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R A Goldsby

Publications and source records attributed to R A Goldsby.

At least 19 recordsLinked to original sources
PubMed2001

Early Vlambda diversification in sheep.

This study examined a number of tissues during early gestation in foetal sheep to determine the earliest site of Vlambda expression and time of generation of the Vlambda repertoire. Tissues, including spleen, liver, gut, blood and bone marrow, were obtained from 48, 55, 60 and 63 gestational day (g.d.) ovine foetuses and cDNA libraries were prepared from them by reverse transcription-polymerase chain reaction. Clones were randomly selected from cDNA libraries and subjected to sequencing. Analysis of these sequences and comparison with a pool of germline genes led to the following conclusions. The expression of Vlambda occurs earlier in spleen (48 g.d.) than in all of the other tissues examined. Also, diversity is seen earlier and at higher levels in early foetal spleen than in all of the other tissues examined. In this regard, it is notable that splenic Vlambda expression is readily apparent even before such gut-associated lymphoid tissue as the ileal Peyer's patch (IPP) has developed. Two germline Vlambda genes, 5.1 and 5.3 predominate in early immunoglobulin lambda light-chain gene rearrangement. Examination of Jlambda usage revealed the existence of a new Jlambda gene and its utilization during the early phases of the development of the ovine antibody repertoire. This study indicates that sites other than the IPP contribute to the diversification of the Vlambda repertoire in sheep. We suggest that it is likely that foetal spleen may provide a partially diversified B-cell repertoire before the IPP becomes active as a major site for massive clonal expansion and extensive diversification of B cells.

Animals↗
PubMed1998

Multiple sites of V lambda diversification in cattle.

Ig repertoire diversification in cattle was studied in the ileal Peyer's patch (IPP) follicles of young calves and in the spleens of late first-trimester bovine fetuses. To investigate follicular diversification, individual IPP follicles were isolated by microdissection; VA diversity was examined by RT-PCR and subsequent cloning and sequencing. When 52 intrafollicular sequences from a 4-wk-old calf were determined and compared, two major groups, one of 23 members and the other of 25, could be delineated. An examination of these groups revealed clear genealogic relationships that implicated in situ diversification of V lambda sequences within the confines of an IPP follicle. V lambda expression was also examined in early (95 and 110 gestational day) fetal bovine spleens. Although earlier studies in cattle and sheep implicated the IPP as a likely site of Ab diversification, a close investigation of V lambda sequences in late first-trimester fetal calves revealed that diversity appears in the early fetal spleen before the establishment of a diverse repertoire in the ileum. When the sequences for the fetal spleen were compared with an existing pool of germline sequences, we found evidence of possible gene conversion events and possible untemplated point mutations occurring in sequences recovered from fetal spleens. We conclude that IPP is not the sole site of VA diversification in cattle. Also, as suggested for rabbits, cattle may use both gene conversion and untemplated somatic point mutation to diversify their primary VA repertoire.

Animals↗
PubMed1997

Immunoglobulin gene diversification in cattle.

Research in several species has revealed that different types of mammals have evolved divergent molecular and cellular strategies for generating immunoglobulin diversity. Other chapters in this text have highlighted the specific characteristics unique to chicken, rabbit, mouse, human and sheep B lymphocyte development; namely indicating differences in the mechanisms of diversity and the site of primary B cell development. Studies of the bovine system have indicated that like the sheep system, the ileal Peyer's patch (IPP) is a likely chicken bursal equivalent, and is a site of primary B lymphocyte development. Substantial investigation in sheep has indicated that Ig diversity is created by untemplated somatic mutation and intense selective pressure (Reynaud et al., 1991). The frequency of alteration in the sheep Ig light chain gene locus also is characteristic of the bovine system, however, recent evidence from sequencing of bovine lambda light chain genes indicates that one mechanism that contributes to diversity is gene conversion, utilizing several pseudogenes located in the Ig locus (Parng et al., 1996). The mechanism by which this hyperalteration of Ig genes occurs in both sheep and cattle is poorly understood and is thus the focus of considerable investigation. The study of events in the IPP may also have informative ramifications for secondary diversification of the Ig repertoire by somatic hyperalteration in germinal centers.

Amino Acid Sequence↗
PubMed1996

Gene conversion contributes to Ig light chain diversity in cattle.

In humans and mice, extensive gene rearrangement is the major mechanism of diversification of the primary Ig repertoire. This study shows that cattle depart from this pattern because rearrangement in the light chain locus is sharply limited. Furthermore, in cattle, gene conversion contributes to the diversification of the primary light chain repertoire. Sequencing of germ-line and expressed Vlambda genes revealed three important features. First, the germ line contained a number of Vlambda pseudogenes. In fact, 14 (70%) of the 20 germ-line genes identified and sequenced were pseudogenes, because they had one or more of the following defects: lack of recombination signal sequences at the 3' end, stop codons within the reading frame or truncations, and/or insertions or deletions that resulted in loss of reading frame. Second, Vlambda cDNA from ileal Peyer's patch B cells demonstrated that the light chain repertoire arises from only a small number of V(J) rearrangements. Even though two J genes were identified in the germ line, all of the expressed Vlambda genes examined contained the same J segment, indicating that only a single J gene participates in rearrangement at the lambda locus. Third, a significant number of departures from the germ-line sequences of rearranged Vlambda can be traced to donor sequences of one or more Vlambda pseudogenes. We conclude that a limited number of rearrangements and gene conversion play a role in contributing to the diversification of the primary lambda repertoire. Furthermore, while clear indications of a role for somatic mutation in lambda diversification was seen, V gene rearrangement was not a major factor.

Amino Acid Sequence↗
PubMed1996

The role of oxygen in thymocyte apoptosis.

Signals generated by T cell receptor (TCR) cross-linking (or phorbol 12-myristate-13-acetate + Ca2+ ionophore), glucocorticoids or ionizing radiation all stimulate apoptotic cell death in thymocytes by signals that are initially distinct from each other. However, when these stimuli were administered to thymocyte cultures that were maintained under an atmosphere containing less than 20 ppm oxygen as opposed to one that contained 18.5% molecular oxygen, cell death was inhibited or abrogated, suggesting that the induction of death by all three different stimuli depend on the presence of molecular oxygen. Studies of the effects of the cysteine analog N-acetyl cysteine (NAC) with normal thymocytes demonstrated that this antioxidant inhibited the induction of death by each of the different stimuli in a manner the paralleled anaerobiosis. Furthermore, studies with thymocytes demonstrated that the induction of nur77, a gene shown to be involved in thymocyte apoptosis signaled through the TCR or its surrogates, is not inhibited by NAC or dependent upon molecular oxygen. The possible implications for negative selection of NAC-mediated inhibition of TCR-signaled thymocyte cell death was examined in thymic organ culture. Treatment of these cultures with NAC provided significant protection against staphylococcal enterotoxin B-mediated deletion of V beta 8-expressing thymocytes.

Acetylcysteine↗
PubMed1996

Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis.

A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Salmonella enteritidis isolates. A total of 65 arbitrary primers were screened with S. enteritidis isolates of different phage types. This allowed selection of a panel of primers capable of detecting DNA polymorphisms among S. enteritidis isolates. This panel was used to examine a panel of 29 isolates of S. enteritidis which had been previously characterized by other subtyping methods, including phage typing (PT) (n = 7), ribotyping (RT) (n = 13), and pulsed-field gel electrophoresis (PFGE). Applied collectively, these three methods resolved the collection into 20 different subtypes. However, by the RAPD fingerprinting method alone, 14 RAPD subtypes were revealed. Eight isolates of S. enteritidis phage type 8 that failed to be discriminated by other typing methods (PT, RT, and PFGE) were resolved into three different subtypes by RAPD analysis. In contrast, isolates that were derived from the same sources were not differentiated by any of the subtyping methods employed, including PT, RT, PFGE, and RAPD analysis. This RAPD approach to S. enteritidis subtyping provided more discriminatory power than did any of several other subtyping methods applied individually. Once the challenging step of primer identification was accomplished, determinations of the appropriate concentrations of arbitrary primer, DNA template, and MG2+ ion were also necessary for optimal discriminatory power. The bacterial DNA used in this RAPD protocol was obtained by boiling the bacterial sample. This simple procedure yielded DNA that produced fingerprint patterns as consistent as those obtained from phenol-chloroform-extracted DNA. Clearly, when appropriately constituted primer sets are identified and employed, RAPD analysis provides a simple, rapid, and powerful subtyping method for S. enteritidis.

Bacterial Typing Techniques↗
PubMed1994

Quantitative analysis of the B cell repertoire by limiting dilution analysis and fluorescent in situ hybridization.

We have made a quantitative and concurrent analysis of B cell frequencies and VH gene family expression to study the influence of tissue type and age on the development and establishment of the primary B cell repertoire. Using LPS-mediated limiting dilution analysis and a panel of antigens we show that the newly generated B cell specificities from bone marrow get distributed without a bias to peripheral tissues such as spleen and Peyer's patches throughout the lifetime of the animal. Comparison of the B cell frequencies in animals of four different age groups (2-4 days old, 3, 12, and 18 months old) reveals that while the neonatal repertoire is comparable to that of adults, there was a selective twofold increase in the generation and distribution of B cells reactive with autologous mouse red blood cells in older mice compared to young ones. By means of a novel technique that employs fluorescent in situ hybridization and flow cytometry, we have also compared the VH gene family usage in large numbers of single B cells from these mice. Analysis of the same cell population (surface Ig+, functional B lineage cells) for expression of 7183, J558, and S107 VH families shows a preferential twofold increase in the use of VH 7183 in neonates compared to adults, while all three families show no significant difference in levels of expression during adult life or between primary and secondary lymphoid tissues. Taken together, our data indicate that specific and selective changes occur in both VH gene usage and antibody frequencies during murine ontogeny.

Age Factors↗
PubMed1992

Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry.

We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.

Animals↗
PubMed1988

Comparative idiotype network interactions: antigen mimicry by an anti-bovine idiotype monoclonal antibody in rats and cattle.

Idiotype/anti-idiotype networks have been extensively investigated in such conventional animal models as the mouse and the rabbit. However, systems of veterinary interest have remained largely unexplored. A monoclonal target idiotype, with which to begin such studies in cattle was provided by LHRB 19.17 an interspecific bovine x mouse hybridoma. This hybridoma was constructed by the fusion of supramammary lymph node cells from S. agalactiae-immunized lactating Holsteins with the Ig synthesis-permissive established cell line, SP 2/0. Two collections of monoclonal anti-idiotype antibodies were generated by fusion of spleen cells from LHRB 19.17-immunized Balb/c or A/J mice immunized with the monoclonal bovine idiotype, LHRB 19.17. Many of the anti-idiotypes inhibited binding of LHRB 19.17 to S. agalactiae, but only one, LHRAID 2.71, proved to be an internal image of a S. agalactiae epitope. Immunization of C/D outbred rats by priming with 100-300 micrograms of LHRAID 2.71 emulsified in CFA followed by a 300 micrograms boost at day 32 elicited anti- S. agalactiae antibody in 4/4 animals tested. Similarly, the injection of two lactating Holsteins with the anti-id resulted in the production of anti- S. agalactiae antibody in serum and milk. In both rats and cattle, the administration of the antigen-mimicking anti-idiotype induces the appearance of S. agalactiae-reactive horseradish peroxidase-streptavidin conjugate; LHRBs, interspecific bovine x mouse hybridomas secreting bovine Ig; LHRAID.X, monoclonal anti-bovine idiotype antibodies derived against LHRB 19.17; PBS, phosphate buffered saline; PBS/BSA, PBS containing 0.1% bovine serum albumin: antibody [AB3] that competes with LHRB 19.17 [AB1] for binding to LHRAID 2.71 [AB2]. It should also be noted that the immunization of C/D rats with S. agalactiae does not result in the appearance of idiotypes which compete with LHRB 19.17 for binding to LHRAID 2.71. We have concluded that immunization of two widely divergent species with the antigen mimicking LHRAID 2.71 induced a S. agalactiae-reactive idiotype which was not detectable in the immune response of rats to S. agalactiae.

Animals↗
PubMed1988

Production and characterization of monoclonal antibodies to bovine beta 2-microglobulin.

In an attempt to isolate monoclonal antibodies specific for bovine lymphocytes, spleen cells from mice immunized with bovine lymphocytes were fused with the mouse myeloma cell line SP-2/0. The resulting hybridoma cell lines were tested for reactivity with bovine lymphocytes, polymorphonuclear neutrophils, RBC, gamma-globulin, kappa-casein, beta-casein, alpha-S1-casein, and beta 2-microglobulin (beta 2m) and with beta 2m from rabbits, goats, and human beings. None of the clones secreted anti-bovine lymphocyte-specific antibody. However, 4 secreted monoclonal antibodies to bovine beta 2m. They also reacted with beta 2m from rabbit, goat, and human being. One monoclonal antibody also was found to be reactive with bovine immunoglobulin. Monoclonal antibodies to beta 2m could serve as a tool to (1) explore the homology of the beta 2m molecule among various species, (2) examine the relationship of beta 2 m with the constant region of the immunoglobulin molecule, (3) quantitate bovine beta 2m in various body fluids and major histocompatibility antigens on cell surfaces, (4) help characterize those antigens in cattle, and (5) be used for tissue typing of those antigens.

Animals↗
PubMed1987

The application of hybridoma technology to the study of bovine immunoglobulins.

Studies are described in which hybridoma technology is used to produce a variety of reagents for the characterization and manipulation of the bovine humoral immune system. Selected members of a set of murine monoclonal antibodies (MAb) specific for each of four major isotypes of bovine Ig constant regions, one specific for anti-bovine Ig constant regions as well as one specific for anti-bovine light chains are discussed. Interspecific fusion of bovine lymphocytes with the established mouse cell line, SP2/0 was used to produce a collection of stable hybridomas among which were found secretors of bovine IgG1, IgG2, IgM, IgA and bovine light chain. Interspecific fusion of SP2/0 with lymphocytes from a multiparous Holstein four days post immunization with Streptococcus agalactiae yielded MAb with specificity for the immunizing antigen. One of these hybridomas, LHRB 19.17, which displayed a particularly stable secretory phenotype, was used as an immunogen for the production of a library of murine monoclonal anti-idiotype antibodies. Competitive antigen binding analysis showed that 15 of the 24 anti-LHRB 19.17 idiotype antibodies isolated blocked the binding of the idiotype to its nominal antigen and so were candidates for evaluation as antigen mimics. Some of the ways in which monoclonal anti-idiotypes in particular, and monoclonal in general, might be of use in problems of animal disease are discussed.

Animals↗
PubMed1987

Immunoaffinity chromatography of bovine FSH using monoclonal antibodies.

Bovine FSH (bFSH) was used to immunize BALB/c mice. Spleen cells were fused to the SP 2/0 cell line to produce hybridomas that secreted monoclonal antibodies to bFSH. One of these antibodies (USDA-bFSH-MC28) was extensively characterized and found to be a gamma 1 with kappa light chains, having extremely low cross-reactivity with other bovine pituitary hormones and with ovine and porcine FSH. The dissociation constant as measured by Scatchard analysis was 4.3 nmol/l, and proved to be in a very useful range for affinity chromatography. In an essentially one-step immunoaffinity chromatography procedure, bFSH was easily isolated in a single chromatographic step from crude anterior pituitary homogenate with better yield and with the same purity as classical chromatographic techniques.

Animals↗
PubMed1984

Production and characterization of monoclonal bovine immunoglobulins G1, G2 and M from bovine x murine hybridomas.

Three bovine x murine hybridomas, which secrete bovine IgG1, IgG2 and IgM, respectively, were derived by the fusion of normal bovine B lymphocytes to the murine cell line SP-2/0. The hybridomas were initially selected by testing the culture supernatant of individual clones with rabbit anti-bovine light chain antibody. The bovine nature of Ig secreted by the bovine x murine hybridomas was confirmed by the ability of bovine serum but not murine serum to inhibit specific adsorption by murine anti-bovine Ig coupled to Sepharose 4B and the inability of sheep anti-mouse Ig (all isotypes) to bind biosynthetically labelled Ig from the respective bovine x murine hybridomas or to react with bovine x murine hybridoma Ig in Ouchterlony analysis. The isotype of the bovine Ig produced by each hybridoma was determined by cRIA using bovine Ig isotype-specific murine mcab, Ouchterlony analysis with guinea pig anti-bovine Ig isotypes and molecular weight analysis of unreduced and reduced affinity purified Ig from the respective bovine x murine hybridomas. Even though the hybridomas in this study showed chromosome loss, they have continued to secrete bovine Ig in culture for over 16 months, indicating that it is possible to isolate and stabilize interspecific hybridomas retaining the chromosome, or at least the genes, encoding an Ig. These hybridomas will provide monoclonal bovine Ig for; 1) serological standards, 2) production of polyclonal and monoclonal antisera to bovine Ig isotypes, 3) sequencing studies, 4) serological and structural studies of bovine Ig classes and subclasses, and messenger RNA for the production of cDNA probes for the cloning of bovine Ig genes and for determining the organization of Ig genes in the bovine genome.

Animals↗