have isolated new transcripts from chromosome 15q11-q13. Two novel transcripts located within 300 kilobases telomeric to the small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN) were paternally expressed in cultured cells, along with SNRPN, defining a large imprinted transcriptional domain. In three PWS patients (two sibs), small deletions remove a differentially methylated CpG island containing a newly described 5' exon alpha of SNRPN, and cause loss of expression for the three imprinted transcripts and altered methylation over hundreds of kilobases. The smallest PWS deletion is familial and asymptomatic with maternal transmission. Our data imply the presence of a paternal imprinting control region near exon alpha.

Angelman Syndrome↗

PubMed1994

Imprinting analysis of three genes in the Prader-Willi/Angelman region: SNRPN, E6-associated protein, and PAR-2 (D15S225E).

In order to identify genes in the Prader-Willi/Angelman syndrome critical region, radiolabeled cDNA probes from poly(A)+ RNA from mouse tissues were used to identify potential exon-containing genomic DNA fragments in cosmid or phage clones from appropriate yeast artificial chromosomes, and these fragments were subsequently used to screen human cDNA libraries. A mouse brain cDNA probe was effective in detecting control genes of various abundance including small nuclear ribonucleoprotein polypeptide N (SNRPN), hypoxanthine-guanine phosphoribosyl transferase, glyceraldehyde-3-phosphate dehydrogenase, and beta-actin. Two genes mapping within the Angelman syndrome critical region were isolated. One gene was found to encode the E6-associated protein (E6-AP; gene symbol HPVE6A), a protein which interacts with the E6 protein of human papilloma virus. The other gene is previously uncharacterized and is designated PAR-2 (D15S225E) for Prader-Willi and Angelman region-gene 2. Imprinting analysis using reverse transcription-polymerase chain reaction of RNA from fibroblasts and lymphoblasts of deletion Prader-Willi and Angelman patients demonstrated imprinting of SNRPN with exclusive expression from the paternal allele, but E6-AP and PAR-2 were not imprinted in these cultured human cells. The ability to analyze for imprinting and expression of SNRPN and other genes in this region in cultured human cells will be a valuable tool for analyzing the molecular basis of the Prader-Willi and Angelman syndromes, although imprinting may differ between cultured cells and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Angelman Syndrome↗

PubMed1994

Somatic gene therapy for cystic fibrosis.

There is considerable potential to ameliorate the pulmonary disease in cystic fibrosis (CF) using somatic gene therapy. Even low levels of expression of the gene in airways epithelium may be beneficial. Adenoviral vectors, DNA-liposome complexes, adeno-associated viral vectors and DNA-ligand complexes have been used effectively in vitro and have been tested in animals to varying extent. Adenoviral vectors and DNA-liposome complexes are being used to deliver the CF gene to patient airways in phase I clinical trials. Transient correction of the electrophysiological defect in human CF nasal epithelium has been achieved. Major goals are (i) to demonstrate that expression of the CF gene in airways epithelium will ameliorate lung disease in CF patients, and (ii) to achieve long-term expression of the introduced gene either through a single delivery with persistent expression or through the ability to use a delivery system repetitively with safety and efficacy.

Cystic Fibrosis↗

PubMed1993

Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1.

Gene targeting was used to produce mice deficient in intercellular adhesion molecule 1 (ICAM-1) or CD54, an immunoglobulin-like cell adhesion molecule that binds beta 2 integrins. Homozygous deficient animals develop normally, are fertile, and have a moderate granulocytosis. The nature of the mutation, RNA analysis, and immunostaining are consistent with complete loss of surface expression of ICAM-1. Deficient mice exhibit prominent abnormalities of inflammatory responses including impaired neutrophil emigration in response to chemical peritonitis and decreased contact hypersensitivity to 2,4-dinitrofluorobenzene. Mutant cells provided negligible stimulation in the mixed lymphocyte reaction, although they proliferated normally as responder cells. These mutant animals will be extremely valuable for examining the role of ICAM-1 and its counterreceptors in inflammatory disease processes and atherosclerosis.

Animals↗

PubMed1993

Gene targeting yields a CD18-mutant mouse for study of inflammation.

CD18 is the common beta subunit for the heterodimeric leukocyte integrins that mediate many inflammatory cell adhesion responses including binding to intercellular adhesion molecules 1 and 2. CD18 deficiency in humans causes a severe granulocyte disorder with susceptibility to bacterial infections and high morbidity and mortality. Gene targeting was used to prepare an insertion mutation in the murine CD18 gene. The insertion mutation resulted in a hypomorphic rather than a null allele due to low expression from a cryptic promoter in the plasmid construct. Homozygous mutant mice are viable and fertile, demonstrate a mild granulocytosis, and have 2 or 16% of normal CD18 expression on granulocytes in the resting or activated state, respectively. Mutant mice show an impaired inflammatory response to a chemical peritonitis and delayed rejection of cardiac transplants. These CD18-mutant mice provide a model of the moderate form of the human disease and should be extremely valuable for in vivo analysis of the role of leukocyte integrin-dependent adhesion in inflammatory disease models.

Animals↗

PubMed1993

Expression of human CD18 in murine granulocytes and improved efficiency for infection of deficient human lymphoblasts.

The CD18 gene encodes the beta 2-subunit of leukocyte integrins, and mutations in this gene cause extreme host susceptibility to bacterial and fungal infection. Because expression of CD18 is restricted to bone marrow-derived cells, this disorder is considered an excellent candidate for somatic gene therapy utilizing ex vivo infection of bone marrow stem cells. We have constructed a retroviral vector expressing CD18 with the Moloney murine leukemia virus (Mo-MLV) long terminal repeat (LTR) as the promoter, and high-titer ecotropic and amphotropic producer cell lines were isolated using the GP+E-86 and GP+envAM12 safe packaging cell lines. Infection of CD18-deficient lymphoblasts resulted both in expression of immunodetectable CD18 at 35-40% of normal levels on 55-60% of cells and in functional restoration of CD18-dependent aggregation. All of 16 mice transplanted with syngeneic bone marrow infected with the CD18 retrovirus expressed human CD18 on 17-36% of granulocytes at 2 weeks after transplantation, and expression was appropriately up-regulated in response to stimulation with zymosan-activated serum. This recombinant retrovirus should prove useful for further studies of somatic gene therapy for CD18 deficiency.

3T3 Cells↗

PubMed1993

A severe phenotype in mice with a duplication of exon 3 in the cystic fibrosis locus.

To develop an animal model for cystic fibrosis (CF), targeted gene disruption in embryonic stem (ES) cells was used to generate a duplication of exon 3 (cftrm1Bay allele) of the mouse CF gene. ES cells containing this mutation were used to generate chimeric animals that transmitted the mutant allele through the germline. Homozygous mutant animals display a severe phenotype, with approximately 40% dying within 1 week from intestinal obstruction. RNAase protection analysis of the cftrm1Bay allele did not detect any normal mRNA (< 1-2% of wild-type) in mutant animals. Pathologic changes in the intestines from mutant mice included mucus accumulation in the crypts and intestinal lumen, dilatation of the bases of the crypts, enlargement of goblet cells, and the presence of concretions in the crypts or between the villi. Changes were also present in the mucosal glands of the pharynx and the minor sublingual glands, where dilatation of acini and accumulation of eosinophilic material were evident. Atrophy of acinar cells that may be secondary to nutritional deficiency and mild inflammation in the main pancreatic duct were present in the pancreas of mutant animals. No changes were noted in the lung, trachea, liver, or male reproductive tract of mutant animals, and mutant males were fertile. Homozygous mutant mice showed defects in cAMP-mediated ion transport both in ileum and in cultured fetal tracheal explants. Thus, an additional mouse model for CF has been generated that should prove useful for the understanding of the pathogenesis and the development of treatments for CF.

Alleles↗