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M J Ratcliffe

Publications and source records attributed to M J Ratcliffe.

At least 37 records · Page 2Linked to original sources
PubMed1994

Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different position within FhuA. Immunoblotting of outer membranes with anti-FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.

Amino Acid Sequence↗
PubMed1994

Survivors of bursal B cell production and emigration.

The bursa of Fabricius is the site of primary B cell generation in the chicken. Analysis of the rate of bursal cell emigration demonstrated that about 1% of the blood B cell pool was replaced per hour by bursal emigrants. Surgical bursectomy distinguished three populations of blood B cells. About 60% of blood B cells in 3-wk-old chickens were short lived, with a lifespan of 2 to 3 d, and included most bursal emigrants. These cells migrate directly from the bursal follicular cortex to the periphery, express the LT2 antigen, and are proposed to represent a diversified repertoire of B cell specificities that have emigrated from the bursa in the absence of interaction with environmentally derived antigens in the follicular medulla. About 35% of blood B cells were much longer lived cells, having a lifespan exceeding 2 wk. These cells represent about 10% of bursal emigrants, which do not express the LT2 antigen and do not divide in the peripheral blood following emigration from the bursa. This population may represent cells that have undergone positive selection in the bursa by interacting with environmentally derived antigens and should, therefore, contain a restricted repertoire of B cell specificities. The third population, about 5% of blood B cells, were short-lived cells that represent the progeny of postbursal B cell production. The frequency of these cells progressively increases with time, taking over from the short-lived bursal emigrants as the bursa involutes, likely as a diversified repertoire of B cell specificities.

Animals↗
PubMed1993

Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T.

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.

Animals↗
PubMed1993

Bursa-dependent subpopulations of peripheral B lymphocytes in chicken blood.

By selective labeling of juvenile chicken bursal cells with colloidal fluorescein isothiocyanate in situ, the emigration rate of bursal lymphocytes to the periphery was estimated at approximately 0.84% and 0.96% of the peripheral blood lymphocyte (PBL) and splenic B cell pool per hour, respectively. Emigrant bursal cells were found primarily in blood and spleen, with very small numbers migrating to thymus, bone marrow, and gut-associated lymphoid tissues. Emigrant bursal cells expressed high levels of both major histocompatibility complex class II antigen and the Ov alloantigen, a phenotype found on a population comprising approximately 4% of bursal cells from which the bursal emigrants may be derived. Surgical bursectomy at 3 weeks of age revealed that peripheral blood B cells could be divided into three distinct populations. Specifically, 60% of the peripheral blood B cells were short lived with a half-life of about 30 h in the blood. These cells accounted for the great majority of emigrants from the bursa to the peripheral blood. Approximately 35% of PBL B cells had a half-life of 12 days following bursectomy and comprised cells which did not divide in the periphery. Consequently, we propose that physiological differences between this population and the majority of bursal emigrants are established intrabursally. The remaining PBL B cells, whose relative proportion increases with age from about 5% of PBL B cells at 2-3 weeks of age, are short lived and are being continually produced from (a) post-bursal site(s) of B cell production.

Animals↗
PubMed1993

Polymorphism of the functional immunoglobulin variable region genes in the chicken by exchange of sequence with donor pseudogenes.

We have isolated a number of new allelic variants of the unique functional genes encoding chicken immunoglobulin heavy and light chain variable regions (VH1 and VL1, respectively). The distribution and nature of nucleotide variation among these and previously identified VH1 and VL1 alleles demonstrates that random point mutations are likely not the predominant cause of allelic variation at these loci. Comparison of the variant nucleotides with sequences from the pseudo-VH and pseudo-VL gene families, which lie 5' to VH1 and VL1, respectively, suggests that the great majority of allelic variants can be accounted for by segmental transfer of sequence from donor pseudogenes into the germ-line VH1 and VL1 genes. These results demonstrate that the chicken VH1 and VL1 genes are susceptible to sequence replacement at the germ-line level as well as somatically during antibody diversification. The limited repertoire of B cell specificities produced by gene rearrangement in the chicken has led to speculation that these specificities may play a critical role in the progression of chicken B cell development. The results presented here do not support this hypothesis since many of the allelic variant nucleotides described here encode non-conservative amino acid substitutions within the antigen-binding sites of the Ig molecule.

Alleles↗
PubMed1993

Flow cytometric analysis of the neutralizing immune response against infectious bursal disease virus using reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Infectious bursal disease virus (IBDV) is a lymphotropic virus with cytocidal effect on B lymphocytes of the bursa of Fabricius. We investigated the susceptibility of clonal populations of reticuloendotheliosis virus-transformed chicken B lymphocytes of both spleen and bursal origin to IBDV infection. The infected cells were metabolically-labelled and the viral polypeptides were analyzed by immunoprecipitation using monoclonal antibodies (MAbs). Virus adsorption and the effects of neutralizing convalescent antisera and MAbs on virus attachment were studied using flow cytometry. The results of the study indicate firstly that the transformed B cells support virus replication and provide an efficient system for studying IBDV-lymphocyte interactions. Secondly, results obtained also showed that the most potent neutralizing antibodies may not be those involved in preventing the receptor-mediated viral attachment but rather those involved in the inhibition of downstream events such as virus penetration or uncoating.

Animals↗
PubMed1992

Stochastic rearrangement of immunoglobulin variable-region genes in chicken B-cell development.

The molecular mechanism by which immunoglobulin (Ig) gene rearrangement occurs is highly conserved between mammalian and avian species. However, in avian species, an equivalent to the mammalian pre-B cell, which has undergone Ig heavy-chain gene rearrangement and expresses mu heavy chains in the absence of Ig light-chain rearrangement, has not been convincingly demonstrated. It is consequently unclear whether an ordered progression of gene rearrangement events leading to functional Ig expression occurs in avian species. To examine the sequence of Ig gene rearrangement events in chicken B-cell development, we transformed day 12 embryo bursal cells with the REV-T(CSV) retrovirus. More than 100 clones were analyzed by Southern blotting and polymerase chain reaction for the presence of Ig gene rearrangements. The majority of these clones contained only germline Ig sequences. Several clones contained complete heavy- and light-chain rearrangements and 13 clones contained only heavy-chain rearrangements analogous to stages of mammalian B-cell development. However, 5 clones contained rearrangements of light-chain genes in the absence of complete heavy-chain rearrangement. Consequently, we conclude that rearrangement of chicken Ig light-chain genes does not require heavy-chain variable-region rearrangement. This observation suggests that chicken Ig gene rearrangement events required for Ig expression occur stochastically rather than sequentially.

Alleles↗
PubMed1992

T cell receptor aggregation, but not dimerization, induces increased cytosolic calcium concentrations and reveals a lack of stable association between CD4 and the T cell receptor.

Exposure of T94, a CD4+ V beta 8-expressing murine Th cell clone, or immediately ex vivo CD4+ T cells to deaggregated, bivalent antibodies specific for either the TCR or CD3 failed to induce an increase in [Ca2+]i, or activation of phosphatidylinositol hydrolysis unless cross-linked with a secondary anti-Ig antibody. In contrast, we show that a combination of two mAb directed against different components of the TCR/CD3 complex (145.2C11, anti-CD3 epsilon and F23.1, anti-V beta 8) successfully induce second messenger formation, that is, without any requirement for a secondary antibody. This requirement for either a secondary antibody or two independent bivalent antibodies to activate second messenger production in T cells suggested that the signal transduction apparatus may be activated by multiple TCR/CD3 complexes being brought together on the T cell surface. This was supported by the observation that conditions inducing increased T cell [Ca2+]i through the TCR/CD3 complex also resulted in aggregation of the TCR/CD3 complex on the T cell surface. Conversely, binding of anti-TCR/CD3 antibodies to the T cell under conditions that did not induce increased [Ca2+]i also failed to induce surface TCR/CD3 redistribution. Cross-linking of the CD4 accessory molecule on T94 also resulted in increased [Ca2+]i, with kinetics similar to those observed after TCR/CD3 oligomerization. CD4 is involved in the recognition of invariant regions of MHC class II during Ag presentation and has been proposed to be associated with TCR/CD3 in the absence of Ag. Aggregation of TCR/CD3 and subsequent second messenger formation was achieved by combinations of mAb to distinct determinants within the complex due to the stable association of these determinants within the T cell membrane. We therefore assessed the functional association of CD4 with the TCR/CD3 complex by examining whether a combination of mAb directed against CD4 and CD3 or TCR induced second messenger formation. We found that anti-CD4 in combination with F23.1 or with 145.2C11 failed to induce increases in [Ca2+]i. Furthermore, mAb to CD4 failed to inhibit the increase in [Ca2+]i observed with the combination of 145.2C11 and F23.1. We therefore conclude that CD4 is not stably associated with TCR or CD3 in the absence of Ag/MHC class II composites.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Biological roles of the major capsid proteins and relationships between the two existing serotypes of infectious bursal disease virus.

Neutralizing monoclonal antibodies (n-MAbs) were produced against infectious bursal disease virus (IBDV) of serotypes 1 and 2. The n-MAbs recognizing the major antigenic proteins VP2 and VP3, were characterized using different strains of IBDV representing the existing two serotypes and a variant subtype of serotype 1. The biological properties of these viral antigens as defined by the MAbs in vitro, were studied utilizing post-adsorption virus neutralization tests and fluorescence-activated cell sorter analysis. The MAbs directed against the immunodominant epitopes on VP2 were capable of enhanced virus neutralization but did not inhibit the virus attachment to susceptible cells. These MAbs were able to neutralize the virus by interfering with an event subsequent to virus adsorption, possibly inhibiting virus penetration or uncoating. On the contrary, a MAb that immunoprecipitated the other capsid protein VP3 was able to prevent virus attachment although it possessed lower neutralization titers. Cross-immunoprecipitations of various virus strains by these MAbs and antisera revealed interrelationships between the two serotypes of IBDV.

Antibodies, Monoclonal↗
PubMed1992

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities.

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.

Amino Acid Sequence↗
PubMed1992

Antigenic sites on porin of Haemophilus influenzae type b: mapping with synthetic peptides and evaluation of structure predictions.

The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.

Amino Acid Sequence↗
PubMed1992

tkl is the avian homolog of the mammalian lck tyrosine protein kinase gene.

We have tested the possibility that tkl, a partially characterized avian tyrosine protein kinase gene, is the chicken homolog of lck, a lymphocyte-specific mammalian gene. Using polymerase chain reactions, we have cloned sequences encoding the previously unidentified amino terminus of the tkl gene product. The newly defined unique domain of Tkl displayed significant identity (68%) to the equivalent region of the mammalian lck gene product, p56lck. This identity included a glycine residue at position 2 (present in all Scr-related tyrosine protein kinases) and a cysteine motif at positions 20 and 23, which allows binding of p56lck to CD4 and CD8 in mammalian T lymphocytes. A specific RNase protection assay revealed that, in contrast to a previous report (K. Strebhardt, J. I. Mullins, C. Bruck, and H. Rübsamen-Waigmann, Proc. Natl. Acad. Sci. USA 84:8778-8782, 1987), tkl expression is restricted to the lymphoid tissues thymus and spleen. Moreover, the absence of tkl transcripts in the bursa of Fabricius suggested that this gene is expressed in avian T lymphocytes but not in B lymphocytes. A polyclonal rabbit antiserum raised against the unique domain of Tkl recognized a 56-kDa polypeptide with associated protein kinase activity from avian thymus-derived cells. Additional studies showed that p56tkl is structurally similar to mammalian p56lck and that it is physically associated with the avian CD4 and CD8 T-cell surface antigens. It was also determined that tkl transcripts have one major type of 5' untranslated region (UTR), which differs greatly from the two known 5' UTRs of mammalian lck mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

High levels of CD45 are coordinately expressed with CD4 and CD8 on avian thymocytes.

In this paper we describe the avian homolog of mammalian CD45. We show that this Ag is expressed on all leukocytes but not on erythroid cells or their immediate precursors. Immunoprecipitations demonstrated that B lineage cells from the bursa of Fabricius expressed a higher molecular mass variant (215 kDa) than did T lineage cells from the thymus (190 kDa), and crucially, these high molecular mass molecules had intrinsic phosphotyrosine phosphatase activity characteristic of mammalian CD45. We show that levels of CD45 expression as detected by mAb LT40 in the avian thymus are heterogeneous and further that mAb LT40 can deplete all phosphotyrosine phosphatase activity from thymocyte membrane preparations. Therefore total levels of CD45 are heterogeneous among avian thymocytes. Specifically, 87 to 89% of thymocytes expressed fourfold higher levels of surface CD45 (CD45hi) than the remaining 11 to 13% (CD45lo). The CD45lo population contained exclusively thymocytes with the phenotype CD3-4-8lo, characteristic of the immediate precursors to the CD3-4+8+ thymic population which are CD45hi. The shift from low to high levels of surface CD45 expression therefore occurred at the same stage as the transition from CD4-8lo to CD4+8+ and before the expression of CD3. The protein tyrosine kinase activity associated with CD4 and CD8 (p56lck) and the phosphatase activity of CD45 have been implicated elsewhere in jointly regulating peripheral T cell signal transduction and subsequent cellular responses. The coordinated expression of high levels of CD45 with both CD4 and CD8 in the avian thymus supports the possibility that these molecules may function together in regulating thymocyte growth and/or differentiation.

Animals↗
PubMed1991

Avian CD4 and CD8 interact with a cellular tyrosine protein kinase homologous to mammalian p56lck.

Functional as well as immunochemical studies have indicated that chicken T lymphocytes express surface antigens homologous to mammalian CD4 and CD8. The results presented in this report demonstrate that the avian CD4 and CD8 homologs are physically associated with a cellular tyrosine protein kinase related to mammalian p56lck. Furthermore, they show that engagement of either avian CD4 or CD8 is associated with a rapid intracellular tyrosine phosphorylation signal, suggesting that the function of these surface molecules may be mediated through alterations of the associated tyrosine protein kinase. Taken together, these findings demonstrate that the associations of CD4 and CD8 with an intracellular tyrosine protein kinase are evolutionarily conserved, further enhancing the likelihood that they are critical for normal immune functions.

Animals↗
PubMed1991

Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.

Animals↗
PubMed1990

Cross-linking of the surface immunoglobulin on lymphocytes from the bursa of Fabricius results in second messenger generation.

The bursa of Fabricius represents the major site of B lymphocyte development in avian species. Although the vast majority of bursal lymphocytes express cell surface immunoglobulin (sIg), it is generally considered that the bursa does not represent a significant site of antigen-induced B cell maturation to Ig secretion. However, the question as to whether antigen, either exogenous or self, can induce positive or negative selection of bursal lymphocytes in such a way as to modify the peripheral B cell repertoire remains open. Clearly, such intrabursal selection would require that bursal lymphocyte sIg have the molecular machinery to transduce signals into the cell as a consequence of its interaction with antigen. In this report we demonstrate that exposure of bursal lymphocytes to antibodies directed against sIg induced a rapid increase in cytosolic free calcium ion concentrations [Ca2+]i. Furthermore, such antibodies also induced a rapid increase in intracellular phosphatidic acid concentrations followed by a rise in intracellular phosphatidylinositol. Increased [Ca2+]i, phosphatidic acid and phosphatidylinositol levels required the cross-linking of sIg and were not induced by antibodies to other bursal cell surface antigens. Thus, cross-linking of the sIg on bursal lymphocytes results in second messenger generation, demonstrating that bursal sIg is a functional signal transduction element.

Animals↗
PubMed1990

The end can justify the means.

The generation of a pre-immune repertoire of B lymphocyte specificities is a prerequisite for subsequent antigen-dependent antibody responses. In avian species the bursa of Fabricius plays a central role in the development of B lineage cells and in the generation of antibody diversity. Apart from its segregation into the bursa, the pathway of avian B cell development differs from that seen in mammalian species by a number of other criteria. In particular the chicken has evolved a remarkable mechanism for the diversification of immunoglobulin V region genes. Here we review some of the key features by which avian and mammalian B cell development differ.

Animals↗