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PubMed2006

Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27.

The immune response to bacterial infections must be tightly controlled to guarantee pathogen elimination while preventing tissue damage by uncontrolled inflammation. Here, we demonstrate a key role of interleukin (IL)-27 in regulating this critical balance. IL-27 was rapidly induced during murine experimental peritonitis induced by cecal ligation and puncture (CLP). Furthermore, mice deficient for the EBI3 subunit of IL-27 were resistant to CLP-induced septic peritonitis as compared with wild-type controls, and this effect could be suppressed by injection of recombinant single-chain IL-27. EBI3-/- mice displayed significantly enhanced neutrophil migration and oxidative burst capacity during CLP, resulting in enhanced bacterial clearance and local control of infection. Subsequent studies demonstrated that IL-27 directly suppresses endotoxin-induced production of reactive oxygen intermediates by isolated primary granulocytes and macrophages. Finally, in vivo blockade of IL-27 function using a newly designed soluble IL-27 receptor fusion protein led to significantly increased survival after CLP as compared with control-treated mice. Collectively, these data identify IL-27 as a key negative regulator of innate immune cell function in septic peritonitis. Furthermore, in vivo blockade of IL-27 is a novel potential therapeutic target for treatment of sepsis.

Animals↗

PubMed1998

Characterization of two monoclonal antibodies against the RON tyrosine kinase receptor.

RON is a receptor protein tyrosine kinase belonging to the hepatocyte growth factor (HGF) receptor family. Using Récepteur d'Origine Nantais (RON) transfected cell lines, Macrophage Stimulating Protein (MSP) was identified as the ligand of RON. RON is synthesized as a single chain precursor, which subsequently is cleaved to yield a disulfide-linked heterodimer, with a 40-kDa alpha chain and a 150-kDa beta chain. Activation of RON by MSP results in cell migration, shape change, and proliferation. The present work centers on the production and characterization of two monoclonal antibodies (MAbs) to RON called ID-1 and ID-2. Antibodies were generated by immunization of mice with Madin-Darby Canine Kidney (MDCK) cells expressing human RON (clone RE7). Both antibodies recognized the mature and precursor form of RON. The specificity of the anti-RON antibodies was confirmed using a hepatocarcinoma cell line HepG2 expressing both task MET and RON receptors. Specific immunoprecipitation with ID-1 and ID-2 or anti-MET antibody followed by Western blotting under reducing conditions with rabbit polyclonal antibodies against RON and MET showed that our anti-RON antibodies recognize specifically the RON receptor. Ligand binding experiments showed that both antibodies are able to block the binding of radiolabeled MSP to RON and showed also that the antibodies recognize two different epitopes in the molecule. The blocking of MSP binding to RON by the anti-RON antibodies was confirmed by inhibition of cell migration induced by MSP in HT-29-D4 cells. Significant immunostaining was not observed in any subpopulation of whole blood with either ID-1 or ID-2. We analyzed the expression of RON receptor in a number of human hematopoietic and nonhematopoietic cells lines by flow cytometry. We found a strong mean of fluorescence intensity (MFI) in colon adenocarcinoma cells SW620 and HT-29-D4, low MFI in SVK14 and HepG2 cells, and no immunostaining in melanoma, lymphoma, and leukemia cells. Immunohistochemistry revealed that RON was expressed in germinal centers of tonsil, in skin, small intestine, and colon. These antibodies defined RON as CDw136 during the last leucocyte typing VI.

Animals↗

PubMed1984

Isolation of milligram quantities of a group of histidine-rich polypeptides from human parotid saliva.

Freshly collected parotid saliva collected from human donors were shown by polyacrylamide gel electrophoresis to continuously secrete a group of low-molecular-weight cationic polypeptides. Up to 14 bands could be identified by Coomassie blue staining, and all bands migrated more rapidly than purified human leukemic lysozyme in cationic polyacrylamide gel electrophoresis. These peptides could be isolated as a group relatively free of other salivary components and recovered in high yields from concentrated parotid saliva by Sephadex G-25 chromatography. In sodium dodecyl sulfate gel electrophoresis, the histidine-rich polypeptide bands appeared as just two bands migrating at the tracking dye and ahead of insulin chain B. Amino acid analysis of the mixture revealed an average content of at least 48% cationic residues, of which half were histidine. When stained bands were eluted from electrophoretic gels, hydrolyzed, and subjected to amino acid analyses, they were found to be enriched in histidine. There was also a correlation of the electrophoretic mobility with the content of basic amino acids. Sephadex G-25 chromatography is a convenient, simple method for preparing milligram quantities of the histidine-rich polypeptides for chemical and biochemical studies.

Adult↗

PubMed1998

Maturation of the arginine-specific proteases of Porphyromonas gingivalis W50 is dependent on a functional prR2 protease gene.

The prpR1 of Porphyromonas gingivalis codes for three distinct enzymes with specificity for arginyl peptide bonds termed RI, RIA, and RIB. These three isoforms comprise the majority of the extracellular, arginine-specific protease activity in P. gingivalis W50. RI is a heterodimer in which the catalytic alpha chain is noncovalently associated with a second chain involved in adherence phenomena. RIA and RIB are both monomeric species. RIA represents the free alpha chain, and RIB is a highly posttranslationally modified form of the alpha chain which is exclusively vesicle or membrane associated and migrates as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In previous studies, insertional inactivation of the prpR1 demonstrated that arginine-specific protease activity can also arise from a closely related second gene, prR2. In the present work, the prR2 was insertionally inactivated in P. gingivalis W50 in order to establish the contribution of this locus to the arginine-specific protease activity of this periodontal bacterium. Loss of prR2 function had several effects on prpR1-derived enzymes. First, the total Arg-X activity was reduced by approximately 50% relative to that of the parent strain. The reduction in total activity was a consequence of decreased concentrations of the monomeric enzymes derived from the prpR1, while the heterodimeric enzyme, RI, was unaffected by this mutation. Second, the chromatographic behavior of both the soluble and vesicle- or membrane-associated monomeric enzymes was radically different from the behavior of RIA and RIB from the parent strain. Finally, the vesicle- or membrane-associated enzyme in the prR2 mutant strain lacked the extensive posttranslational additions which are found on RIB in P. gingivalis W50. These data suggest that the product(s) of the prR2 plays a significant role in the maturation pathway of prpR1-derived enzymes, and this may contribute to the coconservation of these two genes in P. gingivalis.

Adhesins, Bacterial↗

PubMed2004

Accuracy of actuarial procedures for assessment of sexual offender recidivism risk may vary across ethnicity.

Little is known about whether the accuracy of tools for assessment of sexual offender recidivism risk holds across ethnic minority offenders. I investigated the predictive validity across ethnicity for the RRASOR and the Static-99 actuarial risk assessment procedures in a national cohort of all adult male sex offenders released from prison in Sweden 1993-1997. Subjects ordered out of Sweden upon release from prison were excluded and remaining subjects (N = 1303) divided into three subgroups based on citizenship. Eighty-three percent of the subjects were of Nordic ethnicity, and non-Nordic citizens were either of non-Nordic European (n = 49, hereafter called European) or African Asian descent (n = 128). The two tools were equally accurate among Nordic and European sexual offenders for the prediction of any sexual and any violent nonsexual recidivism. In contrast, neither measure could differentiate African Asian sexual or violent recidivists from nonrecidivists. Compared to European offenders, AfricanAsian offenders had more often sexually victimized a nonrelative or stranger, had higher Static-99 scores, were younger, more often single, and more often homeless. The results require replication, but suggest that the promising predictive validity seen with some risk assessment tools may not generalize across offender ethnicity or migration status. More speculatively, different risk factors or causal chains might be involved in the development or persistence of offending among minority or immigrant sexual abusers.

Actuarial Analysis↗

PubMed1986

Suppression of synthesis of pro-alpha 1(I) and production of altered pro-alpha 2(I) procollagen subunits in 4-nitroquinoline-1-oxide-transformed fibroblasts.

The collagen phenotype of a 4-nitroquinoline-1-oxide-transformed line of Syrian hamster embryo fibroblasts, NQT-SHE, was markedly altered from that of normal Syrian hamster embryo cells, which synthesized mainly type I procollagen [pro-alpha 1(I)]2 pro-alpha 2(I). Total collagen synthesis in the transformant was reduced to about 30% of the control level primarily because synthesis of the pro-alpha 1(I) subunit was completely suppressed. The major collagenous products synthesized consisted of two polypeptides, designated as N-33 and N-50, which could be completely separated by precipitation with ammonium sulfate at 33 and 50% saturation, respectively. N-33 migrated similarly to pro-alpha 2(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-50 migrated slightly more slowly. The collagenous regions of these chains were more sensitive to protease than the analogous region of procollagen I, but alpha-chains could be obtained by digestion for 2 h at 4 degrees C with high ratios of protein:pepsin. Staphylococcus V8 protease and cyanogen bromide peptide maps of N-33 alpha and N-50 alpha chains indicated that the chains were homologous with, but different than, alpha 2(I) chains and that they differed from each other. Considering their similarity to pro-alpha 2(I), it was surprising to find that the N-collagens were secreted to the same extent as was type I procollagen from Syrian hamster embryo cells and that there were no disulfide bonds between N-collagen chains. Intrachain disulfides were present. One possible explanation for the unusual collagen phenotype of NQT-SHE cells is that transformation induced one or more mutations in the pro-alpha 2(I) structural gene while suppression of synthesis of the pro-alpha 1(I) subunit may be due to a mutation in the regulatory region of its gene or in a general regulatory gene.

4-Nitroquinoline-1-oxide↗

PubMedDate not supplied

[Nucleoprotein fusion. III. Causes and conditions for development of the multiphase character of the DNA--protein fusion curve].

The influence of proteins reversibly and irreversibly bound to DNA on the shape of melting curve has been considered. It is shown that the melting curve becomes biphasic in two cases: (i) cooperative binding of proteins with DNA (II) STRONG DIFFERENCE IN THE BINDING CONSTANTS WITH HELICAL AND COILED REGIONS. Simple formulae permitting to determine which of two causes stipulate for biphasic profile of a given experimental melting curve are obtained. Melting curves of DNA-basic oligopeptides complexes have been investigated. It is shown that the oligopeptides, when their chain length does not exceed 10, are able to migrate along DNA and biphasic shape of the melting curve is stipulated by the cooperative manner of their binding with DNA.

DNA↗

PubMed1978

Partial characterization of human collagen and procollagen secreted by human corneal stromal fibroblasts in cell culture.

Human corneal stromal fibroblasts were established into cell culture. Fractions highly enriched for collagen and procollagen were purified from the medium and cell layer. Analysis using polyacrylamide gel electrophoresis and DEAE cellulose chromatography indicated the presence of a heterogeneous population of procollagen molecules ranging in molecular weights from 200,000 to 120,000 daltons along with alpha1 and alpha2 collagen chains. Cyanogen bromide peptides of a corneal peptide fraction migrated concomitantly with type I standard cyanogen bromide peptides.

Cells, Cultured↗

PubMed1993

Structure and antigenic properties of Citrobacter freundii lipopolysaccharides.

Citrobacter freundii strain E69366 was detected in a colony immunoassay with a rabbit antiserum prepared to a strain of Escherichia coli belonging to serogroup O157. Lipopolysaccharide (LPS) was shown to contain the epitope(s) involved in antibody binding. LPS prepared from strain E69366, with hot phenol contained core-LPS that migrated to the water-phase and long-chain LPS that separated into the phenol-phase. A total of 36 strains of Cit. freundii were analysed for their LPS profiles by SDS-PAGE. Sixteen could be allocated into five groups (A(3), B(7), C(2), D(2) and E(2)) based on similarities in LPS profile. The remaining strains either expressed unique SDS-PAGE profiles (18) or did not express long-chain LPS (2). All strains with a profile 'A' reacted with the antiserum prepared to E. coli O157, whereas those with other profiles did not. The two strains of Cit. freundii expressing LPS profiles designated 'E' reacted with an antiserum prepared in rabbits to E. coli O45.

Adult↗

PubMed2000

Activation of epidermal growth factor receptor during corneal epithelial migration.

PURPOSE: Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-alpha, heprin-ding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process. METHODS: In the in vivo model, 3-mm debridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-alpha, HB-EGF, and AR were determined using reverse transcription-polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 microM), a specific inhibitor of EGFR kinase activity. RESULTS: In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-alpha, HBEGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner. CONCLUSIONS: These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates.

Amphiregulin↗

PubMed2000

The CD44 receptor of the mouse LB T-cell lymphoma: analysis of the isoform repertoire and ligand binding properties by reverse-transcriptase polymerase chain reaction and antisense oligonucleotides.

CD44 is a cell surface glycoprotein involved in cell migration and cell docking in target organs via interactions with various ligands, including hyaluronic acid (HA), which is the principal ligand of this receptor. Alternative splicing generates many isoforms of CD44, including standard CD44 (CD44s) and CD44 variants (CD44v). LB T-cell lymphoma, which predominantly expresses CD44s, acquires additional CD44v and HA binding capacity after activation with phorbol ester. The HA9 cell line, isolated from parental LB cells, expresses CD44v and constitutively binds HA. Downregulation of CD44v isoforms of HA9 cells, by CD44v specific antisense inhibited their ability to bind HA, indicating that CD44v, rather than CD44s, is associated with the activation status of this molecule. Using the reverse transcriptase polymerase chain reaction, we found that LB cells after infiltrating spleen and lymph nodes of BALB/c mice, contain an enriched repertoire of CD44v, implying that the metastatic cells acquired the activated form of this receptor.

Animals↗

PubMed1997

Capillary electrophoretic separation of structurally similar solutes in noncross-linked poly(acrylamide-co-N-isopropylacrylamide) solution.

Noncross-linked acrylamide (AA)-N-isopropylacrylamide (IPAAm) copolymers have been used as a buffer additive in capillary electrophoretic separation of structurally similar small solutes. Seven kinds of barbiturates and five kinds of dansylated (Dns) amino acids, which have different hydrophobic side chains, were separated on poly(AA-co-IPAAm)-filled columns and on AA polymer-filled columns under the condition of totally eliminating the contribution of electroosmotic flow (EOF). It is known that the copolymer containing IPAAm has thermosensitive properties, and the hydrophobicity of its surface changes with surrounding temperature. In this investigation, therefore, the effects of an isopropyl group in the copolymer on the electrophoretic separation of the small solutes were studied by comparing the two polymer-filled columns at ambient temperature and at elevated temperature. Although slight differences in migration behavior were observed at ambient temperature between the columns filled with these two polymer solutions, obvious differences in the separation of the solutes were observed at elevated temperature. The observed changes on the migration behavior might be caused by the interaction between copolymer chains exhibiting hydrophobic property and the solute.

Acrylamides↗

PubMed1991

Large disulfide-stabilized proteoglycan complexes are synthesized by the epidermis of axolotl embryos.

Proteoglycans (PGs) synthesized by the epidermis during stages crucial to the subepidermal migration of neural crest cells in the trunk of the axolotl (Ambystoma mexicanum, Urodela, Amphibia) embryo were studied. The glycosaminoglycan chains were biosynthetically labeled with [35S]sulfate in vitro during a period corresponding to the onset of migration. After extraction with guanidine HCl, the radiolabeled PGs were separated according to size by molecular-sieve chromatography on Sepharose CL-2B under dissociative conditions. This resulted in the separation of high-molecular-weight PGs, which eluted in the void volume, and low-molecular-weight PGs, eluting in a broad peak with a mean Kav of 0.7. The large PGs were also found to elute in the void volume when chromatographed on a Sephacryl S-1000 column. The low-molecular-weight PGs contained heparan sulfate and chondroitin sulfate (CS) and were not further characterized. The glycosaminoglycan component of the high-molecular-weight PG was completely degraded by chondroitinase ABC, while a large portion was resistant to chondroitinase AC, indicating the presence of dermatan sulfate (DS). These CS/DS chains were of unusually large size (Mr approximately 150,000) as estimated by chromatography on Sepharose CL-4B, relating the elution position to hyaluronan standards. Moreover, the chains were found to have a lower surface charge density than standard CS, and may therefore be undersulfated. After reduction and alkylation the high-molecular-weight PGs were included on both Sepharose CL-2B and Sephacryl S-1000 columns, eluting at Kav 0.2 and 0.4, respectively. Hence, the high-molecular-weight material appears to consist of large PG complexes, stabilized by intermolecular disulfide bonds. A CS/DSPG of similar size as the reduced monomeric form of the high-molecular-weight PG was found in small amounts in the total extract of 35S-labeled material.

Ambystoma mexicanum↗

PubMed2006

Inhibition by mitoxantrone of in vitro migration of immunocompetent cells: a possible mechanism for therapeutic efficacy in the treatment of multiple sclerosis.

BACKGROUND: Damage of the blood-brain barrier and invasion of immunocompetent cells into the central nervous system represent key events in the immunopathogenesis of multiple sclerosis. Mitoxantrone hydrochloride reduces progression of disability and clinical exacerbations in patients with multiple sclerosis. Its precise mode of action is unclear. OBJECTIVE: To investigate the effects of mitoxantrone on the migratory capacity of immunocompetent cells ex vivo and in vitro. DESIGN: Case-control study. SETTING: Department of Neurology, Heinrich Heine University, Düsseldorf, Germany. PARTICIPANTS: Peripheral blood mononuclear cells (PBMCs) were obtained from 11 patients with multiple sclerosis before and after intravenous mitoxantrone treatment; PBMCs from 5 healthy control donors were treated with mitoxantrone in vitro. MAIN OUTCOME MEASURES: The migratory capacity was studied in an in vitro Boyden chamber assay; cells and their rates of migration were analyzed by light microscopy and flow cytometry. To determine the specificity of our findings, PBMCs were treated with perfosfamide in vitro. RESULTS: Mitoxantrone decreased the migratory capacity of CD14(+) monocytes and (to a lesser degree) of CD4(+) and CD8(+) T lymphocytes. These observations were confirmed when control PBMCs were treated with an equivalent dose of mitoxantrone in vitro. Similar effects were seen when PBMCs were preincubated with perfosfamide. The inhibitory effects of mitoxantrone on the migratory capacity of PBMCs were mediated by reduced matrix metalloproteinase 9 activity, as demonstrated by zymography, polymerase chain reaction, and inhibitory studies. CONCLUSION: Mitoxantrone may inhibit the migration of inflammatory cells into and within the central nervous system.

Adult↗

PubMed2005

Neonatal lactic acidosis, complex I/IV deficiency, and fetal cerebral disruption.

Cerebral developmental abnormalities occur in various inborn errors of metabolism including peroxisomal deficiencies, pyruvate dehydrogenase complex deficiency and others. Associations with abnormalities of the respiratory chain are rare. Here we report male and female siblings with microcephaly, a complex neuromigrational disorder including ependymal cysts, leptomeningeal and subcortical heterotopia, polymicrogyria, multifocal cerebral calcifications, agenesis of the corpus callosum, and spongiform changes in brainstem and cerebellum. Intractable lactic acidosis, causing death on the first day of life, was associated with severely reduced activities of complex I and complex IV. The neuropathological and biochemical findings are closely similar to those reported previously. The findings confirm a distinct genetic syndrome of disrupted brain development with TORCH-like calcifications, and a complex neuronal migration disorder associated with a multicomplex disorder of the respiratory chain.

Acidosis, Lactic↗

PubMed1997

Phenotypic characterization of keratinocytes migrated from polymer support - in vitro study.

The keratinocytes are able to migrate from the poly (2- hydroxyethylmethacrylate) disc if it is transferred to the new Petri-dish colonized with irradiated 3T3 mouse fibroblasts, and form a ring-shaped colony around the disc. The phenotypic characterization of human keratinocytes migrated from these discs was studied using a group of monoclonal antibodies. The keratinocytes in the external periphery of the colony of cells which migrated from the disc express the proliferating cell nuclear antigen (PCNA), alpha2, alpha3 chains and alpha5beta1 integrin receptor. A protein of the desmosome complex, desmoplakin-1, was also expressed. Involucrin and cytokeratin-10 were expressed after prolonged cultivation. These results suggest that the migrated keratinocytes are able to proliferate, recognize extracellular matrix molecules important in the process of the re-epitelization of the wound, and terminally differentiate in vitro. They are encouraging for further experiments with respect to the development of a support for keratinocyte cultivation and for grafting in clinical practice.

Journal Article↗

PubMed1976

Purification and translation of an immunoglobulin lambda chain messenger RNA from mouse myeloma.

Here we describe the 500-fold purification of an mRNA encoding an immunoglobulin lambda light chain derived from the mouse myeloma tumor, RPC-20. Purification involves the isolation of membrane-bound polysomes, oligo(dT)-cellulose chromatography, and sucrose gradient centrifugation under conditions favoring denaturation of polynucleotide complexes. The mRNA purified in this way directs the cell-free synthesis of a polypeptide which is five or six amino acids longer than the mature form of RPC-20 light chain. In addition to directing the synthesis of a precursor-like polypeptide, the mRNA migrates on electrophoresis as a band containing approximately 1150 nucleotides, about 500 more than required to encode the mature form of the light chain.

Cell Line↗

PubMed1998

A role for the alphavbeta3 integrin in the transmigration of monocytes.

The beta2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin alphavbeta3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking beta3 integrins revealed weak migratory ability, whereas monocytes expressing beta3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains alphaL, beta2, alphav, or IAP, a protein functionally associated with alphavbeta3 integrin. Transfection of beta3 integrin chain cDNA into monocytes lacking beta3 integrins resulted in expression of the alphavbeta3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in alphaLbeta2-dependent locomotion on recombinant ICAM-1 which was enhanced by alphavbeta3 integrin occupancy. Antibodies against IAP were able to revert this alphavbeta3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of alphavbeta3 integrin could decrease monocyte binding to ICAM-1. In conclusion, we show that alphavbeta3 integrin modulates alphaLbeta2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.

Animals↗