Increased expression of TRPV1 in squamous cell carcinoma of the human tongue.

OBJECTIVES: Recent reports have unambiguously identified the presence and the growth-modulatory role of transient receptor potential vanilloid-1 (TRPV1), a central integrator of pain sensation, on numerous non-neuronal cell types and, of great importance, in certain malignancies. In this study, we have investigated the molecular expression of TRPV1 in the human tongue and its high-incidence malignant (squamous cell carcinoma, SCC) and premalignant (leukoplakia) conditions. METHODS: Immunohistochemistry, Western blotting and quantitative 'real-time' Q-PCR were performed to define the expression of TRPV1. RESULTS: A weak and sparse TRPV1-specific immunoreactivity was identified in the basal layers of the healthy human tongue epithelium. By contrast, we observed a dramatically elevated TRPV1-immunoreactivity in all layers of the epithelium both in precancerous and malignant samples. Furthermore, statistical analysis revealed that the marked overexpression of TRPV1 found in all grades of SCC showed no correlation with the degree of malignancy of the tumours. Finally, the molecular expression of TRPV1 was also identified in an SCC-derived cell line and was shown to be increased in parallel with the accelerated growth of the cells. CONCLUSION: Collectively, our findings identify TRPV1 as a novel, promising target molecule in the supportive treatment and diagnosis of human tongue SCC.

The rapid molecular subtyping and pathotyping of avian influenza viruses.

Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes.

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKCalpha and beta; novel nPKCdelta and epsilon) in the regulation of various keratinocyte functions. cPKCalpha and nPKCdelta stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKCbeta and nPKCepsilon increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.

Phorbol ester treatment inhibits proliferation and differentiation of cultured human skeletal muscle satellite cells by differentially acting on protein kinase C isoforms.

We have previously shown that cultured human skeletal muscle cells express five protein kinase C (PKC) isoforms (PKCalpha, -gamma, -eta, -theta, and -zeta) and that expression levels of various PKC isozymes differentially change during differentiation. In this study we investigated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on differentiation and on PKC isozymes of human skeletal muscle satellite cells. PMA inhibited the growth and fusion of cultured human myoblasts in a dose-dependent manner. In addition, prolonged treatment of cells with PMA suppressed the expression of the myogenic differentiation marker desmin showing similar dose-response characteristics. Furthermore, PMA also induced the intracellular translocation of PKCgamma, -eta, and -theta, whereas cellular localization of PKCalpha and -zeta were not altered. These changes in subcellular localization patterns were of great importance since only those PKC isoforms were translocated that possessed alterations in their expression levels during differentiation. Our findings, therefore, suggest that the PMA-induced inhibition of differentiation of human skeletal muscle cells is mediated by certain PKC isoforms. Moreover, these data strongly argue for differential and isozyme-specific roles of various PKC isoforms in these processes.

Protective effect of two Mycoplasma gallisepticum protein fractions affinity purified with monoclonal antibodies.

Two protein fractions of Mycoplasma gallisepticum (Mg) were affinity purified with monoclonal antibodies A3 and B3, and tested for protective capacity in chickens. One fraction, designated MgP1, appeared as a doublet of 64 and 62kDa bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, while MgP2 consisted of five polypeptides (64, 56, 47, 45 and 43 kDa). The molecular mass, haemagglutination activity and matching amino acid sequence of MgP1 suggest that it is identical to pMGA1.2, the putative haemagglutinin of Mg. Groups of Mg-free chickens were immunized once or twice with 1 or 5 mug MgP1 or MgP2, or a combination of the two, and adjuvanted with immunostimulating complexes. Except for the group given 1 mug MgP1, all vaccinated groups showed a significant (P < 0.01) reduction in air sac lesions after challenge compared with unvaccinated controls. MgP2 appeared more protective than MgP1. Vaccination twice with MgP2 was the only regime that produced a detectable serum antibody response.

Potency test of inactivated Newcastle disease vaccines by monoclonal antibody blocking ELISA.

The potency of 27 different inactivated Newcastle disease (ND) vaccines was determined by immunization and challenge tests. Three- and four-week-old SPF chickens were immunized with 1/50 dose of vaccines. The chickens were bled and challenged three weeks later. The protected/challenged ratio was determined. Serum samples were tested by the haemagglutination inhibition (HI) test and by a monoclonal antibody (MAB) blocking enzyme-linked immunosorbent assay (B-ELISA). Ninety-four percent of the vaccinated chickens (353 birds) with a B-ELISA value above 60% were protected. A strong positive correlation (0.934) was found between 'actual protection' (protection against challenge) and 'estimated protection' (protection calculated from the B-ELISA results). Based upon the results obtained, this B-ELISA seems to be suitable for replacing the challenge test in the potency control of inactivated ND vaccines in the future.

A monoclonal antibody blocking ELISA for the detection of IBV antibodies in fowl.

A murine monoclonal antibody (mAb) reacting with the spike protein of seven field and laboratory strains of Infectious Bronchitis Virus (IBV) was characterised. Neutralisation tests performed in chicken tracheal organ culture showed that the mAb is directed against a conserved neutralising epitope. A monoclonal antibody blocking ELISA (B-ELISA) was developed based on the mAb. The sensitivity and specificity of the test was evaluated by examining sera and egg yolk from IBV-free, vaccinated or naturally infected chickens from different European countries. The comparisons showed that the IBV blocking ELISA was very sensitive and more specific than the commercially available indirect ELISA and the haemagglutination-inhibition (HI) test. As part of the Swedish national health control program, more than 60,000 sera have been examined with the B-ELISA at the National Veterinary Institute (Sweden) since 1993. The test proved to be very specific in detecting the spread of disease during the Swedish IBV outbreaks in 1994.

Detection of Newcastle disease virus-specific antibodies in ostrich sera by three serological methods.

Sera from 211 ostriches were tested for the presence of Newcastle disease virus (NDV)-specific antibodies by the virus neutralisation test, the haemagglutination inhibition (HI) test and a recently developed avian paramyxovirus serotype 1 (PMV-1) specific monoclonal antibody blocking ELISA (b-ELISA). The virus neutralisation test was used as the reference for the estimation of the sensitivity and specificity of the b-ELISA and HI tests. Of the 211 sera, 140 contained NDV-specific neutralising antibodies, 130 were positive by the HI test and 122 by the b-ELISA. The sensitivity, specificity and predictive accuracy of the HI and b-ELISA tests relative to the virus neutralisation test were similar. The good agreement between the HI and b-ELISA test (kappa = 0.85) suggested that the two methods are interchangeable.

Detection of NDV-specific antibodies and the level of protection provided by a single vaccination in young chickens.

Fourteen groups of young commercial chickens were immunized once with a live NDV vaccine using different vaccine doses and routes of vaccination in five experiments. Three to six weeks later, small groups were selected from each flock. Sera were tested by the haemagglutination-inhibition test and a monoclonal antibody blocking ELISA, and the birds were challenged with a virulent NDV strain. Degree of protection was dose-dependent in those groups where the vaccine was administered orally at 3 weeks of age. Aerosol and eye drop vaccinations performed in day-old chicks provided full protection at 5 or 6 weeks of age. There was a good agreement between the two serological methods and positive results in any of the tests were reliable forecasts of protection.

Detection of PMV-1 specific antibodies with a monoclonal antibody blocking enzyme-linked immunosorbent assay.

A highly reproducible monoclonal antibody (Mab) blocking ELISA (B-ELISA) has been developed and evaluated for the detection of NDV-specific antibodies. The Mab utilised is specific for a conserved PMV-1 serotype-specific epitope, as demonstrated by the indirect immunoperoxidase test. It reacted with all strains representing different serogroups within the PMV-1 serotype, but not with any strain belonging to other PMV serotypes. Sensitivity and specificity of the B-ELISA were compared with the haemagglutina-tion inhibition test (HI). Blocking and HI antibodies were detected in sera of chickens 8 days post-experimental infection. The B-ELISA proved consistently more sensitive than the HI test. In another survey, 62 sera from experimentally vaccinated chickens were tested; 95.2% proved positive by B-ELISA, 85.5% by indirect ELISA and 74% by HI test. When 504 field sera from vaccinated chickens and turkeys were tested, 98% were positive by B-ELISA, and 69% by HI. The specificity was evaluated by testing 1066 samples from NDV-free flocks, all of which proved negative by both methods. Other advantages of the B-ELISA include easy standardization and quality control, and ability to test sera from any species (including exotic or wild birds as well as mammals). The use of low dilution serum or egg-yolk samples makes the test quick and easy to perform and suitable for large-scale screening.

Protective immunity induced in chicken by a single immunization with Mycoplasma gallisepticum immunostimulating complexes (ISCOMS).

An experimental immunostimulating complex vaccine has been prepared from detergent (Mega-10) solubilized Mycoplasma gallisepticum (MG) antigens. Sucrose gradient centrifugation, SDS-PAGE and immunoblotting studies demonstrated that the ISCOM vaccine contained virtually all of the immunodominant MG membrane proteins, including p64 and p56. Protective immunity generated by the experimental MG ISCOM vaccine was demonstrated in challenge experiments. Chickens immunized with a single dose containing between 1 and 50 micrograms of MG ISCOMs had significantly reduced lesion scores in the air sac after challenge. The reisolation of the challenge MG strain was significantly less frequent from the chickens in vaccinated groups than from the unvaccinated control group. Presence of humoral antibodies in chickens vaccinated with 1-25 micrograms MG ISCOMs was not detectable by blocking ELISA or haemagglutination-inhibition before challenge. Chickens vaccinated once or twice with a 50 micrograms dose were transitory positive by blocking ELISA and rapid plate agglutination before challenge.

Detection of specific antibodies directed against a consistently expressed surface antigen of Mycoplasma gallisepticum using a monoclonal blocking enzyme-linked immunosorbent assay.

Sera from 14 groups of chickens inoculated with different laboratory and field strains of Mycoplasma gallisepticum (MG) were used to compare the diagnostic potential of the hemagglutination-inhibition (HI) test and a recently developed monoclonal blocking enzyme-linked immunosorbent assay (ELISA). HI was performed with strain A5969, commonly used as hemagglutinating antigen, and it could detect 62.7% of the inoculated chickens as positive. Of all sera, 83% proved to be positive when examined with the blocking ELISA. The difference between the sensitivities of the two methods was due to group-specific insensitivity of the HI test. None of the sera from groups inoculated with strains K 1501, K 1503, K 503, or K 703 and only half of the sera from groups inoculated with K 1453 or 236C could inhibit the activity of the A5969 hemagglutinating antigen, indicating antigenic differences between these challenge strains and the diagnostic strain. ELISA detected MG-specific antibodies in every group of sera, although inoculation with variant strains K 503 or K 703 resulted in lower level of antibody production than inoculation with other strains. The monoclonal blocking ELISA can be useful in the serological diagnosis of MG infections, because it is based on a consistently expressed, specific region of MG.

Detection of Mycoplasma gallisepticum antibodies in turkey blood samples by ELISA and by the slide agglutination and haemagglutination inhibition tests.

Comparative examination of a total of 1,030 blood samples from six turkey flocks of three Eastern Hungarian turkey farms was performed by the conventional haemagglutination inhibition (HI) and slide agglutination (SA) tests and by a competitive ELISA visualizing the inhibition by a positive test serum of the reaction between a monoclonal antibody and the specific epitope of Mycoplasma gallisepticum recognized by it. All the three tests detected the flocks which were certainly infected. The highest rate of positivity (93% of the samples tested) was revealed by the ELISA. By SA and HI the positivity rate was 56% and 55%, respectively. Thirty-five per cent of the positive blood samples reacted in all three tests, 17% of them only by ELISA and HI, another 17% only by ELISA and SA, while 3% only by SA and HI. In the case of positive flocks first the SA test and ELISA, then the HI test and ELISA give parallel results.

Indirect ELISA for the detection of a specific antibody response against Mycoplasma gallisepticum.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for determining Mycoplasma gallisepticum antibodies in chicken sera. The M. gallisepticum antigen was detergent extracted and incorporated into ISCOMs. Sediment of broth medium treated with sarcosyl was used as control antigen. Sera were tested before and after absorption with broth medium components and ELISA titres are expressed as optical density (OD) at 492 nm. Sera from experimentally or naturally infected chickens, those vaccinated with Salsbury Mg bacterin or both vaccinated and experimentally infected were compared with sera from M. gallisepticum free or SPF chickens. A high OD was observed when unabsorbed sera (even from SPF chickens) were tested with control antigen. The non-specific binding of M. gallisepticum negative sera could be removed by absorbing the sera with broth media components before the ELISA was performed. In contrast, ELISA titres obtained with sera from M. gallisepticum positive birds did not decrease significantly after absorption, except in the vaccinated and experimentally infected group. When the OD obtained with control antigen was subtracted from that obtained with ISCOM antigen, the mean value for M. gallisepticum free chickens was 0.083. Higher values were obtained with absorbed sera from experimentally or naturally infected (0.248-0.526), vaccinated (0.506), or vaccinated and infected (0.276-0.930) birds. The use of the ISCOM antigen presentation system in the indirect ELISA, combined with absorption of sera with broth components was demonstrated to be a useful diagnostic assay for M. gallisepticum antibodies.

Monoclonal antibodies to Mycoplasma gallisepticum membrane proteins.

Monoclonal antibodies (MAbs) were prepared to study the immunogenesis of Mycoplasma gallisepticum. Balb/c mice were immunized with M. gallisepticum immunostimulating complexes and the supernatant of heterokaryotes screened with M. gallisepticum and closely related M. synoviae as antigens in indirect enzyme-linked immunosorbent assay. All selected MAbs proved to be M. gallisepticum species-specific when they were tested against 10 different avian Mycoplasma species. After immunoblotting analysis, five polypeptides were identified with estimated molecular weights of 110,000, 66,000, 64,000, 56,000, and 50,000. Cell membrane localization of the recognized polypeptides was studied by immunoelectron microscopy. None of the MAbs inhibited the hemagglutinating activity of freshly prepared M. gallisepticum. However, one MAb (B3) specific for p56 agglutinated the stained M. gallisepticum antigen in the slide agglutination test. Results seemed to correlate with published information on the protein composition and agglutinating activity of Mycoplasma gallisepticum.

Evaluation of a monoclonal blocking enzyme-linked immunosorbent assay for the detection of Mycoplasma gallisepticum-specific antibodies.

A monoclonal blocking enzyme-linked immunosorbent assay (blocking-ELISA) was developed to detect antibodies to Mycoplasma gallisepticum (MG) in poultry sera with the help of a peroxidase-labeled monoclonal antibody (MAb) recognizing an epitope of a 56-kilodalton polypeptide (p56) of MG. Immunoglobulins from undiluted MG-positive sera prevent the MAb conjugate from attaching to its specific binding site on p56, which results in no color development. The opposite result--a strong color reaction--was obtained after incubation with MG-negative sera (or when no serum was added before the MAb conjugate). Results were expressed in percent inhibition or ELISA titers. The blocking-ELISA detected 84.7% positive chickens in an experimentally infected flock and 72.6% of chickens in naturally infected flocks, whereas the hemagglutination-inhibition (HI) test was positive only with 68.4% and 48.6% of these serum samples, respectively. All HI-positive serum samples reacted positively in blocking-ELISA. Of sera negative by the HI test, 51.6% and 46.8% proved to be positive when examined with the blocking-ELISA. Overall agreement between the ELISA and HI test was 76.8%. Infection with closely related M. synoviae did not induce any false-positive reactions in blocking-ELISA. There was a strong positive correlation between HI and blocking-ELISA titers (r = 0.83).

Biochemical and serological study of two mycoplasma strains isolated from geese.

2 mycoplasma strains were isolated, one from the phallic lymph of a gander and the other from a cloacal swab of a laying goose. The strains proved to be different from mycoplasma species isolated from geese before. Strain No. 1223 is a glucose-negative and arginine-negative species belonging to the genus Mycoplasma. In the growth inhibition test, it fails to react with hyperimmune sera raised in rabbits against the presently known mycoplasma species of avian origin nor with sera produced against mammalian mycoplasma species sharing its biochemical properties. Strain No. 1225 belongs to the digitonin resistant Acholeplasmataceae family. It is glucose-positive and aesculin-positive. It is negative by all the other tests and fails to react with sera produced against the presently known acholeplasma species.