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R A Hubbard

Publications and source records attributed to R A Hubbard.

14 recordsLinked to original sources
PubMed1999

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in swab specimens by the Hybrid Capture II and PACE 2 nucleic acid probe tests.

BACKGROUND AND OBJECTIVES: The Digene Hybrid Capture II (HC II) CT/GC Test (Digene Corp., Beltsville, MD) is a new nucleic acid signal amplification-based test for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in specimens from the genital tract. For optimal results, the HC II CT/GC Test employs a special conical shaped brush for cervical specimen collection from nonpregnant women and swabs from pregnant women. GOALS: To validate a protocol for HC II C. trachomatis and N. gonorrhoeae testing of specimens collected for the GenProbe PACE 2 System. STUDY DESIGN: Specimens were collected from 1,746 patients with a swab and placed in GenProbe transport media according to the manufacturer's recommended procedure. The specimens were first tested at two clinical laboratories by the PACE 2 system, and then blindly tested by HC II CT/GC using an adjusted cutoff value. Discrepant specimens were adjudicated by polymerase chain reaction (PCR), and the result common to two of the three testing methods (HC II, PACE 2, and PCR) was defined as the consensus result. RESULTS: Combining the data from both sites, the relative sensitivity of the HC II Test compared with the consensus result for the detection of 1,761 specimens for C. trachomatis and 1,750 specimens for N. gonorrhoeae was 100% for both organisms. The relative specificities for C. trachomatis and N. gonorrhoeae detection were 99.8% and 99.7%, respectively. The relative sensitivities of the PACE 2 CT and GC Systems were 86.5% and 87.1%, respectively, with relative specificities of 99.9% and 100%. The difference in sensitivity between HC II and PACE 2 for C. trachomatis detection was significant (P < 0.016). CONCLUSION: The HC II CT/GC Test can be performed using specimens collected in GenProbe transport media and has a significantly greater sensitivity for C. trachomatis detection than the PACE 2 System.

Cervix Uteri↗
PubMed1992

T-cell receptors of man and mouse studied with antibodies against synthetic peptides.

We used polyclonal rabbit antibodies directed against synthetic peptides predicted from the gene sequence of the human T-cell receptor (TCR) beta-chain YT35 to study the antigen receptor on human helper T-cell leukemia lines and on normal mouse thymocytes. Antibodies were raised to peptides corresponding to joining segment (J beta) and to a conserved stretch of sequence around the first cysteine in the constant region (C beta). These peptides were selected on the basis of homology with corresponding segments of immunoglobulin light chains. The specificity of the antibodies was established using synthetic overlapping peptides that modelled the complete TCR beta-chain. Western blot analysis was performed against detergent lysates of T cells. Both of the antibodies reacted strongly with 2-3 polypeptides in the mass range 40-45 kDa in mouse and human cells. Clearance experiments using monoclonal antibodies against murine TCR alpha- and beta-chains and against human TCR beta-chain and immunoprecipitations with monoclonal antibody to the murine T3 complex established that these components represented the alpha/beta heterodimer. An additional component around 31 kDa was detected by anti-J beta antibodies in murine thymus extracts. The use of the affinity-purified antipeptide antibody in two-dimensional Western blot analyses allows the clear discrimination between the characteristic individual receptors of monoclonal neoplastic T cells and the polydisperse patterns representative of heterogeneous normal populations. Antigenic cross-reactions between T-cell receptor beta-chains of man and mouse observed with monoclonal antibodies and rabbit antisera to peptides are consistent with the homology in gene sequence between the two species.

Amino Acid Sequence↗
PubMed1989

Antibodies to synthetic peptides corresponding to variable-region first-framework segments of T cell receptor. Detection of T cell products and cross-reactions with classical immunoglobulins.

Recent studies at the gene level have shown that T cells express rearranged genes for four types of T cell receptors that are strongly homologous to classical immunoglobulins in the joining region and in the framework 1 (Fr1) and 3 segments of the variable region. Based upon the homologies in gene sequence, it follows that the gene products would show similarities in amino acid sequence and in the folding of the proteins so that cross-reactivities in antigenic determinants would be expected between variable regions of the T cell receptors and classical immunoglobulins. We have synthesized peptides corresponding to predicted protein sequences of the Fr1 residues of T cell receptor alpha, beta- and gamma-chains and have produced antibodies in rabbits against these synthetic peptides. Use of antisera and affinity-purified antipeptide antibodies indicated that high-titer antibodies could be raised that were specific for individual Fr1 peptides. Cross-reactions among Fr1 peptides of T cell receptors and immunoglobulin light chains were observed. In addition, some rabbit antisera raised against classical polyclonal immunoglobulins or affinity-purified immunoglobulin-like T cell receptors were found to exhibit binding activity against Fr1 peptides of T cell receptor beta- and gamma-chains. The sequence homology, although real among the Fr1 of T cell receptors and immunoglobulin light chains, is moderate and the antigenic cross-reaction must reflect the configuration and types of amino acids present. The development of antipeptide antibodies holds promise for the characterization of T cell receptors of various T cell sources and also offers a new means for the identification of molecules related to rearranging immunoglobulins.

Animals↗
PubMed1989

A monoclonal antigen-binding T cell immunoprotein: antigenic relatedness to T cell receptor beta chain FR1 V and J peptide segments: physicochemical distinctiveness from classical immunoglobulins and T cell receptor heterodimers.

The monoclonal murine T cell hybridoma, 51H7D, was previously shown to bind the arsazobenzene hapten and to produce a soluble antigen-binding molecule. In this paper we characterize this antigen-binding immunoprotein for its relationship to known T cell receptors serologically, using antibodies specific for variable region framework, or joining region peptides predicted from gene sequence and by biochemical means. The 51H7D cell expresses a protein with subunit size of approximately 31,000, that reacts antigenically with affinity-purified antibodies directed against synthetic first framework and joining segment peptides, corresponding to the gene sequence of the T cell receptor beta chain, YT35. This molecule does not react with affinity-purified antibodies directed against murine immunoglobulin, framework 1 sequences of alpha and gamma T cell receptors, or with antibodies against synthetic heavy chain joining segments. The subunit of mol. wt. 31,000 can form higher aggregates, notably in the mol. wt range of 60,000-70,000, depending upon extraction conditions. The soluble form of the antigen-binding molecule bears the J beta cross-reactive determinant and occurs predominantly as a charge restricted molecular species of approximate mol. wt 60,000-70,000. The purified molecule has a blocked N-terminus, but quantitative statistical analysis of its amino acid composition indicates a closer relatedness to T cell receptor beta chains and other antigen-binding T cell products, than it has to alpha, gamma or delta TCR chains. No evidence for more than one type of polypeptide chain was found and the polymerization is not dependent upon the formation of disulfide bonds. These studies raise the possibility that antigen-binding soluble T cell molecules might belong to a new family of immunoproteins, that is related to, but distinct from, classical immunoglobulins and alpha beta or gamma delta heterodimers.

Amino Acids↗
PubMed1988

Immunoglobulin epitopes defined by synthetic peptides corresponding to joining region sequence: conservation of determinants and dependence upon the presence of an arginyl or lysyl residue for cross-reaction between light chains and T-cell receptor chains.

Joining or J region sequences of rearranging immunoglobulins and T-cell receptors show considerable sequence homology, particularly in their C-terminal portion corresponding to the fourth framework region of immunoglobulin variable regions. In order to test the question of whether serological cross-reactions between immunoglobulin variable regions and T-cell receptors were due to antigenic similarities in their J regions, we synthesized synthetic peptides corresponding to immunoglobulin J regions and to J regions predicted from gene sequence of the T-cell receptor beta chain. We found that antibodies produced against a synthetic 16-mer J beta sequence reacted with T-cell receptor chains and also with immunoglobulin light chains. The cross-reactivity was dependent upon the J signature sequence FG()GT(R or K)L where the presence of a positively charged lysyl or arginyl residue was essential for cross-reactivity. We were able to classify J region determinants into two distinct antigenic sets; one corresponding to JH and the other corresponding to J kappa, J lambda, J beta and J alpha. Although considerable homology occurs between JH and JL (or J beta) sequences, little cross-reactivity was observed between these two J subsets. Antibodies raised against polyclonal murine IgG immunoglobulins contained antibody subpopulations specifically reactive with either JH or J beta peptides. The serological data derived here using antipeptide antibodies are consistent with computer modeling studies that indicate that the conformations of T-cell receptor variable regions resemble those of classical immunoglobulins. Our data comparing cross-reactivities restricted to the J region indicate that the expression of the J region by intact T-cell receptor beta chains is probably more similar to that of light chains than it is to the corresponding region of heavy chains.

Animals↗
PubMed1987

Conservation among vertebrate immunoglobulin chains detected by antibodies to a synthetic joining segment peptide.

We used affinity purified antibodies produced against a synthetic peptide sequence corresponding to the entire J beta of a human T cell receptor gene to screen sera of man, mouse and other vertebrates to determine the presence of cross-reactive molecules. Little evidence for free alpha/beta heterodimers was found, but the antibody reacted with light chains of many vertebrate species, including characterized myeloma proteins of man and mouse. Some vertebrate orders, notably Aves, lacked polypeptide chains cross-reactive with J beta, but detectable determinants occurred in primitive vertebrates such as the galapagos shark (Carcharhinus galapagensis). In addition to the strong cross-reaction with purified light chains, human heavy chains reacted weakly with the antibody. The cross-reaction correlated with the sequence of the denatured immunoglobulins and was inhibitable with free peptide. These results establish the similarity of T cell receptor beta chains to immunoglobulin chains and support the conclusion that J region sequences were conserved, not only within mammalian immunoglobulins and T cell receptors, but in vertebrate evolution.

Amino Acid Sequence↗
PubMed1985

Antibody-dependent cell-mediated cytotoxicity by human lymphocytes: a regulatory role for the sheep red blood cell receptor.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cytolytic mechanism whereby unimmunized lymphoid cells lyse antibody-sensitized target cells. The cells mediating ADCC are referred to functionally as killer cells (K cells). K cells are a heterogeneous population of lymphocytes based on cell membrane marker criteria but may be morphologically homogeneous. It is well established that K cells must have an Fc receptor (FcR) appropriate for the class of target cell-bound antibody, and that physical interaction between the K cell's FcR and the Fc portion of target cell-bound antibody is required for the initiation of lysis. Relatively little is known about the regulatory mechanisms presumed to exist and modulate ADCC reactions. We have made 2 observations that led us to investigate the possibility that the sheep erythrocyte (SRBC) receptor (ER) on human cells might be involved as a regulator of ADCC. First, SRBC are more efficiently lysed by human lymphocytes than are other erythrocyte target cells, and second, although nonsensitized bystander cells are usually not lysed during ADCC reactions, SRBC are consistently lysed. Removal of E-rosetting capacity of lymphocytes by trypsin treatment selectively decreases lysis of IgG sensitized SRBC. Furthermore, lysis of nonsensitized bystander SRBC is also inhibited by trypsin treatment of effector cells. ADCC is also decreased by blocking the ER with simple sugars or monoclonal antibody so that E-rosettes are inhibited. In contrast to trypsin treatment, which selectively inhibits lysis of SRBC, ER-specific monoclonal antibody and simple sugars decrease cytolysis against all target cells tested.

Animals↗
PubMed1982

Nonspecific human lymphocyte-mediated cytotoxicity induced by immobilized IgG aggregate: regulation by suppressor cells.

Normal human lymphocytes lyse a variety of erythrocytes when effector cells and target cells are incubated on immobilized, normal human IgG aggregates (10). Lymphocyte-mediated cytolysis induced by immobilized aggregate requires an overnight incubation with adherent mononuclear cells. This study was carried out to determine whether preincubation with adherent cells effects the activation of a cytolytic cell or inactivation of a suppressor cell. It was determined that a suppressor cell function is inactivated, and the suppressor cells were characterized as T gamma and T mu lymphocytes.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1981

Antibody-dependent cell-mediated cytotoxicity by human lymphocytes. II. Comparison with polymorphonuclear leukocytes against erythrocyte and nucleated target cells.

Human peripheral blood lymphocytes were compared to neutrophils with respect to their cytotoxic activity against IgG-sensitized erythrocytes (ox; ORBC) and nucleated (Raji) target cells in vitro. Overall, neutrophils were more efficient than lymphocytes in lysing erythrocytes, but lymphocytes were more efficient in lysing Raji cells. The antibody concentrations producing maximal levels of lysis against erythrocyte target cells were similar for both kinds of effector cells and guinea pig complement; however, in order to obtain Raji cell lysis mediated by neutrophils at a level comparable to that produced by lymphocytes a higher concentration of antibody was required. Another difference found between the lytic activity of these effector cell populations was kinetics of lysis against erythrocyte target: a 15 min lag phase was consistently observed only with neutrophil effector cells. Both effector cells lyse Raji cells with similar kinetics and no lag phase. Lysis by lymphocytes and neutrophils was directly proportional to the effector:target cell (E:T) ratio against both target cells. The effector cells were similarly inhibited from lysis of erythrocytes by IgG aggregate, but neutrophil lysis of Raji cells appeared to be more sensitive to inhibition. The neutrophils did not lyse nonsensitized bystander cells, and lymphocytes lysed sheep erythrocyte bystander cells only.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1981

Correlation if circulating immune complexes and disease status in patients with leukaemia.

The occurrence of soluble immune complexes (IC) was investigated in 177 serum samples from 92 patients with various leukaemias using the Raji cell immunoassay. In general, patients with myeloproliferative diseases had a higher incidence and higher quantities of IC than did patients with lymphoproliferative disorders. Elevated levels of IC were found in the sera of patients as follows: 17% with chronic lymphocytic leukaemia (mean value of 13.1 microgram/ml), 67% with acute lymphocytic leukaemia (54.1 microgram/ml), 65% with chronic myelocytic leukaemia (86.7 microgram/ml), 70% with acute myelocytic leukaemia (202.5 microgram/ml) and 56% with acute myelomonocytic leukaemia (41.9 microgram/ml). Patients in terminal blastic crisis of chronic myelocytic leukaemia had the highest levels, with a mean level of 1,364.1 microgram/ml. Serial samples were obtained, as available, from individual patients during the course of the disease in an attempt to relate severity with the incidence and quantity of IC. No significant correlation could be made between the occurrence or levels of IC and the presence of absence of systemic symptoms. Similarly, no correlations could be made between levels of IC and haematological parameters, infection, or therapy. However, the data does indicate a positive relationship between the levels of IC and the progressive state of the leukaemia, especially, the myelocytic leukaemias.

Antigen-Antibody Complex↗
PubMed1979

Antibody--antigen complex stimulated lysis of non-sensitized sheep red cells by human lymphocytes. I. Requirements for IgG complexes.

IgG antibody--antigen complexes stimulated lysis of non-sensitized sheep erythrocytes (SRBC) by normal human peripheral blood lymphocytes (PBL). Heat-aggregated human IgG, rabbit IgG-ovalbumin complexes and rabbit IgG-sensitized ox erythrocytes (ORBC) were effective in the induction of SRBC lysis by PBL. However, IgM-sensitized ORBC and IgM-complement-sensitized ORBC were ineffective. As only SRBC and not ORBC or chicken erythrocytes (CRBC) were lysed under identical experimental conditions, it is conceivable that the SRBC receptor present on the T cell is involved. Furthermore, 45% inhibition of lysis was obtained by pretreating the effector cells with anti-human thymocyte globulin (ATG) and complete inhibition was obtained by adding SRBC stroma to the reaction mixture. The requirement for the inclusion of IgG complexes and the absence of specific anti-target cell antibody distinguish this reaction from natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Immune killer T cells would not appear to be responsible as eight different donors were used and none of these were cytotoxic to SRBC in the absence of IgG complexes. The induction of this cytotoxic reaction appears to require the recognition and interaction by the effector cells of two separate molecular entities, i.e. the SRBC membrane by the T cell and the IgG Fc region by an IgG-Fc receptor-bearing cell.

Animals↗