Antimicrobial efficacy of an innovative emulsion of medium chain triglycerides against canine and feline periodontopathogens.

OBJECTIVES: To test the in vitro antimicrobial efficacy of a non-toxic emulsion of free fatty acids against clinically relevant canine and feline periodontopathogens METHODS: Antimicrobial kill kinetics were established utilising an alamarBlue(®) viability assay against 10 species of canine and feline periodontopathogens in the biofilm mode of growth at a concentration of 0·125% v/v medium chain triglyceride (ML:8) emulsion. The results were compared with 0·12% v/v chlorhexidine digluconate and a xylitol-containing dental formulation. Mammalian cellular cytotoxicity was also investigated for both the ML:8 emulsion and chlorhexidine digluconate (0·25 to 0·0625% v/v) using in vitro tissue culture techniques. RESULTS: No statistically significant difference was observed in the antimicrobial activity of the ML:8 emulsion and chlorhexidine digluconate; a high percentage kill rate (>70%) was achieved within 5 minutes of exposure and was maintained at subsequent time points. A statistically significant improvement in antibiofilm activity was observed with the ML:8 emulsion compared with the xylitol-containing formulation. The ML:8 emulsion possessed a significantly lower (P < 0·001) toxicity profile compared with the chlorhexidine digluconate in mammalian cellular cytotoxicity assays. CLINICAL SIGNIFICANCE: The ML:8 emulsion exhibited significant potential as a putative effective antimicrobial alternative to chlorhexidine- and xylitol- based products for the reduction of canine and feline periodontopathogens.

Characterization of certain inflammatory variables in the peripheral blood of clinically healthy dogs.

Many laboratory techniques have been developed to study and quantify the inflammatory response, including the release of acid hydrolase enzymes, leukotriene B(4) (LTB(4)) production, reactive oxygen species (ROS) production and complement conversion studies. Although extensively studied in human health and disease, the relevance of such tests in the dog is largely unknown. After isolation of the peripheral blood mononuclear cell (PBMC) and polymorphonuclear cell (PMN) fractions from the peripheral blood of 38 clinically healthy dogs, values for ROS production were similar for both cell fractions when measured by luminol-enhanced chemiluminescence (17,853+/-9,695 U/10(6) cells versus 19,138+/-14,569 U/10(6) cells for the PBMC (n=38) and PMN (n=18) fractions, respectively). However, the mean time taken to reach maximum chemiluminescence was noticeably shorter in the PBMC fraction (5.1+/-3.3 versus 10.7+/-2.5 min for PBMCs (n=36) and PMNs (n=18), respectively). Intracellular concentrations of beta-glucuronidase, beta-galactosidase and N-acetyl-beta-glucosaminidase were assayed by spectrofluorometry. Mean values for all three enzymes were higher in PBMCs (n=31-35) than in PMNs (n=10-14). Both cell fractions released 20% of the intracellular enzyme concentration when stimulated with opsonized zymosan. Following incubation with A23187 (1 microM), mean LTB(4) production was higher in PBMCs (4.45+/-2.92 ng/10(6) cells; n=27) than in PMNs (0.96+/-2.22 ng/10(6) cells; n=13) using a validated high performance liquid chromatography (HPLC) assay. Immunoprecipitation studies revealed that the mean percentage conversion of C3 to C3b following stimulation with opsonized zymosan was 57.3+/-13.4% (n=36). The results provide normal values for clinically healthy dogs that may subsequently be used in future studies investigating dogs with various inflammatory disorders.

Increased leukotriene B(4) production, complement C3 conversion and acid hydrolase enzyme concentrations in different leucocyte sub-populations of dogs with atopic dermatitis.

Various markers of the inflammatory response were measured in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) from 31 dogs with atopic dermatitis (AD). The variables assayed included chemiluminescence, acid hydrolase enzyme concentrations, leukotriene B(4) (LTB(4)) production and complement C3 conversion. The results were compared to those derived from a population of clinically healthy dogs. Dogs with AD exhibited a significant increase in median LTB(4) production in PMNs compared to controls (0.94 versus 0.00 ng/10(6) cells; P<0.01). Significant increases in the median concentrations of intracellular beta-galactosidase (PBMC fraction - 0.42 versus 0.25 mU/10(6) cells; P<0.05) (PMN fraction - 0.47 versus 0.12 mU/10(6) cells; P<0.01) and beta-glucuronidase (PBMC fraction - 0.52 versus 0.27 mU/10(6) cells; P<0.05) were also evident in the AD group. Although median maximum chemiluminescence values for both leucocyte sub-populations were higher in controls, the differences recorded were not significant (P>0.05). However, the median time taken to reach maximum chemiluminescence was significantly shorter in the PMN fraction of dogs with AD (7.00 versus 10.00 min; P<0.01). Atopic dogs had a significant increase in the median percentage conversion of complement C3 to C3b (66.0 versus 57.3%; P<0.01). The results of this study indicate a priming of the inflammatory response in dogs with AD. The role of LTB(4) in the pathogenesis of canine AD and the potential efficacy of leukotriene antagonists in the treatment of this disorder warrant further investigation.

Polypyrimidine tract-binding protein represses splicing of a fibroblast growth factor receptor-2 gene alternative exon through exon sequences.

The fibroblast growth factor receptor (FGFR)-2 gene contains two mutually exclusive exons, K-SAM and BEK. We made a cell line designed to become drug-resistant on repression of BEK exon splicing. One drug-resistant derivative of this line carried an insertion within the BEK exon of a sequence containing at least two independent splicing silencers. One silencer was a pyrimidine-rich sequence, which markedly increased binding of polypyrimidine tract-binding protein to the BEK exon. The BEK exon binds to polypyrimidine tract-binding protein even in the silencer's absence. Several exonic pyrimidine runs are required for this binding, and they are also required for overexpression of polypyrimidine tract-binding protein to repress BEK exon splicing. These results show that binding of polypyrimidine tract-binding protein to exon sequences can repress splicing. In epithelial cells, the K-SAM exon is spliced in preference to the BEK exon, whose splicing is repressed. Mutation of the BEK exon pyrimidine runs decreases this repression. If this mutation is combined with the deletion of a sequence in the intron upstream from the BEK exon, a complete switch from K-SAM to BEK exon splicing ensues. Binding of polypyrimidine tract binding protein to the BEK exon thus participates in the K-SAM/BEK alternative splicing choice.

TIA-1 and TIAR activate splicing of alternative exons with weak 5' splice sites followed by a U-rich stretch on their own pre-mRNAs.

TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.

Gene structure of the human receptor tyrosine kinase RON and mutation analysis in lung cancer samples.

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors.

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site.

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.

A processed pseudogene codes for a new antigen recognized by a CD8(+) T cell clone on melanoma.

The M88.7 T cell clone recognizes an antigen presented by HLA B*1302 on the melanoma cell line M88. A cDNA encoding this antigen (NA88-A) was isolated using a library transfection approach. Analysis of the genomic gene's sequence identified it is a processed pseudogene, derived from a retrotranscript of mRNA coding for homeoprotein HPX42B. The NA88-A gene exhibits several premature stop codons, deletions, and insertions relative to the HPX42B gene. In NA88-A RNA, a short open reading frame codes for the peptide MTQGQHFLQKV from which antigenic peptides are derived; a stop codon follows the peptide's COOH-terminal Val codon. Part of the HPX42B mRNA's 3' untranslated region codes for a peptide of similar sequence (MTQGQHFSQKV). If produced, this peptide can be recognized by M88.7 T cells. However, in HPX42B mRNA, the peptide's COOH-terminal Val codon is followed by a Trp codon. As a result, expression of HPX42B mRNA does not lead to antigen production. A model is proposed for events that participated in creation of a gene coding for a melanoma antigen from a pseudogene.

hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer.

Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer's activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.

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.

Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon.

The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5' splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5' splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements.

Macrophage stimulating protein (MSP) binds to its receptor via the MSP beta chain.

Macrophage stimulating protein (MSP) is a 78-kDa disulfide-linked heterodimer belonging to the plasminogen-related kringle protein family. MSP activates the RON receptor protein-tyrosine kinase, which results in cell migration, shape change, or proliferation. A structure-activity study of MSP was performed using pro-MSP, MSP, MSP alpha and beta chains, and a complex including the first two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP beta chain both bound specifically to RON. The Kd of 1.4 nM for MSP beta chain is higher than the reported Kd range of 0.6-0.8 nM for MSP. Pro-MSP, MSP alpha chain, and MSP-NK2 did not bind. Only MSP stimulated RON autophosphorylation. Although the beta chain bound to RON and partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP alpha chain, or MSP-NK2 failed to activate RON, consistent with their inability to bind to the RON receptor. Functional studies showed that only MSP induced cell migration, and shape change in resident macrophages, and growth of murine keratinocytes. Our data indicate that the primary receptor binding domain is located in a region of the MSP beta chain, in contrast to structurally similar hepatocyte growth factor, in which the receptor binding site is in the alpha chain. However, full activation of RON requires binding of the complete MSP disulfide-linked alphabeta chain heterodimer.

Identification of a gp100 epitope recognized by HLA-A3 restricted melanoma infiltrating lymphocytes.

The large majority of known melanoma-associated antigenic peptides presented by MHC class I molecules are presented by the most frequent allele, HLA-A*0201. Thus although a significant percentage of Caucasians express HLA-A3, no melanoma-associated antigenic peptide presented by this allele has yet been identified. We show here that the T cell clone M45-10 isolated from tumor infiltrating lymphocytes recovered from a melanoma biopsy recognizes the gp100-derived peptide ALLAVGATK presented by HLA-A*0301. Since gp100 is expressed on most melanoma cells, our results imply that the gp100-based anti-melanoma strategies developed for individuals expressing HLA-A2 will also be applicable to those expressing HLA-AS (about one Caucasian in four). gp100 is therefore a particularly promising melanoma antigen, as different peptides derived from it can be presented by at least two different frequently encountered HLA class I molecules.

T cell response to Epstein-Barr virus transactivators in chronic rheumatoid arthritis.

Rheumatoid arthritis is a multistep disorder associated with autoimmune features of yet unknown etiology. Implication of viruses such as Epstein-Barr virus (EBV) in rheumatoid arthritis pathogenesis has been suspected on the basis of several indirect observations, but thus far, a direct link between EBV and rheumatoid arthritis has not been provided. Here we show that a large fraction of T cells infiltrating affected joints from a patient with chronic rheumatoid arthritis recognizes two EBV transactivators (BZLF1 and BMLF1) in a major histocompatibility complex-restricted fashion. Responses to these EBV antigens by synovial lymphocytes from several other chronic rheumatoid arthritis patients were readily detectable. Thus these results suggest a direct contribution of EBV to chronic rheumatoid arthritis pathogenesis. They also demonstrate for the first time the occurrence of T cell responses against EBV transactivating factors, which might be central in the control of virus reactivation.

The exon sequence TAGG can inhibit splicing.

The fibroblast growth factor receptor-2 gene contains a pair of alternative exons, K-SAM and BEK, which are spliced in a cell type specific manner. We have shown previously that a 10 nucleotide sequence within the K-SAM exon exerts a negative effect on K-SAM exon splicing independent of cell type. We demonstrate here that this sequence works autonomously, as it can repress splicing of a heterologous exon, the EIIIb alternative exon of the rat fibronectin gene. By introducing point mutations into the 10 nucleotide sequence, we have shown that the functional portion is limited to 4 nucleotides, TAGG, the dinucleotide AG of which is particularly important. This short sequence may participate in the control of splicing of exons carrying it, provided that they carry weak splice sites.

Restricted expression of the ron gene encoding the macrophage stimulating protein receptor during mouse development.

The human ron gene codes for a transmembrane protein tyrosine kinase which is a receptor for the macrophage stimulating protein. The ron receptor, together with the hepatocyte growth factor/scatter factor receptor encoded by the proto-oncogene met, and the product of the c-sea proto-oncogene, make up a family of structurally related receptors. We have cloned murine ron cDNA sequences and used them as probes for in situ hybridization and Northern blot experiments. We show that ron gene expression occurs relatively late in development, and is much more restricted than that of the met gene. ron gene expression is detected in specific areas of the central and the peripheric nervous system, as well as in discrete cells in developing bones, and in the glandular epithelia along the digestive tract.

Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor.

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.

Structure of the promoter for the human macrophage stimulating protein receptor gene.

The macrophage stimulating protein receptor is a receptor protein tyrosine kinase of the met/hepatocyte growth factor receptor family. Binding of the macrophage stimulating protein to its receptor provokes changes in cell morphology and motility. Here we report the structure of the promoter of the gene coding for this receptor. The major transcription start sites have been identified. The 5' flanking region has characteristics of other receptor tyrosine kinase gene promoters, namely several GC boxes but the absence of a TATA box. Deletion analysis shows that multiple elements are needed for full promoter activity.