Suppression of graft versus host reactivity by a single host-specific blood transfusion to prospective donors of hemopoietic cells.
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Animals↗
Biomedical subjects
R Benner
Publications and source records attributed to R Benner.
Analysis of anti-insulin antibodies using time-resolved fluoroimmunoassay.
This paper describes the analysis of both monoclonal and polyclonal murine insulin antibodies with time-resolved fluoroimmunoassay (TRFIA). This assay is based on the binding of antiserum by coated isotype-specific capture antibodies and the detection of insulin binding using europium-labeled human insulin (HI-Eu). This reagent was shown to have a detection limit of 10(-16) mol. Among various applications this technique permitted an isotype-specific determination of the affinity distribution of polyclonal antibodies from individual mice.
Animals↗
Requirements for flare reactions of joint inflammation induced in mice by cloned MT4+, Lyt-2- T cells.
Joint inflammation was induced in C57B1/6 mice by injection of cloned MT4+, Lyt-2- T cells specific for the antigen methylated bovine serum albumin (mBSA), together with mBSA. In this model, after waning of the inflammation, flare reactions can be induced by a rechallenge with the specific antigen. Herein we show that such flare reactions can still be induced several weeks after waning of the joint inflammation, as was demonstrated both in normal C57B1/6 mice and in athymic C57B1 nude mice. The results in the latter group indicate that T cells of the recipient mice are not necessary for the elicitation of flare reactions. On histologic examination, the inflammatory infiltrates in the knee joints of the nude mice appeared to be mainly granulocytic. The cloned T cells persisted and remained functionally reactive in the knee joint for at least 2 weeks in the absence of the antigen, and thus, in the absence of inflammation. In view of the similarities between induced joint inflammation in mice and rheumatoid arthritis in humans, these data may be relevant to our understanding of the processes involved in the latter disease.
Animals↗
B cell repertoire in adult antigen-free and conventional neonatal BALB/c mice. II. Analysis of antigen-binding capacities in relation to VH gene usage.
Hybridomas were derived from lipopolysaccharide-reactive splenic B cells of adult germ-free BALB/c mice fed a chemically defined ultrafiltered "antigen-free" diet (GF-CD) and from splenic B cells of 5-day-old conventional (CV-NEO) BALB/c mice. The monoclonal antibodies (mAb) from both collections of hybridomas were tested for reactivity against a large panel of antigens of exogenous and endogenous origin. As a source of natural exogenous antigens 36 different bacteria and 9 different viruses were used, while as endogenous antigens frozen tissue sections of stomach, liver and kidney, the Hep-2 cell line and the anti-idiotopic mAb Ac38 and Ac146 were used. In both collections of mAb approximately 70% reacted with one or more bacterial antigens, while no reactivity could be detected against the viral antigens. Of the GF-CD and CV-NEO hybridomas, 16% and 19%, respectively, reacted with one or more frozen tissue sections. Overall 56% and 68% of the GF-CD and CV-NEO hybridomas, respectively, were producing multireactive antibodies reactive to several exogenous and/or endogenous antigens. Among the GF-CD hybridomas a correlation was found between multireactivity and the usage of the VH gene family PC7183. In CV-NEO hybridomas, however, the preferential utilization of the VH gene family PC7183 was found among both mono- and multireactive hybridomas. The results suggest (a) that the actual B cell repertoire of neonatal mice consists of a large proportion of multireactive B cells which are reactive with autoantigens and bacterial antigens, but not viral antigens and (b) that in antigen-deprived mice the neonatal repertoire is largely preserved during maturation of the mice.
Animals↗
Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet.
This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.
Animals↗
Effects of topically applied glucocorticosteroids on patch test responses and recruitment of inflammatory cells in allergic contact dermatitis.
The effects of repeated topical application of a strong glucocorticosteroid (GCS) on patch test responses and the inflammatory infiltrate were studied in twenty nickel allergic patients. Patch test responses were strongly inhibited in 18 out of 20 patients. Immunohistochemical analysis revealed marked reductions in CD1+(T6+) Langerhans cells, activated inflammatory T cells and of mast cells in the skin. It is concluded that GCS suppress contact allergic responses by reduction, or functional inhibition of antigen presenting cells. The reduced number of mast cells release less vasoactive mediators, inhibiting recruitment of inflammatory cells.
Administration, Topical↗
Glucocorticoid receptors in alveolar macrophages: methodological aspects of the determination of the number of glucocorticoid receptors per cell.
Among patients with asthma or chronic obstructive pulmonary diseases the response to glucocorticoids varies considerably. To investigate the possible relationship between clinical response and number of glucocorticoid receptors in alveolar macrophages or their KD value a micro receptor assay was developed. Assay conditions were adjusted because of receptor occupancy by endogenous or therapeutically used glucocorticoids and high aspecific binding.
Cell Line↗
Joint inflammation induced by a mature macrophage cell line and cloned MT4+, Lyt-2- T cells in allogeneic mice.
Recently we reported the isolation of the mature macrophage cell line AP284. This line can efficiently present antigen to cloned helper T cells in vitro and in vivo. In this paper we show that AP284 can also present antigen to cloned helper T cells in an in vivo model system in which joint inflammation is induced. It appeared that injection of cloned helper T cells specific for methylated bovine serum albumin (mBSA) together with mBSA and AP284 into the joints of allogeneic mice induced a substantial joint inflammation. This system offers great prospects for studying the involvement of soluble mediators in the induction of joint inflammation as well as regulatory aspects of this inflammation.
Animals↗
Solid state line scan cameras for measuring strain in soft tissues and implants.
The line scan camera, or LSC, is an inexpensive and easily applied technique for optical strain measurement of soft biomaterials. The LSC is based on a linear array of photodiodes; in gauging applications, where measurements between dark/light interfaces are important, the digital nature of the array can be exploited. Advantages of the LSC include low cost, high frequency response, applicability to front- or back-lighted samples, insensitivity to stray and nonuniform lighting as well as to accidental overexposure, ease and linearity of calibration, and lack of temperature sensitivity. With the 1024 element arrays used herein, the relative resolution is theoretically limited to 1 part in 1024, or 0.1%; in practice, the relative resolution is somewhat poorer. LSCs have been successfully used in mechanical tests to measure the diameter of arteries and compliant vascular grafts, the longitudinal strain of vascular grafts, and the dynamic diameter of elastic tube models of graft/artery systems in pulsatile flow visualization experiments.
Animals↗
MHC- and Igh-unrestricted interaction between L3T4+, Lyt-2- Ts inducer cells and L3T4-, Lyt-2+ Ts effector cells is required for T cell-dependent suppression of DTH to histocompatibility antigens.
This paper describes the characteristics of T suppressor inducer (Ts ind) cells which can interact with T suppressor effector (Ts eff) cells and thereby can account for suppression of delayed-type hypersensitivity (DTH) to alloantigens. Adoptive transfer of spleen cells from mice intravenously (i.v.) injected with allogeneic spleen cells one day earlier induced an antigen-specific state of suppression in the recipients. This became apparent when DTH was induced by subcutaneous (s.c.) immunization of the recipients three days after transfer. The induction of suppression after adoptive transfer of spleen cells required Thy-1+, L3T4+, Lyt-1+2- cells. These cells that by themselves did not exert a suppressive effect induced a state of suppression in recipient mice by activation of recipient-type Ts eff cells. Therefore, the former cell type was classified as Ts ind cell. When athymic nude mice were used as recipients, Lyt-2+ precursors of Ts eff cells had to be transferred together with the Ts ind cells to induce a state of suppression in these mice. The Ts ind cells could activate Ts eff cells in MHC- and Igh-incompatible recipients. The results are discussed in relation to previously described immunoregulatory T cell pathways.
Animals↗
Suppression of antigraft immunity by preimmunization. V. Characterization of suppressor T memory cells.
We have previously shown that after intravenous (i.v.) immunization of mice with allogeneic spleen cells, two populations of suppressor T (Ts) cells may occur that can suppress delayed-type hypersensitivity (DTH) to alloantigens: Ts effector cells that transiently occur in the spleen, and long-lived, recirculating Ts cells that occur in thoracic duct lymph and can be transferred by parabiosis. In this study, we investigated whether the latter Ts cells fulfill the criteria for memory T lymphocytes, such as induction by doses of antigen lower than required for T effector cell induction, accelerated onset of activity after reactivation and an increased activity as compared with virgin T cells. The Ts cells accounting for the long-lasting state of suppression of DTH to alloantigens indeed fulfilled these criteria. These Ts memory cells displayed the Thy-1+, L3T4-, Lyt-1+2+ phenotype.
Animals↗
Enhancement and suppression of DTH reactivity to Rauscher murine leukaemia virus induced tumour cell lines.
Delayed-type hypersensitivity (DTH) to Rauscher murine leukaemia virus (R-MuLV) encoded or induced determinants was induced in mice by three syngeneic R-MuLV-induced tumour cell lines, i.e. a myeloid tumour, RMB-1, an erythroid tumour, RED-1, and a lymphoid tumour, RLD-1. DTH to subcutaneously (s.c.) administered RMB-1 cells appeared on day 4, with a maximum DTH response on day 6 or 7. The induction of DTH could be prevented by intravenous (i.v.) pre-immunisation with R-MuLV-induced tumour cells several days before the s.c. immunisation. The three R-MuLV-induced tumour cell lines showed cross-reactivity in the DTH assay, whereas no cross-reactivity was found with syngeneic WEHI-3 cells. This indicates that the three R-MuLV-induced tumour cell lines share a virally encoded or induced antigenic determinant, which activates T-cells. When the RMB-1 cells used for immunisation had been cultured in medium supplemented with interferon-gamma (IFN-gamma), the subsequent DTH response was increased. This coincided with an increased expression of the R-MuLV-specific antigenic determinants on RMB-1 cells as demonstrated by Scatchard analysis. Furthermore, IFN-gamma increased the MHC class I antigen expression on RMB-1 cells, whereas the class II antigen expression remained undetectable.
Animals↗
Blood transfusion-induced suppression of delayed-type hypersensitivity to allogeneic histocompatibility antigens.
In this study delayed-type hypersensitivity against histocompatibility antigens in mice was suppressed by a single donor-specific blood transfusion. Whole blood as well as purified white blood cells and purified red blood cells were capable of inducing suppression. White cells appeared more potent in inducing suppression than red cells. Suppression was dose-dependent, still detectable after administration of as little as 0.001 ml of whole blood and maximal at a dose of 0.1 ml. The suppression was already present a few hours after transfusion and proved to be long-lasting. The suppressive effect could be transferred to naive recipients by Thy-1+, L3T4-, Lyt-2+ spleen cells. This suppressor T cell population was of recipient origin--which excludes the possible involvement of "veto cells" and suppresses the afferent phase as well as the efferent phase of the DTH response.
Animals↗
Modulation of total IgE levels in serum of normal and athymic nude BALB/c mice by T cells and exogenous antigenic stimulation.
Several different grades of T-system impairment were studied for their effects on the total serum IgE concentration in BALB/c mice. Homozygous athymic nu/nu mice and their heterozygous nu/+ littermates were compared for serum IgE levels while kept under either barrier-maintained or conventional conditions. The results show a paradox between the T-cell dependency of the IgE immune response and the increased levels of serum IgE in the absence of T cells. Both barrier-maintained and conventionalized nu/nu mice have at least twofold increased serum IgE levels as compared to nu/+ mice. With age, IgE levels increased faster and reached higher plateau values in nu/nu than nu/+ mice. Moreover, after adult thymectomy of BALB/c mice the serum IgE levels increased up to 15-fold at 4 months of age, while infusion of immunocompetent T cells in nude mice resulted in a 2- to 5-fold decrease of the IgE level.
Aging↗
Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers.
To study oncogene expression in heterogeneous cell populations we developed and optimized a non-radioactive in situ hybridization technique using biotinylated single-stranded RNA probes and combined this technique with immunofluorescent staining of cell surface markers. As a model for our studies we used HL60 cells. In these cells we detected c-myc mRNA molecules by in situ hybridization following staining of the pan myeloid cell surface marker CD33, by a monoclonal antibody. Hybrids were detected by streptavidin-FITC and CD33 by a TRITC-conjugated antibody. Controls involved pretreatment with RNAase, hybridization with sense RNA probes and blocking with an excess of unlabeled antisense probes. The integrity of the RNA in the cell was shown by hybridization with the GAPDH antisense probe. Essential for successful double-labeling was the choice of a fixation procedure that was suitable for the in situ hybridization and mild enough not to destroy the cell surface marker staining. This fluorescent in situ hybridization in combination with cell surface marker staining will be useful for studying gene expression in phenotypically well-defined cell populations.
Antigens, CD↗
Increase of precursor frequency and clonal size of murine IgE-secreting cells by IL-4.
IL-4 is able to preferentially enhance murine IgE levels in the supernatant of LPS-stimulated T cell-depleted splenic B cell cultures. Clonal and quantitative analysis of this response revealed that this is due partly to a 14-fold increased IgE precursor frequency and partly to a three-fold increased clone size of IgE-secreting cells. IL-4 increased the precursor frequency and the clone size of IgM-secreting cells not more than twofold. Both the IgM and IgE response in LPS-stimulated B cells were completely inhibited by the addition of anti-IgM mAb (M41) to the cultures, indicating that the IgE-secreting clones developed as subclones from precursors that express IgM. These cells lacked expression of membrane-bound IgE up to day 5 of the culture. Application of feeder cells in these cultures resulted in an increased precursor frequency of IgE-secreting clones among LPS-reactive B cells that is due, partially, to IL-4 produced by the feeder cells.
Adjuvants, Immunologic↗
Rapid procedure for coupling protein antigens to red cells to be used in plaque assays, by prewashing in chromium chloride.
A rapid and efficient procedure is described for the coupling of proteins (protein A, ovalbumin, albumin and chicken gamma globulin) to sheep red blood cells (SRBC) to be used in antigen-specific or protein A plaque assays. This modification of the original procedure has three distinct features: prewash of the red cells with a low concentration of essentially freshly prepared CrCl3, use of a relatively high concentration of CrCl3 in the reaction mixture and a coupling time of only 4 min. Protein A plaque assays performed with such target cells have the same sensitivity as those employing red cells coupled with protein A according to the original procedure. Studies with hybridomas secreting antibody specific for a protein antigen showed that antigen-specific plaque assays employing target red cells coupled with the protein antigen according to the modified procedure have the same sensitivity as the protein A plaque assay. The modified procedure greatly facilitates cellular studies on antibody formation after immunization with protein antigens.
Animals↗
The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes.
The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.
Animals↗