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Biomedical subjects

C R Stokes

Publications and source records attributed to C R Stokes.

At least 37 records · Page 2Linked to original sources
PubMed1998

Summary of workshop findings for porcine adhesion molecule subgroup.

Fifty-nine monoclonal antibodies (mAb) were assigned to the adhesion section of the Second International Swine CD Workshop. They were analysed for their reactivity to selected lymphoid cell populations, as well as to non-lymphoid cell lines. Cell lysate ELISAS and Western Blot analyses were also carried out. As a result, thirteen separate cluster groups emerged (p > 0.95). Workshop assignments for adhesion molecules were made: wCD29/49 for mAbs UCP1D2 (#133) and FW4-101 (#165), and PNK-I (#194) and MUC76A (#025) could be assigned to wCD18. For one cluster (FQ1D7, #161 and 2F4, #069) the cellular distribution and MW were characteristic for MHC Class II, and another cluster comprising several antibodies which appeared to recognise MHC Class I. Other clusters could not be assigned to cell surface structures known to be linked to cellular adhesion, however, two further antibodies, 335-2 (#112) and FG1F6 (#156), could be added to SWC1, and the new SWC8 was defined by MIL3 (#077) and MUC20A (#029), binding a ligand of 29-32 kDa. Clustering for these two antibodies was confirmed by blocking studies. The cellular distribution is known for MIL3, recognising an epitope present on granulocytes, B cells, and a subset of T cells expressing CD8 at high intensity.

Animals↗
PubMed1998

Assessment of intestinal function in cats with chronic diarrhea after infection with feline immunodeficiency virus.

OBJECTIVE: To identify consistent relevant mechanisms of small intestinal dysfunction in cats with experimentally induced feline immunodeficiency virus infection (FIV) that developed chronic diarrhea during the time they were being used in studies of pathogenicity and transmission of FIV. ANIMALS: 10 cats. PROCEDURE: The following investigative tests and techniques were performed on each of the cats: routine hematologic and serum biochemical analyses; urinalysis; fecal parasitologic and microbiologic examinations; breath hydrogen lactulose (BH2LT) and xylose (BH2XT) tests; intestinal permeability test; endoscopic examination of the intestinal mucosa; bacteriologic culture of endoscopically collected small intestinal juice; and histologic examination of endoscopically obtained intestinal biopsy specimens. RESULTS: Neutrophilia was evident in 3 cats, and lymphopenia was detected in 2 cats. Serum biochemical abnormalities were not observed. Urinalysis results were unremarkable. Fecal bacteriologic and parasitologic results were normal, except for isolation of Campylobacter sp from 1 cat. Abnormal BH2XT values suggestive of D-xylose malabsorption were identified in 2 cats, and BH2LT values indicated evidence of small intestinal bacterial overgrowth in 1 cat. Finally, permeability test results, quantitation of bacterial flora from the proximal part of the small intestine and histologic examination of biopsy specimens did not reveal any abnormalities. CONCLUSIONS: Enteric pathogens did not account for the development of diarrhea in cats with experimentally induced FIV infection, and consistent relevant mechanisms of small intestinal dysfunction were not identified.

Animals↗
PubMed1996

Antigen handling in the gastrointestinal lamina propria.

Enteric infections associated with bacteria, viruses and parasites are a world health problem accounting for at least one billion episodes of diarrhoea and up to 10 million deaths in the Third World each year. The widely held conclusion that responses which are protective in the gastrointestinal tract are best stimulated by mucosal presentation of antigen remains largely unchallenged. Despite this there are but a few effective mucosal vaccines. The mucosal immune system of the gastrointestinal tract is presented with a contrasting array of antigens, ranging from harmless dietary components to highly pathogenic organisms, and has evolved a battery of responses from which an appropriate response may be orchestrated. We have postulated that the microenvironment in which an antigen is processed and presented to the mucosal immune system may profoundly influence the nature of the response and the purpose of this review is discuss a possible role for the lamina propria in this process.

Animals↗
PubMed1996

Vaginal and rectal infection of cats with feline immunodeficiency virus.

The objective of this study was to examine the potential of vaginal and rectal mucosal routes for feline immunodeficiency virus (FIV) uptake and infection, as a model of mucosal HIV infection, and to determine the fate of virus at these mucosal sites following transmission of infection. SPF cats were exposed to FIV isolates (PET, GL-8, T637), administered as either cell-associated or cell-free inocula, via the rectum or vagina. Establishment of infection was confirmed by isolation of infectious FIV from peripheral blood mononuclear cells (PBMC), and by presence of FIV proviral DNA in PBMC using a nested polymerase chain reaction. Fate of virus in tissue taken at necropsy from cats infected for 6-48 weeks was assessed by localizing FIV core and envelope proteins, p24 and gp41, using a biotin-streptavidin linked immunoperoxidase (IP) technique. Cells susceptible to infection were identified by an in situ hybridization technique for FIV viral DNA and RNA. Cell-free, as well as cell-associated, virus was infectious across intact vaginal and rectal mucosal surfaces. Transmission was most successful using cell-associated inocula, and via the rectal route. Cells infected with FIV were detected by IP staining in the colon of 6/9 rectally challenged cats and 1/5 vaginally challenged cats. Virus was predominantly localized within the epithelium at the base of the colonic crypts associated with lymphoid aggregates (follicle associated epithelium; FAE), and within the lymphoid follicle itself. Occasional infected cells were also noted within the lamina propria. The distribution of FIV DNA positive cells in the colon was similar to that for FIV antigen whilst FIV RNA positive cells were found more extensively, including within the lamina propria and lymphoid follicle. FIV infected cells were not detected within the vagina, or colonic and ileac lymph nodes. Similar patterns of infected cells were seen in all of the positive cats, indicating that colonic tissues remain persistently actively infected with FIV. We conclude that the FIV/cat model of rectal and vaginal mucosal infection should prove useful for characterizing the mechanism by which HIV infects mucosal surfaces and as a challenge system for the design of vaccines effective at preventing HIV infection via rectal and vaginal routes.

Animals↗
PubMed1996

Acquired immunity in experimental feline Microsporum canis infection.

Seven adult cats were inoculated cutaneously at a shaved site with Microsporum canis; five (group A) had spontaneously recovered from a previous M canis infection, and two (group B) were dermatophyte-naive. Both of the dermatophyte-naive cats developed typical clinical disease, the lesions reaching maximal size four to five weeks after exposure, and resolving by week 13. Only one of the group A cats developed clinical disease; the lesions were more inflammatory in nature and of shorter duration and had resolved by day 25 after exposure. Blood samples collected from the seven cats before and four weeks after exposure were assessed for M canis-specific cellular (by lymphocyte proliferation assays) and humoral (by IgG and IgM ELISA) immune responses. After four weeks the group A cats had significantly (P < 0.05) increased cellular and IgG-humoral immune responses, and the group B cats also had increased cellular, and IgG and IgM responses but the increases were not significant.

Animals↗
PubMed1996

Antigen presenting cells in the porcine gut.

It has long been known that mucosal responses are most effectively stimulated by local presentation of antigen but the mechanisms whereby the gut immune system is able to distinguish between potential pathogens and harmless dietary antigens are not clear. The gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless food proteins. Historically, most attention has focused on Peyer's patches and there is evidence of their role in the induction of both primary and secondary responses. Fed antigen can also be detected in the intestinal lamina propria (LP) and it has been shown that murine LP cells can stimulate allogenic mixed lymphocyte responses and present KLH to naive T cells. In contrast to guinea pigs, rodents and humans, pig intestinal epithelial cells do not express MHC Class II molecules, but they are present on a number of other cell types in the subepithelial LP. Amongst these are a significant proportion of non-professional APC including endothelial cells and eosinophils. Phenotypically pig LP T cells are a homogeneous population and the majority of CD4 T cells express the low molecular weight isoform of CD45. This is compatible with the suggestion that they are CD45RO-positive cells. A significant proportion of LP CD4 T cells are CD25 (IL-2R) positive, but following activation they secrete IL-4, with little or no IL-2 production. Based upon these observations, we would conclude that the lamina propria is a unique immunological microenvironment, and suggest that it may be of significance not only in surveillance and the provision of help during rapid responses to recall antigens but also in the down-regulation of local responses to food-derived peptides.

Animals↗
PubMed1996

Vaccination with fixed feline immunodeficiency virus (FIV) infected cells: protection, breakthrough and specificity of response.

Infection of cats with feline immunodeficiency virus (FIV), a naturally occurring lentivirus infection of cats which causes an AIDS-like disease, has generated considerable interest as an animal model for HIV vaccination. This paper reports on experiments performed to examine the potential of a fixed infected cell vaccine to confer protection against intraperitoneal challenge with cell-free FIV. The cell vaccine was highly immunogenic and elicited antibody responses to virus core antigen, p24, high virus neutralizing (VN) antibody titres, and antibodies which recognized cellular components of the vaccine. Whilst protection, assessed by the inability to detect infectious virus by virus isolation or polymerase chain reaction, against homologous but not heterologous FIV isolates was apparent up to week 12 post-challenge, when cats were monitored longer up to week 50 post-challenge a breakthrough in vaccine protection against homologous virus was observed. Protection could not be correlated with levels of antibody to p24 or VN antibody titres. In contrast with simian immunodeficiency virus vaccine studies in macaques there was no clear evidence that antibodies recognizing cellular components of the vaccine, including MHC class I and II antigens, conferred any protective effect following challenge. These results indicate that long-term post-challenge monitoring for infection is essential in lentivirus vaccine trials.

Animals↗
PubMed1996

Expression of major histocompatibility complex class II antigens on normal porcine intestinal endothelium.

A novel monoclonal antibody (MIL 11) specific for an antigen expressed on porcine endothelial cells is described. The antigen recognized by MIL 11 is most strongly expressed in the intestine but is also expressed on the capillary endothelium of a wide range of tissues. Using two- and three-colour immunofluorescence microscopy we demonstrated the extensive coexpression of MIL 11 and major histocompatibility complex (MHC) class II antigens on normal porcine capillary endothelium in the intestine, trachea, thymus and small veins, while endothelium of large vessels and the heart were negative for MHC class II. In contrast to humans and rodents, available reagents do not detect MHC class II on the intestinal epithelium of pigs. However, porcine intestinal endothelium expressed both DR and DQ antigens. A population of strongly class II-positive cells was also detected immediately adjacent to the endothelium in the lamina propria. Three-colour immunofluorescence microscopy highlighted the close association between endothelium and intestinal CD4+ T cells. Lamina propria T cells were mainly MHC class II positive, whereas those in the epithelial compartment were MHC class II negative.

Animals↗
PubMed1995

Detection of tetanus toxoid-specific memory T cells in equine lymph nodes but not in peripheral blood.

The use of tetanus toxoid as a recall antigen to investigate equine immune responses would be, in theory, a useful and cost-effective model in vitro. However, by using various regimens for culturing peripheral blood mononuclear cells from horses previously immunised with toxoid no proliferative response to the antigen was obtained in vitro, whereas lymph node mononuclear cells from the same animals proliferated significantly in response to it. The lack of response by the peripheral blood mononuclear cells was not due to the presence of a suppressive factor but to a lack of recognition of the antigen by the T cells of the peripheral blood.

Analysis of Variance↗
PubMed1995

Effect of early weaning on the development of immune cells in the pig small intestine.

The controlled effects of age and weaning on the numbers of CD2+ T cells, subsets (CD4+, CD8+), accessory cells (macrophage/granulocyte) and cells expressing MHC class II (DQw) and IL-2R in the piglet intestine was investigated. At birth low numbers of CD2+CD4-CD8- cells were the only demonstrable T cells in the intestine. Monocyte/granulocyte and MHC class II+ cells were also detected in low numbers and IL-2R+ cells were proportionally quite numerous. All those cell populations, except the IL-2R+ cells, increased thereafter and peaked at Week 7 when the numbers of cells were comparable with those of adult animals. CD4+ cells increased dramatically after Week 1. In contrast, CD8+ remained scarce until after 5-7 weeks of age in unweaned animals. Four days after weaning at 3 weeks old, there were increases in CD2+ (P < 0.001) and macrophage/granulocyte (P < 0.01) cells in proximal small intestinal villi and in CD2+ cells only (P < 0.01) in crypts. No significant changes in cell numbers were demonstrated in the distal small intestine.

Aging↗
PubMed1995

Presentation of soluble and bacterial antigens by milk-derived cells to unprimed bovine T cells in vitro.

The ability of cells isolated from bovine milk and peripheral blood to present soluble protein and particulate bacterial antigens to peripheral blood T lymphocytes was compared using a culture system which consistently supports antigen-specific, primary, proliferative responses. The present study shows that cells from blood and from milk can present antigen to unprimed T cells. Major histocompatibility complex class II restriction of the responses was demonstrated by abrogation of proliferation by the addition of anti-bovine class II monoclonal antibody to cultures. Although cells derived from blood or milk were shown to be capable of presenting antigen to T cells, differences in optimal culture conditions and kinetics of the resulting response were observed.

Animals↗
PubMed1995

Ultrastructural, histochemical and immunohistochemical features of porcine intestinal lamina propria macrophages, peripheral blood monocytes and splenic adherent cells.

Ultrastructural, histochemical and immunohistochemical features of porcine intestinal lamina propria macrophages (LPMs), peripheral blood fibronectin-adherent cells (FACs) and splenic-adherent cells (SPACs) were compared. Freshly isolated FACs and SPACs were small and showed small cytoplasmic processes, little evidence of endocytic vacuoles, few lysosomes and sparse rough endoplasmic reticulum (RER). Fresh FACs were negative for acid phosphatase, non-specific esterase (NSE) and beta-galactosidase activity. Of the SPACs, 20-40% were positive for acid phosphatase, < 5% for NSE and 5-10% for beta-galactosidase. Pre-cultured FACs and SPACs were large and showed an abundance of endocytic vacuoles; they possessed dilated and prominent RER and > 95% were positive for the three enzyme activities. LPMs exhibited abundant endocytic vacuoles or vesicles and lysosomes but sparse RER, and > 85% were positive for the three enzymes. LPMs (24%), FACs (49%) and SPACs (40%) expressed MHC (major histocompatibility complex) class II glycoproteins. Macrophage-granulocyte antigens were detected in LPMs (14%), FACs (50%) and SPACs (33%). The results thus suggest that freshly isolated FACs differ from LPMs morphologically and in enzymic features, and the differences may represent part of the cell maturation process.

Acid Phosphatase↗
PubMed1994

Characterization of monoclonal antibodies specific for monocytes, macrophages and granulocytes from porcine peripheral blood and mucosal tissues.

A panel of four monoclonal antibodies produced in our laboratory, MIL1, MIL2, MIL3, MIL4, and the type-specific monocyte/granulocyte marker 74-22-15 were used to isolate and to discriminate between monocytes, macrophages and granulocytes derived from porcine peripheral blood, lung and gut lamina propria. Two-colour flow cytometry and cell sorting showed that while no monoclonal antibody was specific for just a single cell population, each cell type had a unique and characteristic combination of surface antigens. These differences could be used to identify and purify monocytes, macrophages, neutrophils, eosinophils and basophils from the three different sites. The study also demonstrated similarities and differences within cell types from the same site and from different sites: polymorphonuclear neutrophils (PMN) from peripheral blood were subdivided into two subpopulations by the presence or absence of the surface antigen recognized by MIL4, while PMN from alveolar lavage did not express this antigen. Peripheral blood eosinophils were also divided into subpopulations by the presence or absence of the same surface antigen. Lamina propria eosinophils strongly expressed the MIL4 marker and differed morphologically from blood eosinophils. Peripheral blood basophils and lamina propria mast cells were morphologically similar and expressed similar antigens. Monocytes and alveolar macrophages also expressed the same surface antigens.

Animals↗
PubMed1994

SDS-PAGE separation of dermatophyte antigens, and western immunoblotting in feline dermatophytosis.

Water-soluble antigens liberated from the disrupted mycelium of nine dermatophytes (seven isolates of Microsporum canis, one each of Microsporum gypseum and Trichophyton mentagrophytes) were compared by analytical slab SDS-PAGE. No substantial differences were observed between the protein bands of the M. Canis isolates, but certain distinctive bands were apparent in the other two species examined. Western immunoblotting using M. canis-derived antigens separated by SDS-PAGE was used to investigate the humoral immune response in 79 cats with naturally-occurring dermatophytosis (72 with M. canis, six with M. gypseum and one with T. mentagrophytes) and this information was compared to results of immunoblots from 46 control (non-dermatophyte exposed) cats. Seven dominant bands (bands which occurred frequently and stained heavily) were identified in immunoblots from the dermatophyte-infected cats with apparent molecular weights varying between 39 and 120 kD. None of these bands were totally specific markers for dermatophytosis as a variable proportion of the control cats showed reactivity to all these proteins. However, most (73%) of the dermatophyte-infected cats showed reactivity to six or seven of the identified bands whereas most (80%) of the control cats showed reactivity to between zero and three of these bands (p < 0.005). Western immunoblotting could be used to select individual immunodominant antigens for further evaluation of protective (cell-mediated) immunity.

Animals↗
PubMed1994

MHC Class II positive cells and T cells in the equine endometrium throughout the oestrous cycle.

The quantity and distribution of MHC Class II positive cells and T cells in the equine endometrium was investigated throughout the oestrous cycle. Significantly more MHC Class II positive cells were detected in the stratum compactum and stratum spongiosum of endometria from naturally cycling mares during the follicular than during the luteal phase of the oestrous cycle. Significantly more T cells were also detected in the stratum compactum, but not stratum spongiosum, of these mares during the follicular phase. Furthermore, there was a marked increase in the number of MHC Class II positive cells and T cells in the endometria of ovariectomised mares treated with oestradiol compared with progesterone. An increase in the expression of MHC Class II antigen by endometrial epithelial cells was also observed in mares treated with oestradiol and in some naturally cycling mares during the follicular phase.

Animals↗
PubMed1994

Immunology of the porcine gastrointestinal tract.

The gastrointestinal immune system is presented with a contrasting array of antigens, ranging from harmless dietary components to highly pathogenic microorganisms. The mucosal immune system has the ability to recognise different groups of antigens and has evolved a battery of responses from which an appropriate response may be orchestrated. The question as to how the mucosal immune system categories antigens and selects a particular response is central to this process but it remains largely unanswered. The solution to this question is likely to hold the key to the development of safe and effective mucosal vaccines as well as suggesting methods for the prevention and control of allergic responses. Enteric diseases resulting from antigens (microbial and dietary) presented via the gastrointestinal tract are a major cause of morbidity and mortality. In addition to being of substantial economic importance, growing public awareness on animal welfare and food quality confirms the urgent need for new methods of disease control.

Animals↗