Biomedical subjects

F A Zuckermann

Publications and source records attributed to F A Zuckermann.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop.

As a result of the first-round cluster analysis, a panel of 16 novel monoclonal antibodies (mAbs) was assigned for detailed analysis to the CD45 subgroup of the Third International Swine CD Workshop. The specificity of the mAbs was initially determined by examining their reactivity with Chinese hamster ovary (CHO) cells engineered to express individual isoforms of porcine CD45. These analyses indicated that seven of the mAbs (PG77A, PG96A, PG167A, PGB78A, 3C/9, MIL13, NHT 101) recognized the portion of the CD45 molecule encoded by the A exon (CD45RA), while one (MIL15) was specific for that portion encoded by the C exon (CD45RC). In each case, the designation was supported by the demonstration that the molecular weight(s) of the recognized antigen(s) in porcine mononuclear cells, as determined by immunoprecipitation, corresponded to the predicted size(s) according to their specificity. As expected, a similar correlation was obtained for five standard mAbs whose specificity for either common or restricted epitopes of porcine CD45 had been established in previous workshops. Screening of the remaining 174 mAbs that comprised this workshop but were excluded from the CD45 subgroup by cluster analysis failed to detect any additional ones reactive with the porcine CD45-expressing cells.

Animals↗

PubMed2001

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Animals↗

PubMed2000

Reduction in inflammation following blockade of CD18 or CD29 adhesive pathways during the acute phase of a spirochetal-induced colitis in mice.

Colitis develops in mice infected with Brachyspira (Serpulina) hyodysenteriae. Numerous granulocytes (PMNs) are evident in cecal tissue sections 24-48 h post-infection. The role of PMNs was assessed by utilizing monoclonal antibodies specific for CD18 or CD29 to block PMN recruitment. Macroscopic lesions were less severe in mice treated with either monoclonal antibody compared to lesions observed in isotype control-treated mice. While these monoclonal antibodies may inhibit extravasation of other leukocytes, the central role of PMNs was further demonstrated in that colitis was reduced following neutrophil depletion. There was less edema and epithelial erosions in ceca of mice receiving anti-Ly6G, -CD18 or -CD29 monoclonal antibody compared to mice receiving the control. Moreover, there was a significant reduction in PMN infiltration in tissues of mice treated with anti-CD18. The reduction in infiltrating PMNs did not result from downregulation of neutrophil chemoattractant MIP-2 expression in anti-CD18-treated mice. In contrast, PMN recruitment into the cecum was apparently CD29-independent. It is noteworthy that the number of PMNs observed in anti-CD18-treated mice was significantly higher than observed in non-infected mice. The data provide evidence for a threshold number of PMNs necessary for lesion development and indicate that CD18, but not CD29, adhesive pathways are crucial for PMN recruitment in bacterial colitis.

Acute Disease↗

PubMed2000

Antigen-specific proliferation of porcine CD8alphaalpha cells to an extracellular bacterial pathogen.

A vaccine inducing protective immunity to a spirochaete-induced colitis of pigs predominantly stimulates expansion of CD8+ cells in vivo and in antigen-stimulated lymphocyte cultures. CD8+ cells, however, are rarely considered necessary for protection against extracellular bacterial pathogens. In the present study, pigs recovering from colitis resulting from experimental infection with Brachyspira (Serpulina) hyodysenteriae had increased percentages of peripheral blood CD4- CD8+ (alphaalpha-expressing) cells compared with non-infected pigs. CD8alphaalpha+ cells proliferated in antigen-stimulated cultures of peripheral blood mononuclear cells from B. hyodysenteriae-vaccinated pigs. Proliferating CD8alphaalpha+ cells consisted of CD4-, CD4+ and gammadelta T-cell receptor-positive cells. CD4- CD8alphabeta+ cells from vaccinated or infected pigs did not proliferate upon in vitro antigen stimulation. Of the CD8alphaalpha cells that had proliferated, flow cytometric analysis indicated that the majority of the CD4+ CD8+ cells were large (i.e. lymphoblasts) whereas the CD4- CD8+ cells were predominantly small. Addition of monoclonal antibodies (mAb) specific for either porcine major histocompatibility complex (MHC) class I or class II antigens diminished B. hyodysenteriae-specific proliferative responses whereas addition of mAb to porcine MHC II, but not porcine MHC I, reduced the CD8alphaalpha response. In vitro depletion of CD4+ cells by flow cytometric cell sorting diminished, but did not completely abrogate, the proliferative response of cells from vaccinated pigs to B. hyodysenteriae antigen stimulation. These results suggest that CD8alphaalpha cells are involved in recovery and possibly protection from a spirochaete-induced colitis of pigs; yet, this response appears to be partially dependent upon CD4+ cells.

Animals↗

PubMed1999

Extrathymic CD4/CD8 double positive T cells.

Mature T lymphocytes expressing the alphabeta T cell receptor are generally classified as either CD4+ or CD8+, based on the mutually exclusive expression of these two lymphocyte coreceptors. Contrary to this conventional division, there is considerable evidence that significant numbers of CD4/CD8 double positive (DP) lymphocytes exist in the peripheral blood and secondary lymphoid tissues of swine, chickens and monkeys. Although CD4/CD8 DP T cells are rarely present in human peripheral blood the relative percentage of this lymphocyte population can increase spontaneously in healthy individuals and in persons suffering from certain disease conditions. DP can also be found among those T cells infiltrating arthritic joints, rejected kidney grafts and certain tumors. In humans, and rats, CD4/CD8 DP T cells appear transiently following activation of their progenitors. Murine DP cells have been described as a subset of intraepithelial lymphocytes (IELs). However, the relationship of IELs to DP cells in the peripheral blood of other species is unknown. Because of their unconventional phenotype and rarity in human and mice, most immunologists have ignored extrathymic CD4/CD8 DP lymphocytes. Nevertheless, their abundance in the peripheral blood of swine, monkeys and chickens makes it impossible to dismiss this lymphocyte population. Here are reports that have described extrathymic lymphocytes exhibiting a CD4+CD8dim phenotype in several species reviewed. Swine and monkey lymphocytes with this phenotype are represented by small resting cells that simultaneously express CD4 and CD8alpha molecules. The available evidence favors the notion that such DP T cells in swine are comprised predominantly of MHC class II restricted memory CD4+ helper T cells that after activation have acquired the ability to express the CD8alpha chain and then to maintain this DP phenotype. Moreover, porcine CD4/CD8 DP T cells appear to be comprised of memory cells due to their ability to respond to recall antigen, resilience to thymectomy, increase in proportion with age, expression of memory T cell markers, production of interferon-gamma and localization to inflammatory sites. Some of these characteristics are also descriptive of human and monkey CD4/CD8 DP T cells. Thus, in swine, humans and monkeys, these phenotypically distinct lymphocytes appear to represent a primed T cell subset. The possible functional significance of the simultaneous expression of the CD4 and CD8 co-receptors on mature T cells is discussed.

Animals↗

PubMed1999

Cellular immune responses of pigs induced by vaccination with either a whole cell sonicate or pepsin-digested Brachyspira (Serpulina) hyodysenteriae bacterin.

Brachyspira (Serpulina) hyodysenteriae infection of pigs (swine dysentery) causes a mucohemorrhagic diarrhea resulting in significant economic losses for producers. A commercial vaccine consisting of a proteinase-digested bacterin has shown efficacy in the reduction of disease due to B. hyodysenteriae. Vaccines consisting of whole cell bacterins, however, generally fail to protect pigs from disease. In the present study, cellular immune responses induced by a proteinase-digested bacterin were compared to responses induced by a whole cell sonicate antigen preparation. In addition, usage of either squalene or Freund's incomplete adjuvants in combination with each antigen preparation was also compared. Both antigen preparations induced significant cellular immune responses as measured by in vitro (IFN-gamma production and T cell proliferation) and in vivo methods (DTH responses). No significant differences were detected in proliferative, interferon-gamma (IFN-gamma), or delayed type hypersensitivity (DTH) responses by pigs receiving either adjuvant or antigen preparation. T cells (CD3(+)) but not B cells from vaccinated animals proliferated in response to in vitro stimulation with B. hyodysenteriae antigen. CD8(+) (single positive and CD4/CD8 double positive) and gammadelta(+) T cells were particularly responsive. In addition, high percentages of both CD8 single positive and CD4/CD8 double positive cells were detected in antigen-stimulated cultures. These findings demonstrate the unique sensitivity of porcine CD8(+) T cells to priming for recall response by vaccination with a proteinase-digested B. hyodysenteriae bacterin.

Animals↗

PubMed1999

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin.

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-gamma (IFN-gamma) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-gamma responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-gamma responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-gamma responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-gamma production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.

Animals↗

PubMed1999

Molecular cloning and characterization of a novel CD1 gene from the pig.

Much effort is underway to define the immunological functions of the CD1 multigene family, which encodes a separate lineage of Ag presentation molecules capable of presenting lipid and glycolipid Ags. To identify porcine CD1 homologues, a cosmid library was constructed and screened with a degenerate CD1 alpha3 domain probe. One porcine CD1 gene (pCD1.1) was isolated and fully characterized. The pCD1.1 gene is organized similarly to MHC class I and other CD1 genes and contains an open reading frame of 1020 bp encoding 339 amino acids. Expression of pCD1.1 mRNA was observed in CD3- thymocytes, B lymphocytes, and tissue macrophages and dendritic cells. The pCD1.1 cDNA was transfected into Chinese hamster ovary cells, and subsequent FACS analysis demonstrated that mAb 76-7-4, previously suggested to be a pig CD1 mAb, recognizes cell surface pCD1.1. Structurally, the pCD1.1 alpha1 and alpha2 domains are relatively dissimilar to those of other CD1 molecules, whereas the alpha3 domain is conserved. Overall, pCD1.1 bears the highest similarity with human CD1a, and the ectodomain sequences characteristically encode a hydrophobic Ag-binding pocket. Distinct from other CD1 molecules, pCD1.1 contains a putative serine phosphorylation motif similar to that found in human, pig, and mouse MHC class Ia molecules and to that found in rodent, but not human, MHC class-I related (MR1) cytoplasmic tail sequences. Thus, pCD1.1 encodes a molecule with a conventional CD1 ectodomain and an MHC class I-like cytoplasmic tail. The unique features of pCD1.1 provoke intriguing questions about the immunologic functions of CD1 and the evolution of Ag presentation gene families.

Amino Acid Sequence↗

PubMed1999

Use of interleukin 12 to enhance the cellular immune response of swine to an inactivated herpesvirus vaccine.

Vaccination is the single most successful medical measure against infectious disease. However, the major barrier for achieving the full protective effect or immunization is how to render attenuated, killed, or subunit vaccines as immunogenic as the fully infectious versions of these microbes (Hughes and Babiuk, 1995; Rabinovich et al., 1994). In the case of PrV, infection with wild-type virus induces an immune response superior to vaccination with a live modified vaccine. After primary intranasal infection with wild-type PrV, the replication of a homologous secondary virus challenge is completely inhibited, and the much sought "sterile immunity" is generated (Kimman et al., 1994). In contrast, the immune response of pigs similarly exposed to PrV mutants, which have been attenuated by removal of the thymidine kinase (TK) and the envelope glycoprotein gE gene (McGregor et al., 1985; Zuckermann et al., 1988), is insufficient for preventing the replication of a homologous wild-type virus challenge (Kimman et al., 1994). Furthermore, inactivated PrV vaccines are even less effective at inducing protective immunity than are live modified PrV vaccines (de Leeuw and Van Orischot, 1985; Stellman et al., 1989; Vannier, 1985). The importance of inactivated and subunit vaccines resides in their stability and safety, since no infectious microbe is being introduced into the animal. However, because of the recognized lower effectiveness of inactivated vaccine types, they usually fall in disfavor when a modified live vaccine alternative is available. There is a critical need to develop strategies to enhance the immunogenicity of live, inactivated, and sub-unit vaccines for human and veterinary use (Hughes and Babiuk, 1995; Rabinovich et al., 1994). Although the inoculation of an animal with a virulent microbe is obviously not the desired method to produce sterile immunity, the immune response generated to infection with wild-type PrV clearly demonstrates that this type of immunity is possible. Research directed at devising strategies to increase the immunogenicity of different types of vaccines is necessary. Because of the wealth of information available on PrV immunity (reviewed by Chinsakchai and Molitor, 1994; Nauwynck, 1997), on PrV vaccines (Kimman et al., 1992, 1994; Mettenleiter, 1991; Scherba and Zuckermann, 1996) and increasingly on the porcine immune system (Lunney, 1993; Lunney et al., 1996; Saalmüller, 1995), the swine herpesvirus model is ideal for investigating the development of vaccine formulations with enhanced immunogenicity. Among the strategies currently being examined for the enhancement of the immunogenicity of inactivated and subunit vaccines is the use of recombinant cytokines administered together with antigen (Hughes and Babiuk, 1995; Rabinovich et al., 1994). The ability to regulate the development of an immune response by cytokines such as IL-12 provides the theoretical basis to use these cytokines as adjuvants to immunopotentiate the response to an inactivated vaccine. More importantly, it provides a model to investigate the mechanisms behind the induction of protective immunity and the components of a vaccine necessary for stimulating such a response. By providing cytokines such as IL-12 or IFN-gamma in combination with the vaccine inoculum, it is reasonable to expect that they will be able to direct the differentiation of T cells during the primary immune response. Modulation, in a predictable and desired manner of the quality and quantity of the induced protective immunity, should be achievable. The ability to manipulate a vaccine-induced immune response in the direction of a predominantly cellular response (Th1-like) instead of a predominantly humoral one (Th2-like) is perhaps best illustrated by the need to develop an effective vaccine against the porcine reproductive and respiratory syndrome (PRRS) virus, whose infectivity can be significantly enhanced in vitro and in vivo by antibody induced by vaccination

Animals↗

PubMed1999

Weaning anorexia may contribute to local inflammation in the piglet small intestine.

Compromising alterations in villus-crypt structure are common in pigs postweaning. Possible contributions of local inflammatory reactions to villus-crypt alterations during the weaning transition have not been described. This study evaluated local inflammatory responses and their relationship with morphological changes in the intestine in 21-d-old pigs (n = 112) killed either at weaning (Day 0) or 0.5, 1, 2, 4 or 7 d after weaning to either milk- or soy-based pelleted diets. Cumulative intake averaged <100 g during the first 2 d postweaning, regardless of diet. During this period of weaning anorexia, inflammatory T-cell numbers and local expression of the matrix metalloproteinase stromelysin increased while jejunal villus height, crypt depth and major histocompatibility complex (MHC) class I RNA expression decreased. Upon resumption of feed intake by the fourth d postweaning, villus height and crypt depth, CD8(+) T cell numbers, MHC class I RNA expression and local expression of stromelysin returned to Day 0 values. Together the results indicate that inadequate feed intake during the immediate postweaning period may contribute to intestinal inflammation and thereby compromise villus-crypt structure and function.

Animals↗

PubMed1999

Malnutrition modifies pig small intestinal inflammatory responses to rotavirus.

Infectious diarrheal diseases and malnutrition are major causes of child morbidity and mortality. In this study, malnutrition was superimposed on rotavirus infection in neonatal piglets to simulate the combined intestinal stress of viral enteritis in malnourished infants. Two-day-old piglets were assigned to three treatment groups as follows: 1) noninfected, fully nourished; 2) infected, fully nourished; and 3) infected, malnourished. Intestinal indices of inflammation were monitored over the subsequent 2-wk period. Intestinal damage and diarrhea were observed within 2 d of rotavirus infection and began to subside in nourished piglets by d 9 but persisted through d 16 postinfection in malnourished piglets. Rotavirus upregulated small intestinal expression of major histocompatibility complex (MHC) class I and class II genes; malnutrition intensified MHC class I gene expression and suppressed MHC class II expression. Jejunal CD4(+) and CD8(+) T-lymphocyte numbers were elevated for infected, nourished piglets on d 2, 9 and 16 postinfection. Malnutrition did not significantly affect the local expansion of T cell subsets in response to rotavirus. Intestinal prostaglandin E2 (PGE2) concentrations were elevated early after rotavirus infection independent of nutritional state. By d 9, PGE2 concentrations returned to baseline in infected, nourished piglets but remained elevated in malnourished piglets, corresponding to diarrhea observations. Together, the results identify intestinal indices of inflammation that are modulated by malnutrition and prompt reconsideration of current models of rotavirus pathophysiology.

Animals↗

PubMed1999

Effect of transportation and feed withdrawal on shedding of Salmonella typhimurium among experimentally infected pigs.

OBJECTIVE: To determine whether stress associated with transportation or feed withdrawal increased fecal shedding of Salmonella Typhimurium among pigs experimentally infected with the organism. ANIMALS: 86 healthy pigs. PROCEDURE: Pigs were challenge exposed with Salmonella Typhimurium at 4 weeks old and reared conventionally. When pigs reached market weight, they were assigned to groups and subjected to various combinations of transportation and feed withdrawal. Ileocecal contents were collected after slaughter and tested for Salmonella Typhimurium. RESULTS: Salmonella Typhimurium was not detected in feces collected from pigs just prior to slaughter. When feed was withheld for 24 hours prior to slaughter, the proportion of transported pigs with Salmonella Typhimurium in ileocecal contents at the time of slaughter was not significantly different from the proportion of nontransported pigs. However, when feed was not withheld prior to slaughter, the proportion of transported pigs with Salmonella Typhimurium in ileocecal contents at the time of slaughter was significantly higher than the proportion of nontransported pigs. CONCLUSIONS AND CLINICAL RELEVANCE: When carrier pigs remained on feed, transportation stress increased the proportion positive for Salmonella sp. On the basis of results reported here, it is suggested that producers withhold feed from pigs for 24 hours prior to transportation to a slaughter plant.

Animal Feed↗

PubMed1998

Interleukin-12 enhances the virus-specific interferon gamma response of pigs to an inactivated pseudorabies virus vaccine.

Cell-mediated immunity is a major component of the host defense system against viral infections. Since interleukin (IL)-12 has been shown to be a potent stimulus for the in vivo generation of interferon-gamma (IFN-gamma)-producing T cells (i.e. Th-1 cells) in laboratory animals, we evaluated the effect of IL-12 on the cellular immune response of pigs to vaccination against pseudorabies virus (PrV), a herpesvirus of swine. The magnitude of the cellular immune response was measured by IFN-gamma ELISPOT analysis of peripheral blood mononuclear cells (PBMC) from pigs which had been immunized twice, at 2-week intervals, with either, modified live virus (MLV) alone or with a commercial inactivated PrV vaccine with or without the coadministration of human recombinant IL-12 (HrIL-12). No significant differences in the titer of virus-neutralizing antibodies or in the intensity of the virus-specific lymphoproliferative response among the different treatment groups was observed. However, the number of virus-specific IFN-gamma-producing cells among PBMC isolated from animals receiving the MLV vaccine was on average 3.5 times more than animals immunized with the inactivated vaccine (P = 0.01). Administration of the inactivated vaccine and IL-12 induced a two-fold higher frequency of virus-specific IFN-gamma-producing cells from that induced by the inactivated vaccine alone (P < 0.05). Despite this enhancement, the level of protection from lethal PrV challenge provided by the inactivated vaccine in combination with IL-12 was the same as that induced by the inactivated vaccine alone. Both of these vaccination regimes provided significantly lower levels of protection than those afforded by the MLV vaccine. This study demonstrates that an inactivated PrV vaccine is a poor inducer of virus-specific IFN-gamma-producing cells and that this response can be enhanced by administration of exogenous IL-12. The data provides evidence of a dichotomy in the humoral and cellular immune responses of pigs to a viral antigen and implies the existence of a Th-1/Th-2 type regulation of the anti-viral immune response in pigs.

Adjuvants, Immunologic↗

PubMed1998

Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen.

Five monoclonal antibodies (mAbs) specific for porcine interferon-gamma (PoIFN-gamma) were isolated and utilized to develop a PoIFN-gamma sandwich ELISA. Specific reactivity of each mAb with E. coli derived recombinant PoIFN-gamma, but not with rPoIL-2 or rPolL-10, was confirmed in an indirect ELISA and in Western blots. Competitive ELISAs showed that mAbs P2A4 and P2C11 bound an epitope which was not recognized by mAbs P2G10, P1B7 or P2F6. The latter three mAbs were able to neutralize the ability of natural and recombinant PoIFN-gamma to induce the de novo expression of class II MHC antigens on porcine endothelial cells. To simplify the detection of biologically active porcine IFN-gamma, a sandwich ELISA was developed using the mAb P2G10 as a capture antibody and mAb P2C11 as the detecting reagent. The sensitivity of the assay for PolFN-gamma ranged from 1 to 50 ng/ml. Peripheral blood mononuclear cells (PBMC) from all pigs tested produced IFN-gamma when stimulated with either mitogen (PHA) or superantigen (SEB). In contrast, only PBMC obtained from pigs which had previously been vaccinated against PrV produced IFN-gamma in response to stimulation with this virus. Interestingly, cultures with the highest lymphoproliferative response did not necessarily have the highest level of IFN-gamma production.Furthermore, for recall viral antigen, the lymphoproliferative response decreased with time after immunization, whereas the IFN-gamma response increased. Thus, measurement of IFN-gamma production appears to be a good indicator of anti-viral immunological memory.

Animals↗

PubMed1998

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop.

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8+ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8+ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8low and CD8high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8low and CD8high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4-/CD8high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8+ lymphocyte subsets in health and disease.

Animals↗

PubMed1998

Analysis of monoclonal antibodies that recognize gamma delta T/null cells.

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine gamma delta TcR established the majority of null cells are gamma delta T cells. Use of this mAb in further comparisons demonstrated the gamma delta T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2- gamma delta T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.

Animals↗

PubMed1998

Definition of the specificity of monoclonal antibodies against porcine CD45 and CD45R: report from the CD45/CD45R and CD44 subgroup of the Second International Swine CD Workshop.

Swine cell binding analyses of a set of 48 monoclonal antibodies (mAbs), including eleven standards, assigned to the CD44 and CD45 subset group of the Second International Swine CD Workshop yielded 13 clusters. Although none of these corresponded to CD44, seven mAbs formed a cluster which was identified as being specific for restricted epitopes of CD45 (CD45R). In addition, a T-cell subset specific cluster comprised of four mAbs was also identified. Two mAbs (STH106 and SwNL 554.1) reacted exclusively with CD8 bright lymphocytes, the other two (2B11 and F01G9) with a subset of CD4 lymphocytes. The other 10 clusters were either specific for MHC-class I like molecules or overlapped with clusters identified by the adhesion molecule subgroup and are therefore just briefly discussed in this report. The specificity of all the mAbs in the CD45R cluster was verified by their ability to immunoprecipitate distinct proteins and to react with CHO cells expressing individual isoforms of CD45. Three CD45R mAbs (3a56, MIL5, -a2) did react with a 210 kDa isoform(s), while another three (STH267, FG2F9, 6E3/7) only recognized a 226 kDa isoform(s). The remaining one (MAC326) precipitated both a 210 and 226 kDa protein. The specificity of all the mAbs in the CD45R cluster, and of the CD45 common mAbs, was confirmed by their reactivity with CHO cells transfected with cDNAs encoding the extracellular and transmembrane portions of distinct CD45R isoforms. Those mAbs recognizing a 210 kDa protein reacted with CHO cells expressing the CD45RC isoform, while those capable of precipitating a 226 kDa, but not the 210 kDa, polypeptide recognized CHO cells expressing either the CD45RAC and the relatively rare CD45RA isoform. MAC326 was unique in its inability to react with CHO cells engineered to produce the CD45RC and CD45RAC isoforms. Thus, three mAbs (6E3/7, STH267, and FG2F9) appear to be specific for an epitope(s) encoded by the A exon, while one (MAC326) recognizes a determinant encoded by the C exon. The remaining three mAbs (3a56, -a2, MIL5) are apparently specific for an epitope(s) which results from the fusion of the C exon to the invariant leader sequence and is destroyed by inclusion of the A exon. All three CD45 common mAbs, K252.1E4, MAC323 and 74.9.3, did react with the CHO cells lines expressing either the CD45RA, CD45RC, CD45RAC or CD45RO isoforms, but not with untransfected CHO cells. When the natural expression of CD45 isoforms was examined by reacting lymphocytes with CD45R mAbs, a high level expression of isoforms containing the A exon-generated domain was detected in all B cells while the majority of CD4+ T cells had undetectable or lower expression density of this protein than B cells. In contrast, the density of expression of the CD45 isoform(s) containing the C exon-generated domain ranged from undetectable to high in CD4+ T cells whereas the amounts were approximately ten-fold lower in B cells. Overall this panel of CD45 mAbs will be very useful in analyzing the maturation and differentiation of swine lymphoid cells subsets.

Animals↗

PubMed1998

Determination of the specificity of CD45 and CD45R monoclonal antibodies through the use of transfected hamster cells producing individual porcine CD45 isoforms.

The exclusive presence of the tyrosine phosphatase, CD45, on the surface of hematopoietic cells coupled with the differential expression of its various isoforms has enabled the selection of lymphocyte subsets based on their reactivity with monoclonal antibodies (mAbs) specific for one or more CD45 species. As a prelude to defining the specificity of anti-porcine CD45 mAbs for this purpose, Chinese hamster ovary cells were transfected with constructs containing cDNAs encoding the extracellular and transmembrane domains of four pig CD45 isoforms. Cells expressing only one of the three predominant types (CD45RO, CD45RC, and CD45RAC) or the minor species (CD45RA) of porcine CD45 on their surface were sorted based on positive reactivity with the CD45 mAb K252.1E4. Initially, these CD45+ cells were used as a source of antigen when determining the specificities of nine mAbs, which had been identified during the First and Second International Swine CD Workshops as being reactive with porcine CD45. Later, cloned cell lines were established and phenotypically verified for the production of the correct CD45 transcript by RT-PCR. Binding of two more mAbs (74-9-3 and 10-14-1) in addition to the original mAb panel to these cell lines was assessed by using a cell ELISA in lieu of one-color flow cytometry. Despite differences in detection methodology, identical mAb binding results were obtained. As anticipated, CD45 mAbs K252.1E4, MAC 323, and 74-9-3 which recognize an epitope(s) in the common portion of porcine CD45, reacted with cells expressing any one of the four isoforms but not with the parental CHO cells. In contrast, none of the restricted (CD45R) mAbs bound to cells producing the CD45RO isoform which lacks any of the alternate extracellular regions. However, three of these mABs (6E3/7, FG2F9 and STH267) did react specifically with the CD45RA and CD45RAC isoforms, indicating their specificity for an epitope(s) encoded by the CD45 A exon. The other four CD45R mAbs (MAC326, 3a56, MIL5, and -a2) recognized the CD45RC isoform. Interestingly, only CD45R mAb MAC326 also bound to cells expressing the CD45RAC isoform, suggesting that the epitope(s) recognized by the other three may have arisen due to the juncture of the invariant 5' leader sequence with the CD45C exon. The eleventh mAb (10-14-1) was unique in that it did not react with any of the expressed CD45 isoforms. This inability coupled with the previously demonstrated recognition of a 240 kDa protein suggests that it may be specific for CD45RABC. Overall, this panel of CD45R mAbs should prove useful for obtaining functionally distinct subpopulations of B and T lymphocytes.

Animals↗