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R H Stevens

Publications and source records attributed to R H Stevens.

At least 55 records · Page 3Linked to original sources
PubMed1985

Subclasses of human IgG anti-Fab antibodies: parameters for optimum detection.

In this study we have defined the parameters needed for the optimum detection of anti-Fab antibodies in the serum of patients with rheumatoid arthritis. We have found that the majority of the anti-Fab antibodies are of the IgG3 and IgG4 subclasses which were not optimally detected using polyclonal heterologous anti-human IgG antisera; subclass-specific antibodies instead were needed. Additionally we determined that dissociation of circulating immune complexes by dialysis against urea for 3-7 days was also needed for the detection of these antibodies. Lastly we have shown that the dissociated complexes can recombine with their target Fab molecules, and therefore separation of the anti-Fab antibodies from the other immunoglobulins by chromatofocusing may enhance the detection of these antibodies. When the above conditions were fulfilled it was determined that IgG anti-Fab antibodies could be detected in rheumatoid arthritis and normal sera and that acidic IgG3 and IgG4 subclasses predominated. However, IgG3 levels were 10.5-fold higher in rheumatoid arthritis sera (p less than 0.05) and IgG4 levels 5-fold higher (p less than 0.01) than in normal sera.

Antibodies, Anti-Idiotypic↗
PubMed1985

Anti-Fab antibodies in humans. Predominance of minor immunoglobulin G subclasses in rheumatoid arthritis.

Isoelectric focusing analyses of sera from patients with rheumatoid arthritis (RA) demonstrate two populations of antibodies directed against the Fab portion of pooled human IgG. One population is composed of polyclonal alkaline anti-Fab antibodies (alpha FABA) and the other, acidic alpha FABA which are more clonally restricted. In this study we have identified the immunoglobulin classes and subclasses of these antibodies in RA sera. Enzyme-linked immunosorbent assays (ELISA) demonstrated alpha FABA in RA sera to be predominantly IgG. A large portion of IgG alpha FABA existed as immune complexes, inasmuch as dialysis of RA sera against 6 M urea before ELISA analysis was necessary for maximal detection of alpha FABA activity. Chromatofocusing of RA sera isolated alpha FABA of different charges and revealed the acidic clonally restricted alpha FABA to be IgG4 and IgG3, whereas the polyclonal alkaline group contained IgG1, IgG2, and IgG3. Overall, acidic IgG3 and IgG4 comprised 70% of IgG alpha FABA, and high levels of IgG4 were seen in most RA sera. When alpha FABA were elevated in normal sera, they were primarily of the IgG4 subclass, and also existed as immune complexes. Serum anti-Fab activity was removed by adsorption of sera with Fab fragments. Anti-Fab antibodies of both kappa and lambda light-chain types were present in RA sera, and F(ab')2 fragments of RA serum immunoglobulin were found to possess anti-Fab activity. These studies indicate that alpha FABA in RA sera are limited to the IgG class, and that most of these antibodies exist as immune complexes and display clonal and minor IgG subclass restriction.

Antibodies, Anti-Idiotypic↗
PubMed1984

Clonally restricted anti-IgG antibodies in rheumatoid arthritis.

Clonally restricted anti-IgG antibodies were detected, by isoelectric focusing (IEF) and chromatofocusing techniques, in the sera of patients with rheumatoid arthritis (RA). Anti-Fab antibodies were predominantly acidic proteins with isoelectric points of 4.5-6.5 and displayed restricted spectrotype patterns. Proteins reactive with the Fc portion of IgG showed polyclonal spectrotype patterns with alkaline pI of 7.5-9.0. A limited array of anti-Fab spectrotypes was consistently detected in RA sera when analyzed by IEF on 6M urea gels. Additional anti-Fab antibody bands were detected when the RA sera were dialyzed against 4-6M urea prior to IEF analysis, indicating that some anti-Fab antibodies exist in a complexed form in serum. Under these dissociating conditions, anti-Fab antibodies could also be detected in normal subjects, but the spectrotype patterns were more restricted than those in RA sera. Because anti-Fab antibodies may regulate normal immune responses, the increased quantity of clonally restricted anti-Fab antibodies in RA may indicate an abnormality of this immunoregulation.

Antibodies, Anti-Idiotypic↗
PubMed1984

12-O-Tetradecanoylphorbol-13-acetate (TPA) directly inhibits spontaneous immunoglobulin secretion by in vivo antigen-induced human lymphoblastoid B cells.

Tetanus toxoid (Tet) booster immunization induces the transient appearance in the circulation of lymphoblastoid (LB) B cells which spontaneously produce anti-tetanus toxoid IgG antibody (IgG-Tet) during a 3-day in vitro culture. In this study we have examined the effects of TPA on the ability of LB cells to secrete antibody and have found that as little as 10 ng/ml of TPA provoked a marked inhibition of the induced LB cells' IgG-Tet production. This inhibitory effect was observed only when TPA was added early in the culture and could be achieved by pretreating the B cells with TPA for as little as 1 hr. Only marginal inhibition of IgG-Tet production was observed if the addition of TPA was delayed 14-24 hr. The TPA inhibition was not mediated by contaminant T cells as the addition of increasing numbers of T cells to LB cell cultures proportionally reversed the TPA inhibitory effect. Likewise, the inhibition of antibody synthesis was not due to a monocyte-dependent mechanism since (1) substantial depletion of adherent cells did not reverse the inhibition of antibody synthesis, (2) the addition of a monocyte-enriched population to monocyte-depleted B cells did not enhance, but in fact partially reversed, the inhibition caused by TPA, and (3) the addition of monocyte populations pretreated with TPA to monocyte-depleted B-cell fractions did not inhibit subsequent IgG-Tet production by the LB cells.

Antibodies, Bacterial↗
PubMed1984

Involvement of the transferrin receptor in the production and NK-induced suppression of human antibody synthesis.

Earlier studies have shown that the antibody-secreting lymphoblastoid (LB) B cells which arise in the circulation after immunization can be down-regulated by NK cells and an unusual T suppressor cell. Because the LB B cells require division in vitro before maximal antibody secretion, and as such may express the transferrin receptor, we wished to determine if this receptor is recognized by the NK and/or T suppressor cells. The addition of the antitransferrin receptor antibody B3/25 to cultures of LB cells resulted in a partial inhibition of antibody secretion. This inhibition was not seen with the addition of monoclonal anti-Dr or OKT-8 antisera, and indicated that the LB cells expressed the transferrin receptor and that its activity was required for antibody secretion. The addition of free Fe ions to the culture medium, however, allowed the secretion of antibody by the LB cells even in the presence of B3/25 antibody. Under these conditions, the inhibition of antibody secretion mediated by interferon-activated NK cells was no longer observed. The inhibition induced by pokeweed mitogen-stimulated T suppressor cells was not affected by the antibody treatment, suggesting that these two inhibitory cells recognize different target structures. These results indicate that LB B cells expressed the transferrin receptor at a stage of the in vitro culture, and that this structure is involved in the NK- but not the T cell-mediated inhibition of antibody synthesis.

Antibodies, Bacterial↗
PubMed1984

NK and T cell subsets regulate antibody production by human in vivo antigen-induced lymphoblastoid B cells.

This study demonstrates the existence of two different suppressive systems for the regulation of antitetanus toxoid antibody production by human lymphoblastoid (LB) B cells. These B cells appear in the circulation 5 to 7 days after in vivo immunization and spontaneously secrete antibody during a 3-day in vitro culture. One suppressive system was mediated by large granular lymphocytes that exhibited high natural killer activity. This suppressive cell subset spontaneously inhibited the antibody production by autologous LB cells, and this effect could be enhanced by the addition of interferon. This inhibition of antibody synthesis could be readily reversed by the addition of as few as 10(2) K-562 cells to the culture. Additionally, the activity of this suppressive cell population could be reduced by complement (C)-mediated lysis with Leu-7 antibody. These results strongly suggest that this autologous suppression was mediated by NK cells. The other suppressor system was contained in the fraction of high density T cells depleted of Fc receptor-bearing cells, which was low in NK activity. This subset inhibited LB function in the presence of pokeweed mitogen but not interferon, and even the addition of up to 10(6) K-562 NK target cells only minimally reversed this inhibition. These results indicate that two distinct subsets of cells share regulatory functions on the in vivo induced B lymphoblastoid cells. The observation that NK cells can inhibit these highly differentiated B cells expands our view of the spectrum of natural targets recognized by NK cells.

Antibodies, Bacterial↗
PubMed1984

Human in vivo antigen-induced lymphoblastoid B cells are capable of cyclical antibody production in vitro.

In vivo immunization of normal volunteers with tetanus toxoid induces the formation of a circulating B cell subset that has the capacity to secrete specific antibody in vitro without the need for T cell help or mitogen stimulation. From earlier studies it was not clear whether the spontaneous antibody-secreting lymphoblastoid (LB) B cell was at a terminal stage of differentiation or if it had the capacity to give rise to additional B cell subsets, such as memory cells or more fully mature antibody-producing cells. In this study we have shown that at least two distinct waves of spontaneous antibody secretion can occur in vitro when cultures are initiated with the lymphocytes from individuals immunized 6 days earlier. The first production of antibody was completed by 3 days of in vitro culture and the second production of antibody did not initiate until day 7 or 8 of culture and was completed by day 12. The B cells responsible for the second stage of antibody production appeared derived from a portion of the antibody-secreting cells present on day 3 in that 1) treatment of the cultures on day 0 with BuDr and light equally inhibited the first and second rounds of antibody synthesis; 2) when isolated from the blood, both B cell subsets were in the large cell fraction after 1 X G sedimentation; and 3) under conditions of limiting numbers of cells, the cells responsible for the second wave of antibody production were almost exclusively found in cultures positive for a B cell that had produced antibody on days 1 to 3. Although only a portion (10 to 30%) of the LB B cells present on day 0 had the capacity to again produce antibody on days 8 to 12, the two cells were capable of producing similar quantities of antibody on a per cell basis. These results indicate that the mature circulating LB cell induced in vivo by immunization is not terminally differentiated, but under appropriate conditions has the capacity to give rise to additional antibody-secreting cells.

Antibody-Producing Cells↗
PubMed1983

Inhibition of human antigen-induced lymphoblastoid B-cell function by an in vivo-induced suppressor T cell.

Lymphoblastoid (LB) B cells which spontaneously produce antitetanus toxoid IgG antibodies (Tet-IgG) in short-term cultures (3 days) appear in the circulation 5-7 days after immunization with tetanus toxoid. Addition of pokeweed mitogen (PWM), normally a stimulator of antibody production, caused instead a reduction in the in vitro synthesis of Tet-IgG by the LB cells. In order for this inhibition of antibody production to occur, T cells had to be present, and the inhibition was proportional to the number of T cells added to the culture, demonstrating the existence of PWM-inducible suppressor cells. The cells mediating the suppression had the OKT8 phenotype and also exhibited the following characteristics: (1) a PWM pretreatment period as little as 14 hr was enough to complete activation; (2) conventional inhibitors of suppressor T cells as hydrocortisone and cyclosporin A only partially reversed its effect; and (3) DNA synthesis was not required. The T-suppressor activity was detectable in the circulation before immunization, increased two- to fourfold by 5-12 days after boosting, and waned after 3 weeks. The mechanism of action of this suppression does not appear to involve conventional cytotoxic T cells as (1) the suppression was mediated across allogeneic barriers and (2) the suppression could not be reversed by inclusion of anti-Leu-2a antibodies in the culture. These results suggest that this suppressor T-cell subset may be important in the normal regulation of activated stages of human B lymphocytes.

Antibodies, Monoclonal↗
PubMed1983

T suppressor cells are required for the maintenance of the antigen-induced B-cell unresponsive state in humans.

Tetanus toxoid immunization of humans generates circulating B cells which secrete IgG anti-tetanus toxoid antibodies (IgG-Tet) when stimulated in vitro with T cells and pokeweed mitogen (PWM). A unique property of these cells is the inhibition of maturation into antibody-secreting plasma cells following a 1-hr in vitro pulse with tetanus toxoid. Studies were undertaken to determine if different T-cell subsets could modulate the in vitro generated B-cell unresponsive state. The addition of OKT4+/OKT8- cells to antigen-treated B cells resulted in a partial reversal of the antigen-induced inhibition of IgG-Tet synthesis. The addition of OKT4-/OKT8+ cells to the treated B cells caused a suppression of IgG-Tet synthesis comparable to that seen in cultures containing unfractionated T cells. These results indicate that (1) the B-cell unresponsive state generated by antigen treatment is not absolute, (2) the degree of B-cell unresponsiveness results from a balance of suppressor and helper signals, and (3) T-suppressor cells need to be present to induce and maintain the B-cell unresponsive state.

Antibodies, Monoclonal↗
PubMed1983

Immunoregulatory T cells in men with a new acquired immunodeficiency syndrome.

We have evaluated the functional properties of the OK-T8+/OKT4+ T-cell subpopulations in nine patients with a new syndrome of acquired immune deficiency (AIDS). Despite polyclonal hypergammaglobulinemia in the sera of these patients, their peripheral blood lymphocytes (PBL) produced negligible quantities of immunoglobulin (Ig) when cultured in vitro for 8 days in the presence of pokeweed mitogen (PWM). Patient B cells, however, synthesized normal quantities of immunoglobulin when cocultured with T cells from healthy donors, indicating preservation of B-cell function. Unfractionated PBL or T cells of patient origin mediated marked suppression of pokeweed mitogen-driven immunoglobulin production by T and B cells from healthy donors. The suppressive activity was contained within the population of T cells bearing the OKT8 antigen and was sensitive to in vitro irradiation. On a per-cell basis, patient OKT8+ cells appeared to have greater suppressive activity than normal control OKT8+ cells. In addition, OKT4+ cells from patients had less helper activity for induction of immunoglobulin synthesis than control OKT4+ cells. Increased T suppression and reduced T help are probably a consequence of one or more viral infections and may contribute to progressive immune deficiency and susceptibility to malignancy in patients with the acquired immuno deficiency syndrome.

Acquired Immunodeficiency Syndrome↗
PubMed1983

Cytotoxicity of the bacterium Actinobacillus actinomycetemcomitans extracts in human gingival fibroblasts.

Filter-sterilized sonic extracts (SE) of strains of Actinobacillus actinomycetemcomitans were shown to inhibit the proliferation of human gingival fibroblasts in vitro. The inhibition was dose-dependent: a 50 per cent inhibitory dose of 2 micrograms protein/ml was found for A. actinomycetemcomitans strain Y4. The inhibitory activity could be neutralized by homologous antiserum and was heat inactivated by temperatures of 80 degrees C or greater. The fibroblast-inhibitory activity was present in SEs of both leukotoxic-producing and non-leukotoxic strains of A. actinomycetemcomitans, suggesting that a separate agent is responsible for leukotoxicity and fibroblast inhibition. A short (10 min) exposure of the fibroblasts to the A. actinomycetemcomitans SE was sufficient to inhibit irreversibly cell proliferation, provided that serum was present at the time that the cells were exposed to the SE. SE-challenged fibroblasts exhibited a marked decrease in the rate of DNA synthesis, but no inhibition of RNA or protein synthesis. Although the SE-treated cells did not proliferate, they appeared to remain intact and viable; and displayed no gross morphological alterations.

Actinobacillus↗
PubMed1983

Identification of carcinogens by measurement of cell-mediated immunity. IV. Antitumor immunity following perinatal exposure to 1,2-dimethylhydrazine.

This study was initiated to investigate the possible perinatal carcinogenic effects of the colon carcinogen 1,2-dimethylhydrazine (DMH) in Fischer F344 inbred rats. Pregnant female animals during their 16-18th day of gestation were administered the chemical by intraperitoneal injections, and beginning at 4 months postparturition, the antitumor cell-mediated immunity (CMI) was delineated in the dams and pups as an indirect measure of carcinogenesis. The CMI status was established by the ability of peripheral blood lymphoid cells obtained from the rats to injure and kill target tumor cells derived from an X-ray-induced rat small bowel adenocarcinoma cell line with the degree of damage being reflected in the quantity of loss of radioiodinated peripheral and integral membrane proteins from the target cells. A significant antitumor CMI was observed in the exposed offsprings although there was no apparent difference between the immunoresponsiveness observed in either the males or the female siblings. Unexpectedly, the mothers exhibited little such antitumor cellular immunity following the carcinogenic insult; even though all previous investigations of adult animals always demonstrated such an immunological response following exposure to the quantities of DMH that were administered (0.1 to 20 mg per kg body wt). As a consequence, these findings tentatively implied that the state of pregnancy alters a female's response to chemical carcinogenic insults and may actually serve as a device for protection from environmentally caused cancer. The threshold detection level for DMH exposure utilizing immune measurements was found to be approximately 10 times smaller for the perinatal susceptibility to the chemical insult intimating that such tests might usefully be incorporated in those bioassays utilized for determining the cancer-causing potential of weak carcinogens. Our findings now suggest that DMH may indeed be a perinatal carcinogen and that immune responsiveness may be readily employed for identifying such substances. However, the definitive studies of actually identifying cancer following such in utero exposures remain to be accomplished.

1,2-Dimethylhydrazine↗
PubMed1983

Cell-mediated cytotoxicity expressed by lymphoid cells from rats with asbestos-induced peritoneal mesothelioma towards rat fetal cell.

Cell-mediated immunity (CMI) directed towards rat fetal cells was evaluated in Fischer F344 young inbred male rats having asbestos-induced peritoneal mesothelioma. The tumors were induced by exposure to Canadian chrysotile B fibers and the CMI delineated by the injury and destruction brought about to 6- to 10-day-old primary fetal cell cultures by the so-called educated peripheral blood lymphoid-cells (PBLC) obtained from the cancer-bearing rats. A significant cytotoxicity was found to be expressed by the PBLCs, suggesting that during the development of mesothelioma, a cellular retrodifferentiation occurs, thereby educating the effectors to recognize a common determinant existing in both the tumor and fetal cells. Educated PBLCs were produced from rats having endodermal tissue cancers (adenocarcinomas of the small bowel, colon and pancreas) and were found to also be cytotoxic to the fetal cultures, yet no injury was apparently inflicted upon cultured mesothelioma target cells by these effectors. These results suggested that the tumor education was specific and that probably a unique and different fetal component was being recognized by the effector cells obtained from the rats with lesions arising either in the mesodermal or endodermal tissue. Further support for this concept was the failure of an antibody, specific to an oncofetal protein existing in endodermal lesions, to apparently recognize any common oncogenic proteins in the mesothelioma. Preliminary studies have also been accomplished which suggests the existence of natural killing immune responses existing to the mesothelioma target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Isoelectric focusing of human anti-diphtheria toxoid antibodies: identical spectrotypes of anti-fragment A antibodies with the same IgG subclass and light chain constant regions are expressed in multiple individuals.

The serum antibodies from humans booster-immunized with diphtheria toxoid were analyzed by isoelectric focusing. To restrict the antibody response, we visualized those antibodies that reacted to the Fragment A moiety of diphtheria toxin. Of the 100 normal human donors examined, 20 were estimated to be nonresponders to Fragment A. Sixty-three of the 80 donors who responded to Fragment A had restricted IgG-anti-Fragment A spectrotypes that consisted of three to eight distinct bands. Six series of repeat or shared spectrotypes were delineated among our donor population with repeat frequencies ranging from 0.025 to 0.312. We determined that the repeat spectrotypes were IgG1-kappa anti-Fragment A antibodies. Three percent of our donors had complex IgG-anti Fragment A spectrotypes consisting of greater than 10 bands. In some instances, the complex spectrotypes were composed of simple spectrotypes found among other donors. The complex IgG-anti Fragment A spectrotypes of these donors consisted of both IgG1 and IgG4-anti-Fragment A antibodies. From a statistical analysis, we calculate that only a limited number of IgG-anti-Fragment A spectrotypes are expressed in vivo after booster immunization. Our analysis suggests two groups of IgG-anti-Fragment A spectrotypes exist in our donor population. One IgG-anti-Fragment A spectrotype group is expressed randomly, and a second group is preferentially expressed among individuals in our donor population.

Antibodies, Bacterial↗
PubMed1982

Human IgA antibody and immunoglobulin production after in vivo tetanus toxoid immunization: size and surface membrane phenotype analysis.

The in vitro production of IgA-anti-tetanus toxoid antibodies (IgA-Tet) by human peripheral blood lymphocytes (PBL) was assessed at various times after in vivo intramuscular tetanus toxoid immunization. Five days after immunization, T cell- and mitogen-independent synthesis of in vitro IgG-Tet, but not IgM-Tet or IgA-Tet, was detected. Two to six weeks after immunization, pokeweed mitogen (PWM)-stimulated cultures of B and T cells produced IgM-Tet and IgG-Tet but not IgA-Tet. We did, however, find that 75% of the serum samples of immunized individuals showed increases in IgA-Tet. Mitogen-dependent in vitro total IgA synthesis was detected in human PBL. Cells synthesizing the majority of PWM-induced IgA bear the phenotype Ig+ CR+ Fc gamma R+-. The majority of PPD-induced IgA synthesis was by cells lacking surface membrane IgD, similar to PPD-reactive IgG secretors. These results indicate that intramuscular immunization does not result in circulating B cells that can secrete antigen-specific IgA in mitogen-driven in vitro cultures. Moreover, multiple B-cell subsets are responsible for in vitro IgA production.

Antibody Specificity↗