Biomedical subjects

B M Goddeeris

Publications and source records attributed to B M Goddeeris.

106 records · Page 6Linked to original sources

PubMed1987

Bovine mononuclear phagocytic cells: identification by monoclonal antibodies and analysis of functional properties.

Monoclonal antibodies (mAb) which react with bovine monocytes have been produced. These include three mAb (P8, IL-A22 and IL-A24) that recognize the majority of monocytes and granulocytes in peripheral blood; two of these mAb were also shown to react with 30-40% of cells in bone marrow, including both monocytic and granulocytic cells, and with variable percentages of tissue macrophages. Thus these mAb can act as markers for myeloid cells in haemopoietic tissues and for monocytes in cell populations devoid of granulocytes. A further two mAb (IL-A23 and IL-A25) recognize monocytes and/or macrophages. The reactivity of one of these mAb (IL-A25) appears to be mainly restricted to pulmonary macrophages. The other mAb reacts with a variable proportion of blood monocytes and generally with a higher percentage of tissue macrophages, suggesting that its expression may relate to activation or maturation of monocytes. In order to study the functional properties of peripheral blood monocytes, techniques were developed for obtaining populations of peripheral blood mononuclear cells (PBM) depleted of monocytes to less than 0.2% and monocyte populations of greater than 97% purity. Removal of monocytes from PBM abrogated the capacity of the cells to proliferate in response to Con A and PBS, although addition of 2-mercaptoethanol to the cultures restored proliferation. In both allogeneic and autologous mixed leukocyte cultures (MLC), monocytes were required in the stimulator cell populations for induction of the proliferative responses, and both responses could be elicited with purified monocytes. However, proliferation in the autologous MLC occurred only with responder cell populations that were depleted of monocytes. Moreover, it was shown that addition of more than 5% unirradiated monocytes to the autologous MLC suppressed proliferation. These findings indicate that monocytes play an important role in the induction and regulation of cellular immune responses in cattle. Two of the mAb that react with monocytes and granulocytes were tested for their capacity to inhibit proliferative responses of PBM to mitogens, alloantigens or the soluble antigen, KLH.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

The bovine autologous mixed leukocyte reaction: a proliferative response of non-T cells under the control of monocytes.

The culture of bovine monocyte-depleted peripheral blood mononuclear cells (PBM) (responder) with irradiated autologous undepleted PBM (stimulator) resulted in a strong proliferative response, which was called the bovine autologous mixed leukocyte reaction (MLR). A comparative study was made between this autologous MLR and the bovine allogenic MLR, with respect to the inductive requirements and the nature of responding and stimulator cells. In both the autologous and allogenic MLR, the monocyte was responsible for inducing proliferation: low concentrations of purified monocytes (greater than 95% pure) induced strong proliferative responses, while monocyte-depleted PBM (less than 0.2% monocytes) were unable to induce proliferation. Both MLRs were blocked by monoclonal antibodies specific for class II antigens of the major histocompatibility complex. The magnitude of the autologous MLR was regulated by monocytes: unirradiated monocytes induced or suppressed autologous proliferative responses depending on their concentration in the culture. Proliferating cells, identified by quantifying DNA with chromomycin A3, were all within the population of large cells. In the allogenic MLR, 50% of these large cells were T cells, as defined by monoclonal antibodies. In contrast, all of the proliferating cells in the autologous MLR were non-T cells.

Animals↗

PubMed1987

Bovine helper T-cell clones specific for lymphocytes infected with Theileria parva (Muguga).

T-cell clones specific for lymphocytes infected with Theileria parva were derived from animals immunized by infection with T. parva (Muguga). These clones were non-cytolytic and had the BoT4+ BoT8- surface phenotype, BoT4 and BoT8 being the bovine analogues of human CD4 and CD8 molecules. The clones proliferated in response to irradiated autologous lymphoblasts infected with T. parva (Muguga) but not to autologous uninfected lymphoblasts or monocytes. They were parasite strain-specific, in that they did not respond to autologous lymphoblasts infected with another parasite stock, T. parva (Marikebuni). The clones proliferated in the absence of exogenous T-cell growth factor (TCGF) and produced TCGF when stimulated with concanavalin A. Induction of proliferation of the cloned T-cells was genetically restricted, and evidence was obtained which indicated that they were restricted by determinants on class II major histocompatibility complex (MHC) molecules. These findings demonstrate that infections with T. parva stimulate antigen-specific MHC-restricted T-cells with the properties of T-helper cells. The results also provide further evidence for the expression of a parasite strain-specific antigen on the surface of T. parva-infected lymphocytes.

Animals↗

PubMed1987

Cytotoxic T-cells elicited in cattle challenged with Theileria parva (Muguga): evidence for restriction by class I MHC determinants and parasite strain specificity.

The MHC restriction and parasite strain specificity of cytotoxic cells elicited in a group of Theileria parva (Muguga)-immunized cattle following homologous challenge, were investigated. The cytotoxic cells were specific for parasitized target cells and in 9 of the 10 animals examined, they were clearly genetically restricted. Cytotoxicity could be inhibited by monoclonal antibodies (MoAb) to class I MHC molecules but not by MoAb to class II molecules, indicating that a large component of the response was restricted by class I MHC determinants. Low levels of inhibition of cytotoxicity were also obtained with a MoAb to the T-cell subset marker BoT8, suggesting that at least part of the response was mediated by BoT8+ lymphocytes. When cytotoxic cells from individual cattle were assayed on panels of parasitized target cells, there was a close correlation between susceptibility of the target cells to lysis and sharing of BoLA-A locus-encoded specificities with the effectors. This observation, taken together with the knowledge that within several of the sets of BoLA-A-matched targets the relevant BoLA-A specificities were on different MHC haplotypes, indicated that the responses were restricted predominantly by BoLA-A products. In individual cattle there was a striking bias in the restriction of the response to one or other BoLA-A specificity. Among the six specificities represented, responses restricted by w6, w8 and KN18 consistently predominated over responses restricted by w7, w10 and w11. In the three cattle tested for parasite strain specificity, two showed complete specificity and one partial specificity for cells infected with the parasite stock used for immunization, T. parva (Muguga).

Animals↗

PubMed1987

The bovine autologous Theileria mixed leucocyte reaction: influence of monocytes and phenotype of the parasitized stimulator cell on proliferation and parasite specificity.

In the autologous Theileria mixed leucocyte reaction (MLR), irradiated Theileria parva-infected cells induce proliferative responses in autologous peripheral blood mononuclear leucocytes (PBM), irrespective of the immune status of the donor animal. In this paper we have analysed the cellular basis of this response in naive and immune cattle to determine the Theileria specificity of the response. The magnitude of proliferation is dependent on two parameters, namely the presence or absence of monocytes in the responder population, and the phenotype of the parasitized stimulator cells, both of which appeared to be independent of the immune status of the donor animal. Monocyte-depleted responders invariably gave stronger proliferative responses but generated cytotoxicity from immune cattle that tended to be less genetically restricted. Marked differences were observed in the stimulatory capacity of cloned parasitized T-cell and non-T cell lines. At least part of this variation was associated with differences in the capacity of the parasitized cells to secrete soluble suppressive factors and possibly also stimulatory factors. Two observations indicated that, in immune cattle, part of the proliferative response in the autologous Theileria MLR is parasite-specific. First, stimulator cells fixed with glutaraldehyde stimulated proliferative responses in monocyte depleted PBM from immune animals but not naive animals. Second, in autologous Theileria MLRs with intact PBM, genetically restricted cytotoxic cells were generated from immune but not naive animals. While monocytes seem not to be required for induction of the parasite-specific component of the response, their absence from the assay when viable stimulator cells are utilized appears to enhance the non-specific component of the proliferative and cytotoxic responses.

Animals↗

PubMed1987

A new method for fixation and preservation of trypanosomal antigens for use in the indirect immunofluorescence antibody test for diagnosis of bovine trypanosomiasis.

A new method for fixation of trypanosomes for use in the indirect immunofluorescent antibody test (IFAT) is described. The method involves fixation of live trypanosomes, in suspension, using a mixture of 80% cold acetone and 0.25% formalin in saline. The fixed trypanosomes were stored in suspension at -60 degrees C, 4 degrees C or at room temperature for at least one year without loss of antigenicity. Using trypanosomes prepared this way as antigens in IFAT, species-specific antibodies were detected within 2 weeks of infection in sera of cattle infected with T. brucei or T. vivax. Thereafter, antibodies recognizing antigens common to the two species as well as T. congolense were detected. The antibody levels to common antigens of the three species declined 2-3 months post-infection, leaving only the species-specific antibodies. The sera obtained from T. congolense infected animals, did not react with T. vivax or T. brucei antigens. The non-specific fluorescence commonly associated with this assay was eliminated by prior absorption of the test sera with normal bovine lymphocyte lysate. This treatment of serum did not affect the specific antibodies to trypanosomal antigens. Analysis of bovine sera prepared from cattle in Kisiwani and Muhaka, along the Kenya coast, using the fixed trypanosomes revealed that some animals had antibodies to one trypanosome species only while others had antibodies to 2 or all 3 trypanosome species.

Animals↗

PubMed1986

Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4.

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.

Animals↗

PubMed1986

Alloreactive bovine T lymphocyte clones: an analysis of function, phenotype, and specificity.

A bovine alloreactive cell population was subjected to complement-dependent lysis with monoclonal antibody (mAb) IL-A11. The original population and the population depleted of cells bearing the determinant recognized by mAb IL-A11 were cloned. Parent cultures and 21 clones were examined for cytolytic function and for expression of determinants recognized by mAb IL-A11 and two additional mAb, IL-A12 and IL-A17. Clones could be classified according to maximal achievable levels of cytolysis by using Theileria parva-infected bovine lymphoblastoid target cells. In this way, three groups were identified--one capable of high level cytolysis, one of intermediate levels, and one group comprising apparently noncytolytic clones. The clones in the first group reacted with mAb IL-A17; those in the second and third groups, with mAb IL-A11 and IL-A12. It was shown that cytotoxicity effected by IL-A17+ clones could be inhibited by this mAb and also by a mAb directed to MHC class I determinants on target cells. Conversely, cytotoxicity effected by IL-A11+/IL-A12+ clones could be inhibited by mAb IL-A11 and by a mAb directed to MHC class II determinants on target cells. The levels of expression of class I and class II determinants on target cells correlated with the levels of killing by clones of the IL-A17+ phenotype and clones of the IL-A11+/IL-A12+ phenotype, respectively. The results indicate that cytotoxic bovine T lymphocyte clones specific for class I MHC antigens and both cytotoxic and noncytotoxic clones specific for class II MHC antigens can be obtained. Further, their specificity for class I or class II antigens can be determined by phenotyping with mAb.

Animals↗

PubMed1986

Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells. Functional evaluation of monocytes in responses to lectins.

We have compared different techniques for the enrichment and depletion of monocytes from bovine peripheral blood mononuclear cells. Adherence to plasma-coated gelatin was the most efficient and reproducible method for enrichment of monocytes (80% monocytes), whereas depletion of peripheral blood mononuclear cells of monocytes (0.3% monocytes and less) was best achieved by defibrination of the blood from which the PBM were separated. In both instances, purity of the cell population could be improved further by an additional step, namely, FACS sorting with a monocyte-specific monoclonal antibody to purify monocytes (97% monocytes and more), and adherence to polystyrene to remove residual monocytes from defibrinated PBM (0.1% monocytes and less). Depletion of monocytes abolished the response of PBM to concanavalin A and phytohaemagglutinin. The lectin-induced response could be restored by adding gelatin/plasma purified monocytes. This activity of monocytes could be replaced by 2-mercaptoethanol.

Animals↗

PubMed1986

Generation of bovine cytotoxic cell lines, specific for cells infected with the protozoan parasite Theileria parva and restricted by products of the major histocompatibility complex.

Cytotoxic cell lines specific for cells infected with the protozoan parasite Theileria parva were generated in vitro by repeated stimulation of peripheral blood mononuclear cells (PBMC) from immune animals with autologous parasitized cells from continuously growing cell lines. In PBMC of 5/7 immune animals tested, maximal levels of killing (80-100%) were obtained at effector to target ratios of 10/1 following 3 to 5 stimulations. No killing of uninfected lymphoblasts was observed. Cytotoxic activity was contained in the BoT4- lymphocyte population. The results of analyses of cytotoxicity on panels of target cells of diverse major histocompatibility (MHC) phenotypes indicated that the effectors were restricted by class I MHC antigens. Only autologous targets and allogenic targets sharing one or other bovine lymphocyte antigen A locus-encoded specificity with the cytotoxic cell line were killed. In addition, two of the three cytotoxic cell lines, when tested on targets infected with different parasite stocks, exhibited a high degree of specificity for the stock used for immunization and stimulation of the cultures. The capacity to generate highly cytotoxic, MHC-restricted effectors from peripheral blood was dependent on the immune status of the donor animals. This was confirmed by comparing cytotoxicity generated from the same animal before and after immunization using the same autologous infected cell line as a stimulator. These results indicate that these effector cells are analogous to the cytotoxic cells detected in vivo in cattle undergoing immunization or challenge with T. parva.

Animals↗

PubMed1986

Monoclonal antibodies identify phenotypically and functionally distinct cell types in the bovine lymphoid system.

Monoclonal antibodies were produced against bovine lymphoid cells. The reactivities of the antibodies for membrane determinants were examined on both cell suspensions and cryostat tissue sections prepared from bovine blood, thymus, spleen and lymph nodes. The antibodies were putatively grouped into sets which reacted with monomorphic and polymorphic determinants associated with bovine class I and class II major histocompatibility complex (MHC) antigens (MAbs P12 and P3, and R1 and P2 respectively), or associated with differentiation antigens expressed on T cells and monocytes (MAb P5) or exclusively on monocytes (MAb P8). The antibodies were used to identify the surface phenotypes of cells which stimulate (R1+ P5+ P8+) and proliferate (R1- P5+ P8-) in the bovine mixed leukocyte cultures, and cells which proliferate in response to the mitogen, concanavalin A (R1- P5+).

Animals↗

PubMed1986

Bovine cytotoxic T-cell clones specific for cells infected with the protozoan parasite Theileria parva: parasite strain specificity and class I major histocompatibility complex restriction.

We present information on the specificity of three bovine cytotoxic T-cell clones reactive with lymphoblasts infected with the protozoan parasite Theileria parva. The clones were derived from peripheral blood mononuclear cells of an animal immunized with T. parva (Muguga stock), after five stimulations in vitro with an autologous parasitized cell line. The three clones belonged to the BoT8+ subset of T cells, which is similar to the human CD8+ T-cell subset. On the basis of analysis on a panel of infected target cells originating from cattle of different major histocompatibility complex (MHC) phenotypes, killing by all three clones was found to be restricted to targets bearing the class I MHC specificity KN104, which is defined by alloantiserum KNA104 and monoclonal antibody IL-A4. This class I MHC restriction was confirmed by blocking of target cell lysis with these antibodies and monoclonal antibody w6/32, which reacts with a nonpolymorphic determinant on bovine class I MHC molecules. The three clones were parasite strain specific, in that they did not kill cells of the appropriate MHC type infected with T. parva (Marikebuni stock). These findings, taken together with previous observations that immunization of cattle with T. parva (Muguga) does not provide protection against challenge with T. parva (Marikebuni), suggest that the cytotoxic T cells recognize a cell surface antigen that may be important in induction of immunity to the parasite.

Animals↗

PubMed1986

Characterization by a monoclonal antibody and functional analysis of a subset of bovine T lymphocytes that express BoT8, a molecule analogous to human CD8.

Monoclonal antibody (mAb) IL-A17 characterizes a subset of bovine T lymphocytes. IL-A17 recognizes a 34,000-35,000 MW doublet, designated BoT8, which is expressed on the surface of approximately 20% of peripheral blood mononuclear leucocytes (PBM), a subpopulation of lymphocytes in T-dependent areas of lymph nodes and spleen, and about 70% of thymocytes. This molecule is not expressed on B lymphocytes, monocytes/macrophages or granulocytes. Double labelling of PBM showed that the BoT8+ population is distinct from the T lymphocyte subset expressing BoT4. BoT8+ lymphocytes purified with a fluorescence-activated cell sorter (FACS) proliferated poorly in response to mitogenic and alloantigenic stimulation in the absence of exogenous growth factors. IL-A17 had no inhibitory effect on proliferation of PBM to mitogens (Con A and PHA) or alloantigens and no measurable effect on the in vitro generation of cytolytic effector cells. However, in some experiments IL-A17 was found to block partially allospecific cytolytic function mediated by bulk effector cell populations when included in 51Cr-release assays. Fractionation of effector cells generated in an allogeneic mixed leucocyte culture (MLC) demonstrated that cytotoxic cells specific for class I major histocompatibility complex (MHC) antigens reside within the BoT8+ population. Based on these data, and information reported elsewhere on alloreactive bovine T-cell clones, BoT8 is considered to be analogous to CD8 in humans and equivalent molecules in other species.

Animals↗

PubMed1985

Bovine alloreactive cytotoxic cells generated in vitro: target specificity in relation to BoLA phenotype.

Cytotoxic cells of bovine origin were generated in primary MLC using stimulator cells of BoLA w8/w11 phenotype. Bovine lymphoblasts transformed by the protozoan parasite Theileria parva parva acted as target cells in studies of the specificity of cytotoxicity. When responder cells in MLC did not share w8 or w11 with stimulator cells, cytotoxicity was evident with all targets bearing w8 or w11, or both, and was almost entirely restricted to these products of the BoLA-A locus. When responder and stimulator cells shared both w8 and w11, cytotoxicity was also generated. Whether this was specific for the products of other putative Class I loci in cattle, or for the products of a Class II region, remains to be determined. These results suggest that the determinants recognized by appropriately generated bovine alloreactive cytotoxic cells are identical with, or closely related to, determinants characterized by BoLA w8 and w11 defining alloantisera.

Animals↗

PubMed1985

New approaches to immunization of cattle against Theileria parva.

The protozoan parasite Theileria parva is the causal agent of an acute, usually fatal disease of cattle characterized by widespread parasitism and destruction of cells of the lymphoid system. Immunity against the parasite, can be induced by various infection and treatment regimes. Because of the necessity to use live organisms, these methods of immunization are cumbersome and there is the added problem of parasite strain heterogeneity. Present evidence suggests that immunity may operate at two levels of the infection, namely, the sporozoite and the macroschizont-infected cell. Antibodies with the capacity to neutralize the infectivity of sporozoites have been produced and have been used to identify a sporozoite surface antigen of approximately 68,000 daltons. The feasibility of using such an antigen for immunization is currently being explored. In cattle undergoing immunization against T. parva, cytotoxic T cells are produced which are specific for parasitized target cells and are restricted by class I major histocompatibility antigens. Similar effectors can now be generated and maintained in vitro and work is underway to obtain cloned populations of these cells. It is hoped that such effector cells may be of use, in conjunction with molecular biological techniques, in identification of the target antigens on the surface of parasitized lymphocytes.

Animals↗

PubMed1982

Indirect fluorescent antibody test for experimental and epizootiological studies on East coast fever (Theileria parva infection in cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension.

A schizont antigen for the indirect fluorescent antibody test against Theileria parva was prepared from a T parva-infected bovine lymphoblastoid cell line by fixing the cells in suspension with a mixture of acetone and formaldehyde. The antigen was stored in suspension in phosphate buffered saline for one and a half years at -60 degrees C without loss of activity; the antigen could also be lyophilised. The fluorescence of the intracellular schizonts was bright and specific with T parva positive bovine control serum and absent with negative bovine control serum and Theileria mutans positive bovine control serum. Fluorescence of the lymphoblastoid cell itself was observed with Trypanosoma brucei positive control serum and some bovine test sera: this fluorescence, which masked the intracellular schizonts, was eliminated by absorbing the sera in the supernatant of sonicated lymphocytes obtained from bovine lymph nodes. The antigen was evaluated with sera from cattle experimentally infected with T parva. In an epizootiological study on East Coast fever in the Coast Province of Kenya, there was good correlation between the serological responses of cattle to T parva schizont antigen and the distribution of Rhipicephalus appendiculatus ticks.

Animals↗