RESUMO
Renal cell carcinoma is an immunogenic tumour with a prominent dysfunctional immune cell infiltrate, unable to control tumour growth. Although tyrosine kinase inhibitors and immunotherapy have improved the outlook for some patients, many individuals are non-responders or relapse despite treatment. The hostile metabolic environment in RCC affects the ability of T-cells to maintain their own metabolic programme constraining T-cell immunity in RCC. We investigated the phenotype, function and metabolic capability of RCC TILs correlating this with clinicopathological features of the tumour and metabolic environment at the different disease stages. Flow cytometric analysis of freshly isolated TILs showed the emergence of exhausted T-cells in advanced disease based on their PD-1high and CD39 expression and reduced production of inflammatory cytokines upon in vitro stimulation. Exhausted T-cells from advanced stage disease also displayed an overall phenotype of metabolic insufficiency, characterized by mitochondrial alterations and defects in glucose uptake. Nanostring nCounter cancer metabolism assay on RNA obtained from 30 ccRCC cases revealed significant over-expression of metabolic genes even at early stage disease (pT1-2), while at pT3-4 and the locally advanced thrombi stages, there was an overall decrease in differentially expressed metabolic genes. Notably, the gene PPARGC1A was the most significantly down-regulated gene from pT1-2 to pT3-4 RCC which correlated with loss of mitochondrial function in tumour-infiltrating T-cells evident at this tumour stage. Down-regulation of PPARGC1A into stage pT3-4 may be the 'tipping-point' in RCC disease progression, modulating immune activity in ccRCC and potentially reducing the efficacy of immunotherapies in RCC and poorer patient outcomes.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Recidiva Local de Neoplasia , Progressão da Doença , ImunidadeRESUMO
Current European Commission (EC) surveillance regulations require discriminatory testing of all transmissible spongiform encephalopathy (TSE)-positive small ruminant (SR) samples in order to classify them as bovine spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests, including characterization by bioassay in mouse models. Since 2005, naturally occurring BSE has been identified in two goats. It has also been demonstrated that more than one distinct TSE strain can coinfect a single animal in natural field situations. This study assesses the ability of the statutory methods as listed in the regulation to identify BSE in a blinded series of brain samples, in which ovine BSE and distinct isolates of scrapie are mixed at various ratios ranging from 99% to 1%. Additionally, these current statutory tests were compared with a new in vitro discriminatory method, which uses serial protein misfolding cyclic amplification (sPMCA). Western blotting consistently detected 50% BSE within a mixture, but at higher dilutions it had variable success. The enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only when it was present as 99% of the mixture, with variable success at higher dilutions. Bioassay and sPMCA reported BSE in all samples where it was present, down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at 0.1%. While bioassay is the only validated method that allows comprehensive phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears to offer a fast and cost-effective alternative for the screening of unknown isolates when the purpose of the investigation was solely to determine the presence or absence of BSE.
Assuntos
Coinfecção/diagnóstico , Testes Diagnósticos de Rotina/métodos , Encefalopatia Espongiforme Bovina/diagnóstico , Príons/análise , Animais , Bioensaio/métodos , Bovinos , Cabras , Imunoensaio/métodos , Camundongos , Patologia Molecular/métodosRESUMO
We reported the expression of the homeodomain-containing transcription factor Engrailed-2 (EN2) in prostate cancer and showed that the presence of EN2 protein in the urine was highly predictive of prostate cancer. This study aimed to determine whether patients with prostate cancer have EN2 autoantibodies, what the prevalence of these antibodies is and whether they are associated with disease stage. The spontaneous immunoglobulin (Ig)G immune response against EN2 and for comparison the tumour antigen New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1), were tested by enzyme-linked immunosorbent assay (ELISA) in three different cohorts of prostate cancer patients as well as a group of men genetically predisposed to prostate cancer. Thirty-two of 353 (9·1%) of the SUN cohort representing all stages of prostate cancer demonstrated EN2 IgG responses, 12 of 107 patients (11·2%) in the advanced prostate cancer patients showed responses, while only four of 121 patients (3·3%) with castrate-resistant prostate cancer showed EN2 autoantibodies. No significant responses were found in the predisposed group. Anti-EN2 IgG responses were significantly higher in patients with prostate cancer compared to healthy control males and similarly prevalent to anti-NY-ESO-1 responses. While EN2 autoantibodies are not a useful diagnostic or monitoring tool, EN2 immunogenicity provides the rationale to pursue studies using EN2 as an immunotherapeutic target.
Assuntos
Autoanticorpos/imunologia , Proteínas de Homeodomínio/imunologia , Proteínas do Tecido Nervoso/imunologia , Neoplasias da Próstata/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/sangue , Biomarcadores Tumorais , Neoplasias da Mama/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/terapiaRESUMO
The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5-, 10-, 25-, 50- and 100-µm-wide repeat grating. MG63 bone cells were seeded at 50,000 cells per 25 cm(2) and cell alignment was determined from micrographs taken at time-points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin-patterned cover slips especially to the 50- and 100-µm-wide patterns, although in this case cells did not position themselves in the middle of each fibronectin-coated region, but instead aligned to the small features associated with the edges of the fibronectin-coated regions. Patterned and un-patterned cells also had quite different morphologies. The un-patterned cells had a more rounded morphology and lengths of 25 to 35 µm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50-70 µm. The widefield surface plasmon resonance imaging indicated that cells on un-patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin-patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin-coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.
Assuntos
Adesão Celular , Proteínas da Matriz Extracelular/metabolismo , Microscopia/métodos , Osteócitos/citologia , Osteócitos/fisiologia , Ressonância de Plasmônio de Superfície/métodos , Linhagem Celular , Humanos , Processamento de Imagem Assistida por Computador/métodosRESUMO
The relevance of age on serotonergic involvement in the control of alimentary contractility has not been pharmacologically described. Experiments were performed to investigate the effects of acetylcholine, atropine, 5-hydroxytryptamine (5-HT) and its related drugs on intestinal segments taken from the neonatal and adult ileum. 5-HT induced concentration-dependent contractions of ileum irrespective of age; however, these contractions were diminished by pretreatment with atropine only in neonatal tissues. In tissues taken from both the neonatal and adult ileum, methysergide (5-HT(1/2/5-7) receptor antagonist), ritanserin (5-HT(2) receptor antagonist), and RS23597-190/SB204070 (5-HT(4) receptor antagonists) all differentially reduced 5-HT-induced contractions at a concentration <100 µmol/l. At higher concentrations, the contractions were comparable to those in control tissues. Granisetron and ondansetron (5-HT(3) receptor antagonists) significantly reduced contractions induced by 5-HT at concentrations >30 µmol/l in both neonatal and adult ileum. Combined treatments with ritanserin, granisetron, plus RS23597-190 reduced or abolished contraction responses induced in neonatal ileum by 5-HT. SB269970A (5-HT(7) receptor antagonist) and WAY100635 (5-HT(1A) receptor antagonist) failed to influence contractile responses induced by 5-HT or 5-HT receptor agonists. Pretreatments with WAY100635 and SB267790A also had no influence on the contractile responses induced by 5-HT(1A/7) receptor agonist, 5-CT, and 5-HT(1A) receptor agonist, 8-OH-DPAT, which itself failed to induce a measurable response. It is concluded that the 5-HT-induced contractions in segments taken from both the neonatal and adult rat ileum were mediated via 5-HT(2) receptors, 5-HT(3) receptors and 5-HT(4) receptors. However, the effect of atropine on the neonatal rat intestine indicates that the mechanism of serotonergic involvement in ileal contractility is influenced by age.
Assuntos
Animais Recém-Nascidos/fisiologia , Íleo/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Receptores de Serotonina/fisiologia , Agonistas do Receptor de Serotonina/farmacologia , Serotonina/farmacologia , Fatores Etários , Animais , Atropina/farmacologia , Feminino , Íleo/fisiologia , Masculino , Antagonistas Muscarínicos/farmacologia , Ratos , Ratos Sprague-Dawley , Antagonistas da Serotonina/farmacologiaRESUMO
We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.
Assuntos
Membrana Celular/ultraestrutura , Ressonância de Plasmônio de Superfície/instrumentação , Linhagem Celular , Células/ultraestrutura , Adesões Focais/ultraestrutura , Humanos , Microscopia de Vídeo , Nanotecnologia , Ressonância de Plasmônio de Superfície/métodosRESUMO
Attempts were made to develop a simplified procedure for long-term cryopreservation of intestinal smooth muscle cells (ISMC). ISMC were collected from the ileum of Sprague-Dawley neonatal rats through cellular dissociation in trypsin. Cryopreservation method comprised of a rapid 1-step (protocol 1) and a slow 3-step (protocol 2) freezing of ISMC for 1week. Preparations were thawed and single ISMC were assessed via the comet assay and damaged DNA was quantified through comet tail moment. The control unfrozen ISMC exhibited DNA damage of 2.34+/-0.35 compared to ISMC cooled via protocol 2 (2.62+/-0.36) and protocol 1 (10.15+/-0.72). Thereafter, protocol 2 freezing method was adopted and ISMC were cryopreserved for 1-week, 1-month, and 4-months to analyse the temporal and long-term cryopreservation of ISMC. This revealed a DNA damage of 2.62+/-0.36 (1-week), 3.81+/-0.72 (1-month), and 5.1+/-0.9 (4-months). Gradual cooling is suitable for continuing storage of ISMC and although fluctuation in cryoinjury is observed with time this is considered to reflect cell-to-cell variability.
Assuntos
Criopreservação/métodos , Intestino Delgado/citologia , Miócitos de Músculo Liso/citologia , Animais , Sobrevivência Celular , Células Cultivadas , Ensaio Cometa , Crioprotetores , Dano ao DNA , Dimetil Sulfóxido , Congelamento , Técnicas In Vitro , Miócitos de Músculo Liso/metabolismo , Ratos , Tempo , Azul TripanoRESUMO
AIMS: To audit the safety of differing protocol-driven early-discharge policies, from two sites, for low-risk acute upper gastrointestinal (GI) bleeding and determine if default early (<24 h) in-patient endoscopy is necessary. METHODS: All patients with low-risk acute upper GI bleeding presenting to two separate hospital sites in Leeds from August 2002 to March 2005 were identified. Both hospitals operate nurse-led process-driven protocols for discharge within 24 h, but only one includes default endoscopy. Relevant information was obtained from patients' notes, patient administration systems, discharge letters and endoscopy records. RESULTS: 120 patients were admitted to site A and 74 to site B. Median length of stay on the clinical decisions unit was 12.6 h at site A and 9.4 h at site B (p = 0.045). Oesophagogastroduodenoscopy was performed on 89/120 (74%) patients at site A compared with only 7/74 (9%) at site B (p<0.001). Six of 120 (5%) patients from site A were admitted to hospital for further observation compared with 6/74 (8%) from site B (p = 0.38). Of the remaining patients, all were discharged within 24 h, and 8/114 (7%) at site A vs 17/68 (25%) at site B were given hospital clinic follow-up (p<0.001). None of the 194 patients had further bleeding or complications within 30 days. CONCLUSIONS: Patients admitted with a low-risk acute upper GI bleeding can be managed safely by a nurse-led process-driven protocol, based on readily available clinical and laboratory variables, with early discharge <24 h. Avoiding in-patient endoscopy appears to be safe but at the price of greater clinic follow-up.
Assuntos
Endoscopia do Sistema Digestório/normas , Hemorragia Gastrointestinal/diagnóstico , Alta do Paciente , Doença Aguda , Adolescente , Adulto , Idoso , Endoscopia do Sistema Digestório/enfermagem , Feminino , Hemorragia Gastrointestinal/etiologia , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Medição de Risco , Fatores de RiscoRESUMO
An electronic survey of 188 acute NHS hospitals was carried out to assess the provision of out-of-hours services for gastrointestinal emergencies in England. The response rate was 167/188 (89%) for the main questionnaire and 157/188 (84%) for a supplementary questionnaire. The survey revealed that the majority of gastroenterologists (135/157, 86%) participate in acute general medicine. A rota for out-of-hours endoscopy was in place in only 82/167 (49%) of hospitals. Trained nurse endoscopy assistance was available in 51/82 (62%) of those hospitals with a formal rota. Two thirds of gastroenterologists were telephoned up to five times each month for advice when not on call; 64% felt their emergency endoscopy service provision was unsatisfactory and 38% thought it was unsafe. This paper concludes that there is serious under provision of services for patients presenting with gastrointestinal emergencies in England.
Assuntos
Serviços Médicos de Emergência/organização & administração , Gastroenterologia/organização & administração , Sociedades Médicas , Inquéritos e Questionários , Carga de Trabalho , Endoscopia Gastrointestinal/estatística & dados numéricos , Inglaterra , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/terapia , Humanos , Corpo Clínico Hospitalar/normas , Competência Profissional , Estudos RetrospectivosRESUMO
gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.
Assuntos
Doenças dos Suínos/imunologia , Suínos/imunologia , Subpopulações de Linfócitos T/imunologia , Viroses/veterinária , Adaptação Fisiológica , Animais , Quimiocinas/genética , Citocinas/genética , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Imunidade Inata , Cooperação Linfocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Vacinas Virais/imunologia , Vacinas Virais/farmacologia , Viroses/imunologiaRESUMO
We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.
Assuntos
Técnicas de Cultura de Células/métodos , Queratinócitos/citologia , Cristais Líquidos , Engenharia Tecidual , Linhagem Celular , Sobrevivência Celular , Imunofluorescência , Humanos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Fifty-three patients with biliary obstruction caused by unresectable malignancy were treated by attempted insertion of an endoprosthesis by the percutaneous-endoscopic route. This was successful in 50 patients. A single endoprosthesis was inserted in each case. Both right and left hepatic duct decompression were obtained in 31 patients, but only unilateral or segmental drainage was achieved in 19 patients. Procedure-related complications occurred in 18 (36%) patients, and 15 (30%) patients died within 30 days of the procedure. Satisfactory resolution of jaundice was obtained in 26 (84%) patients with bilateral decompression and in 12 (63%) of those with unilateral drainage. The 30-day mortality rate was 26% for patients with bilateral and 37% for those with unilateral drainage. The morbidity rate from cholangitis after endoprosthesis insertion was 10% after bilateral and 32% after unilateral drainage. None of these differences was statistically significant. Surviving patients with satisfactory bile drainage were relieved of symptoms such as pruritus. The combined percutaneous-endoscopic technique enables difficult biliary strictures to be intubated. Although bilateral duct drainage is preferable, the palliation is often worthwhile even when segmental ducts alone are drained.
Assuntos
Adenoma de Ducto Biliar/cirurgia , Neoplasias dos Ductos Biliares/cirurgia , Colestase/cirurgia , Neoplasias da Vesícula Biliar/cirurgia , Neoplasias Pancreáticas/cirurgia , Próteses e Implantes , Adenoma de Ducto Biliar/complicações , Neoplasias dos Ductos Biliares/complicações , Colestase/etiologia , Drenagem , Endoscopia , Neoplasias da Vesícula Biliar/complicações , Humanos , Cuidados Paliativos , Neoplasias Pancreáticas/complicaçõesRESUMO
The control of neuronal cell position and outgrowth is of fundamental interest in the development of applications ranging from cellular biosensors to tissue engineering. We have produced rectangular networks of functional rat hippocampal neurons on silicon oxide surfaces. Attachment and network formation of neurons was guided by a geometrical grid pattern of the adhesion peptide PA22-2 which matches in sequence a part of the A-chain of laminin. PA22-2 was applied by contact printing onto the functionalised silicon oxide surface and was immobilised by hetero-bifunctional cross-linking with sulfo-GMBS. Geometric pattern matching was achieved by microcontact printing using a polydimethylsiloxane (PDMS) stamp. In this way the produced grid pattern ranged from 3 to 20 microm in line width and from 50 to 100 microm in line distances. As shown by atomic force microscopy (AFM), line widths and line distances of the peptide pattern differ less than 0.5 microm from the used PDMS stamp. The height of the layer of immobilised PA22-2 was approximately 3.5 nm implying the layer to be monomolecular. Immobilised PA22-2 was capable of binding anti-PA22-2 antibodies indicating that the function of the peptide was not compromised by immobilisation. Rat hippocampal neurons, cultured at low density in serum-free medium, were applied to the growth matrix of PA22-2-coated substrates and, within 1-3 h of culture, formed a network-like pattern that more or less matched the printed grid. Reliability and reproducibility of neuronal network formation depended on the geometry, line width and node diameter of the grid pattern. The immobilised neurons showed resting membrane potentials comparable with controls and, already after 1 day of culture, were capable of eliciting action potentials. The suitability of the immobilised neurons for the study of man-made neural networks and for multi-site recordings from a functional neuronal network is discussed.
Assuntos
Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Eletrofisiologia/métodos , Hipocampo/fisiologia , Rede Nervosa/fisiologia , Neurônios/fisiologia , Óxidos/farmacologia , Compostos de Silício/farmacologia , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Adesão Celular/fisiologia , Técnicas de Cultura de Células/instrumentação , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Eletrofisiologia/instrumentação , Feto , Hipocampo/citologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Rede Nervosa/citologia , Rede Nervosa/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , RatosRESUMO
Antigenic differences within equine-1 and equine-2 isolates of influenza were studied by haemagglutination inhibition tests, indirect ELISA and competition ELISA, using the same antisera. Better differentiation was obtained with the competition ELISA than with the other two tests. All three methods produced similar relationships within the equine-1 isolates but differed in their ability to differentiate the equine-2 isolates where the competition ELISA was superior and produced epidemiologically sensible results. In all three tests, post-infection ferret and horse sera were more useful in discriminating isolates than post-vaccination sera.
Assuntos
Antígenos Virais/análise , Vírus da Influenza A/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Testes de Inibição da Hemaglutinação , Análise de RegressãoRESUMO
The use of pig blood samples dried on paper discs for the detection of antibodies against FMDV by the liquid phase blocking ELISA has been evaluated. The average volume of whole heparinised blood required to fully saturate 6.0 mm discs was 7.65 microliters (range 7.2 to 8.1; variation = 0.24, P = 0.05). When 200 clinically healthy animals were assessed by virus neutralisation (VN) titres up to 1/22 were recorded against types O, A and C, 97% being 1/11 or less. Using ELISA, results were more skewed. Overall, 91% showed titres of 1/32 or less, and there were occasional high non-specific reactors with types O and A. Using small groups of sera from experimental animals, a VN titre of 1/16 was found to be equivalent to an ELISA titre of approximately 1/100 (log10 2.0 +/- 0.2) with type O and 1/56 (log10 1.75 +/- 0.1) for type A. Although some loss in sensitivity from disc dried sera was found in selected negatives on testing at a single dilution of 1/32, sera from vaccinated animals with VN titres of 1/16 to 1/708 all gave strong positive results. A monoclonal antibody (Mab) assay was successfully developed to detect different swine anti-FMDV antibody isotypes.
Assuntos
Anticorpos Antivirais/análise , Aphthovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Febre Aftosa/diagnóstico , Animais , Anticorpos Monoclonais , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Testes de Neutralização , SuínosRESUMO
A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/análise , Suínos/imunologia , Subpopulações de Linfócitos T/fisiologia , Animais , Ativação Linfocitária , Ovalbumina/imunologiaRESUMO
Monoclonal antibodies (mAbs) submitted to the workshop B cell panel were screened for reactivity with bovine surface immunoglobulin positive (SIg+) cells from tonsil, mesenteric lymph nodes (MLN), ileal Peyer's patches (IPP), thymus and peripheral blood by fluorescence activated cell sorter (FACS) analysis. All mAbs within B cell panel reacted with B cells except the negative control mAb VPM61. Although most mAbs stained the majority of B cells from different tissues, 14 mAbs did not stain B cells from IPP. Detailed FACS analysis of the 24 mAbs in preliminary clusters PC17, PC28, PC29, PC30, PC35, PC41 and PC43 showed all preliminary clusters (PC) to contain mAbs with similar and variable specificities. This absence of clear-cut clusters therefore did not permit a simple classification of B cell surface antigens via the B cell mAbs and suggests considerable complexity and variability of the B cell surface.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Animais , Bovinos , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
Infection of pigs with many strains of African Swine Fever Virus (ASFV) has been shown to cause a loss or marked decrease in the ability of splenocytes to respond to mitogens. These observations have been extended by cell fractionation and reconstitution experiments to show that the mitogen stimulated proliferative capacity of both the CD4+ and CD8+ T cells is affected. Similarly, monocytes which are directly infectable by virus, are functionally defective as antigen presenting cells when added to mitogen stimulated normal T cells. Interestingly, the same T cells which respond poorly in mitogenic assays can be activated by stimulation through the CD3 receptor. In contrast to the defective mitogenic response of T cells, B cell function, as assessed by stimulation through the CD40 ligand in vitro remains intact. There is no evidence for apoptosis in either the T cells or the B cells recovered from the spleens of ASFV infected animals 1-5 days following infection. Although the number of leucocytes which can be recovered from the infected spleen decreases rapidly with progression of the disease, the proportion of the different cell phenotypes remains constant. Thus decreased activity of lymphocytes in lymphoid tissue from ASFV infected animals appears to be directly attributable to infection of the monocytes.
Assuntos
Peste Suína Clássica/imunologia , Monócitos/imunologia , Baço/imunologia , Linfócitos T/imunologia , Vírus da Febre Suína Africana/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Linfócitos B/imunologia , Linfócitos B/patologia , Complexo CD3/metabolismo , Peste Suína Clássica/patologia , Peste Suína Clássica/virologia , Tolerância Imunológica , Técnicas In Vitro , Ativação Linfocitária , Mitógenos/farmacologia , Monócitos/patologia , Fenótipo , Baço/patologia , Suínos , Porco Miniatura , Linfócitos T/patologiaRESUMO
The light-addressable potentiometric sensor (LAPS) measures localized photo-induced currents from a silicon wafer, which are dependent on the local surface potential and on the intensity of the light pointer. In this study the ability of the LAPS to record extracellular potentials of adherent cells was investigated. Time dependent LAPS photocurrent signals that correlated in time with contractions were recorded from beating cardiac myocytes cultured on LAPS surfaces. Signals could be recorded both when the LAPS was biased to working points where the photocurrent was maximally sensitive to potential changes and when it was biased to working points where the photocurrent was insensitive to changes in surface potential. Therefore, signals could not be predominantly created by changes in extracellular potential and might be related to mechanical contractions. One possible explanation might be, that the cell-induced modulation of photocurrents arose as a result of cell shape changes. Such alterations in cell shape might have focused and defocused the light pointer and, thus, modulated its intensity. To further test this hypothesis, height changes of beating cardiac myocytes were measured with an atomic force microscope (AFM). They were found to match well with signals derived from LAPS measurements. Therefore, it can be concluded, that LAPS signals were mainly determined by the periodic changes in shape of beating heart cells, and this interference precludes the measurements of extracellular electrophysiological potentials from these cells.
Assuntos
Coração/fisiologia , Potenciometria/instrumentação , Animais , Engenharia Biomédica , Tamanho Celular , Células Cultivadas , Embrião de Galinha , Técnicas In Vitro , Luz , Potenciais da Membrana , Miocárdio/citologia , RatosRESUMO
This study examined the behavior of rat tendon fibroblasts, baby hamster kidney fibroblasts, macrophage-like P388D1 cells, and neurons from rat dorsal root ganglia, cultured on fibronectin strands 0.2-5 micrograms in diameter. We investigated cell spreading, orientation, formation of focal contacts, the speed of cell movement, and the speed of neurite outgrowth in cells cultured on fibronectin strands, glass covered with fibronectin, and plain, nontreated glass. Fibronectin strands significantly promoted cell spreading and caused a marked alignment of all kinds of cells to the direction of the fiber. The fibers caused the alignment of actin filaments in fibroblasts and focal contacts in fibroblasts and macrophages and increased polymerization of F-actin in cells. Fibronectin fibers also increased the speed and persistence of cell movement and the rate of neurite outgrowth. Macrophages grown on fibronectin fibers produced numerous actin-rich microspikes and adopted a polarized, migratory phenotype. These findings indicate that fibronectin strands, resembling natural components of the extracellular matrix, are more effective in activating various types of cells then two-dimensional, fibronectin-covered substrata. The results also confirm the suitability of the three-dimensionally oriented fibronectin form for use in clinical practice.