Studies on the syngeneic mixed lymphocyte reaction. III. Development of a monoclonal antibody with specificity for autoreactive T cells.

Monoclonal antibodies with specificity for autoreactive murine T cells have been developed. These antibodies inhibit proliferative response of splenic T cells activated by syngeneic spleen cells. These antibodies have no effect on the proliferative response of T cells activated by allogeneic spleen cells or PHA. The number of splenic T cells that react with these monoclonal antibodies is comparable in several normal mouse strains.

Production of auto-antiidiotypic antibody during the normal immune response. VII. Analysis of the cellular basis for the increased auto-antiidiotype antibody production by aged mice.

We have previously shown that old mice produce more hapten-augmentable plaque-forming cells (PFC) than do young animals, suggesting a greater auto-antiidiotype antibody (auto anti-Id) component in their immune response. In the present studies this is confirmed serologically. The marked auto-anti-Id response of aged mice can be transferred to lethally irradiated young recipients with spleen but not bone marrow cells from old donors, suggesting that it is an intrinsic property of their peripheral B cell population and that the distribution of Id arising from the bone marrow of old and young mice is similar. In contrast with young mice the auto-anti-Id response of old animals is relatively T cell-independent and old donors do not show an increase in their ability to transfer an auto-anti-Id response after priming with TNP-F. These observations suggest that old mice behave as if already primed for auto-anti-Id production. Irradiated mice reconstituted with bone marrow cells from either young or old donors together with splenic T cells from old donors generate a relatively large auto-anti-Id response, whereas mice reconstituted with bone marrow from either young or old donors together with splenic T cells from young donors produce few hapten-augmentable PFC. It is suggested that differences in Id expression and auto-anti-Id production are the consequences of the interaction of Id (and anti-Id) arising from the marrow with anti-Id (and Id) present in the peripheral T cell population which serves as a repository of information about shifts in Id distribution, resulting from lifelong interactions with environmental and self-antigens.

Temperature-sensitive binding of solid phase C1q to aggregated human immunoglobulin G.

The first component of complement (C1q) coupled to Sepharose by cyanogen bromide was found not to bind aggregated human gamma-globulin or immune complexes at room temperature, whereas at 4 degrees C binding was nearly complete. The temperature sensitivity of solid phase C1q binding was reversible. Elution of aggregated human gamma-globulin bound at 42 degrees C was possible by raising the temperature to 23 degrees C. However, free C1q or C1q adsorbed onto polystyrene balls could bind immune complex-like material at both 23 and 4 degrees C. The conformational restraints of C1q covalently coupled to a solid support may not allow functional activity at elevated temperatures.

Characterization of RNP and Sm ribonucleoprotein nuclear antigens.

Antibodies to the RNase-sensitive RNP and to the RNase-resistant Sm nuclear antigens were used to affinity purify these antigens from a saline extract of rabbit thymus acetone powder. Determination of the protein subunits recovered by either glycine-HCl, pH 2.8, or 2.5 M MgCl2 elution on gradient sodium dodecyl sulfate-polyacrylamide electrophoresis containing mercaptoethanol revealed that RNP was composed of five proteins with mol. wts from 10,000 to 15,000 whereas Sm contained the same or similar five chains plus six additional subunits with mol. wts from 21,000 to 42,000. RNase treatment of the thymus extract increased the recovery in Sm of the same bands compared to untreated extract. Thus, RNP and Sm appear to have different numbers of protein components and RNP may be a subset of Sm. Sucrose gradient centrifugation of the 125I-labeled, pH 2.8 eluted antigens gave peaks of 3 and 6S for RNP and Sm, respectively. Sucrose gradient centrifugation of the crude untreated thymus extract followed by quantitative single radial immunodiffusion analysis of each fraction produced a broad peak from 16S to the top of the gradient while pretreatment of the extract with RNase resulted in a discrete 6S peak. These results indicate that in rabbit thymus acetone powder native RNP and Sm exist as larger polydisperse complexes with additional material including RNA and that after acid elution or RNase treatment the antigens are found in a smaller monodisperse form.

mTOR, a novel target in breast cancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models of breast cancer.

The mammalian target of rapamycin (mTOR) is a central regulator of G1 cell cycle protein synthesis that precedes commitment to normal cellular replication. We have studied the effect of cell cycle inhibitor-779 (CCI-779), a rapamycin ester that inhibits mTOR function, on the proliferation of a panel of breast cancer cell lines. Six of eight lines studied were sensitive (IC(50)< or = 50 nM) and two lines were resistant (IC(50)>1.0 microM) to CCI-779. Sensitive lines were estrogen dependent (MCF-7, BT-474, T-47D), or lacked expression of the tumor suppressor PTEN (MDA-MB-468, BT-549), and/or overexpressed the Her-2/neu oncogene (SKBR-3, BT-474). Resistant lines (MDA-MB-435, MDA-MB-231) shared none of these properties. CCI-779 (50 nM) inhibited mTOR function in both a sensitive and a resistant line. In nu/nu mouse xenografts, CCI-779 inhibited growth of MDA-MB-468 (sensitive) but not MDA-MB-435 resistant tumors. Treatment of sensitive lines with CCI-779 resulted in a decrease in D-type cyclin and c-myc levels and an increase in p27(kip-1) levels. There was good correlation between activation of the Akt pathway and sensitivity to CCI-779. Amplification of mTOR-regulated p70S6 kinase, which is downstream of Akt, may also have conferred CCI-779 sensitivity to MCF-7 cells. Taken together, the data suggest that mTOR may be a good target for breast cancer therapy, especially in tumors with Akt activation resulting from either growth factor dependency or loss of PTEN function.

Isolation and purification of human C1q from plasma.

C1q was purified to homogeneity from human plasma by a 3-step purification procedure. Plasma was euglobulin precipitated, and the redissolved precipitate chromatographed on a rabbit IgG-Sepharose column. The 1 M NaCl buffer eluate was passed directly through a rabbit anti-human IgG-Sepharose affinity column. C1q freed of IgG was present in the flow through. The rationale for this scheme to remove IgG free and that bound to C1q is discussed. Overall recovery of C1q was about 40% with IgG less than 4 microgram/mg C1q. In SDS-polyacrylamide electrophoresis with non-reducing conditions bands at 52,000 and 42,000 daltons were demonstrated while with reducing conditions bands at 26,000, 24,000 and 20,000 daltons were found as reported by others. C1q was found to be stable at 4 degrees C for 1 year in a 1 M NaCl, 0.4 M Tris, 10% sucrose, 0.005 M EDTA, 0.02% NaN3, pH 8.6 buffer.

Production of auto-anti-idiotypic antibody during the normal immune response. XII. An enzyme-linked immunosorbent assay for auto-anti-idiotype antibody.

An enzyme-linked immunosorbent assay (ELISA) to detect anti-idiotype (Id) antibody is described. Using this assay auto-anti-Id was detected in the serum of aged mice immunized with 2,4,6-trinitrophenylated-Ficoll (TNP-F). Hapten eluates from anti-TNP-F immune spleen cells also contained readily detectable auto-anti-Id.

Testicular circulatory isolation: a phase I study.

Antineoplastic agents may damage germinal epithelium. Testicular circulatory isolation (TCI) is a regional drug exclusion technique designed to minimize this. We evaluated the technical and anaesthetic aspects of TCI in 10 patients who underwent bilateral orchiectomy immediately thereafter. A modified aortic clamp was placed trans-scrotally across the left testicular pedicle without pre-medication or anaesthesia and left in place for 1 h, occluding testicular blood flow. Minimal pain and anxiety were reported during the procedure. There were no complications related to TCI in any patient. This study supports the institution of trials of TCI in young men about to receive fertility-threatening chemotherapy.

RSD score: criteria for the diagnosis of reflex sympathetic dystrophy and causalgia.

No standardized criteria for the diagnosis of reflex sympathetic dystrophy (RSD) are in common use. An RSD score scoring system of diagnostic criteria is proposed. Diagnosis of reflex sympathetic dystrophy by RSD score correlated well with clinical diagnosis in a group of 25 patients who had treatment for chronic upper extremity pain. Standardization of diagnostic criteria with the RSD score should enhance comparison of the outcomes of studies concerning the treatment of RSD.

Interscalene blocks for chronic upper extremity pain.

A study of 25 patients was carried out to determine the efficacy of interscalene block (ISB) for the treatment of chronic upper extremity pain. An RSD score was used to categorize these patients. Seventeen of the 25 patients had less pain after ISB, and 14 also had increased range of motion of the affected limb. Patients with reflex sympathetic dystrophy (RSD)/causalgia, as well as other chronic pain conditions, improved. ISB was compared with stellate ganglion block (SGB) in patients undergoing both treatments. ISB seemed to be at least as effective as SGB for treatment of RSD/causalgia and may have some advantages over SGB. The role of somatic and sympathetic blockade is discussed.

Immunomodulation by low-dose methotrexate. I. Methotrexate selectively inhibits Lyt-2+ cells in murine acute graft-versus-host reactions.

We have studied the effect of methotrexate in murine acute graft vs host (GvH) disease at concentrations analogous to those used in human rheumatoid arthritis. The GvH reaction was induced by i.v. injection of parental spleen cells into a normal F1 recipient. The acute suppression of T cell function in GvH mice was prevented by methotrexate given orally for 10 days at 1.0 or 0.5 mg/kg but not at 0.25 mg/kg. T cell mitogen response and IL-2 secretion that were inhibited in GvH mice were restored by methotrexate. Protection from immunosuppression in drug-treated GvH mice lasted at least 3 wk after drug dosing was stopped. The mechanism of the protective effect appears to be a preferential inhibition of donor and host Lyt-2+ Ts cell proliferation. In mixing experiments we found that methotrexate inhibited Ts function in GvH mice. By dual fluorescence labeling we showed that the engraftment of donor Lyt-2+ cells was prevented by drug treatment. This was not true of donor L3T4+ cells which were clearly present in the spleens of GvH mice after methotrexate treatment. These donor L3T4 cells were functional in that they induced the production of anti-DNA autoantibodies in the methotrexate-treated GvH mice.

Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function.

Novantrone mitoxantrone, an antineoplastic agent with antiproliferative properties, is under investigation as an immunomodulating agent. The impact of mitoxantrone treatment on B lymphocyte reactivity is presented here. Administered i.p. in H2O at a dose of 0.5 mg/kg, daily for 14 days, mitoxantrone abrogated both the in vivo antibody response (to ovalbumin) and the in vitro plaque-forming cell (PFC) response (to SRC). In addition to the effects on thymus-dependent reactivity, PFC responses to the thymus-independent antigens TNP-LPS and TNP-Ficoll were also inhibited when tested in vivo or in vitro. B cells were identified as a target for the suppressive activity of mitoxantrone by using T cell-replacing factor to reconstitute the in vitro anti-SRC PFC response of a T lymphocyte-depleted spleen cell preparation. LPS-induced B cell mitogenesis was largely inhibited by mitoxantrone treatment. However, depletion of Sephadex G-10-adherent cells significantly restored the proliferative response. Flow cytometric analysis revealed a dramatic decrease in splenic B lymphocyte content. Therefore, mitoxantrone exerted a potent suppressive influence on the humoral immune system through a direct reduction in B cell number augmented by macrophage-mediated inhibition of B cell proliferation.

GLVR1, a receptor for gibbon ape leukemia virus, is homologous to a phosphate permease of Neurospora crassa and is expressed at high levels in the brain and thymus.

The human gene GLVR1 has been shown to render mouse cells sensitive to infection by gibbon ape leukemia virus. This indication that the GLVR1 protein acts as a virus receptor does not reveal the protein's normal physiological role. We now report that GLVR1 is homologous to pho-4+, a phosphate permease of Neurospora crassa, at a level sufficiently high to predict that GLVR1 is also a transport protein, although the substrate transported remains unknown. To characterize the gene further, we have cloned cDNA for the mouse homolog of the gene, Glvr-1. The sequence of the murine protein differs from that of the human protein in 10% of residues, and it may be presumed that some of these differences are responsible for the inability of gibbon ape leukemia virus to infect mouse fibroblasts. Glvr-1 RNA is most abundant in mouse brain and thymus, although it is present in all tissues examined. The pattern of RNA expression found in mouse tissues was also found in rat tissues, in which the RNA was expressed at high levels in all compartments of the brain except the caudate nucleus and was expressed most abundantly early in embryogenesis. Thus, high-level expression of Glvr-1 appears to be restricted to specific tissues and may have developmental consequences.

Acid dissociation constants and pH values for standard "bes" and "tricine" buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and -25 degrees C.

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H2O mixed solvents at subzero temperatures from -20 to 0 degrees C, with the intention of establishing a pH (designated pH*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid ("bes") and N-tris(hydroxymethyl)methylglycine ("tricine"), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H2(g,l atm) /Bes, Na Besate, NaCl / AgCl;Ag and Pt;H2(g,l atm) /Tricine, Na Tricinate, NaCl /AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Buffer)+/- in equilibrium with(Buffer)- + H+.

Selective immunomodulation by the antineoplastic agent mitoxantrone. II. Nonspecific adherent suppressor cells derived from mitoxantrone-treated mice.

Mitoxantrone exerts a potent suppressive influence upon humoral immune responses. The B cell is a likely target for this inhibitory effect, and we have reported evidence supporting this possibility. The impact of mitoxantrone upon T lymphocyte reactivity was assessed as a second mode of action of this novel antineoplastic drug. TH and TS lymphocyte induction were tested in the in vitro anti-sheep erythrocyte response, and a surprising differential effect of mitoxantrone was observed. Helper activity was abrogated and suppressor function was enhanced. In apparent disagreement with this result, mitoxantrone inhibited the in vivo induction of TS cells using trinitrophenylated spleen cells. Macrophages were investigated as potential mediators of these effects upon immunoregulatory function. Replacement of macrophages in mitoxantrone-treated spleen cell preparations by normal adherent cells allowed the induction and complete expression of TH lymphocyte function. Conversely, replacement of mitoxantrone-treated macrophages with normal adherent cells before induction of TS cells failed to generate TS cell function. Thus, TH cells were resistant and TS cells were completely susceptible to mitoxantrone. Furthermore, supplementation of normal TH cell cultures with splenic macrophages from mitoxantrone-treated mice inhibited the induction of helper function. Production of the lymphokines IL 2 and TRF in mitoxantrone-treated mice was normal. This is consistent with the retention of functional TH cells in drug-treated spleens. Macrophages in the spleens of mitoxantrone-treated mice were responsible for the abrogated helper function and the enhanced suppressor activity. Although TS cell induction was directly inhibited by the drug, the effect upon TH cell function was secondary to the action of mitoxantrone-induced suppressor macrophages. Mitogen-stimulated lymphokine production was normal. Thus, mitoxantrone is a selective immunomodulator. The macrophage-mediated suppression of TH cell induction and humoral immunity investigated in spleens from mitoxantrone-treated mice is an intriguing finding that may have significant implications for immunotherapy.

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells.

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.

A comparison of two epidural alpha 2-agonists, guanfacine and clonidine, in regard to duration of antinociception, and ventilatory and hemodynamic effects in goats.

Epidural clonidine produces analgesia in humans with acute and chronic pain. Its use is limited because of short-lasting analgesia, hemodynamic depression, sedation, and respiratory depression. Intrathecal guanfacine has a longer duration of action than intrathecal clonidine. The present study compares these two drugs administered epidurally. Pulmonary artery, carotid artery, and epidural catheters were inserted into five goats. Each animal received guanfacine 5 mg/10 mL, clonidine 750 micrograms/10 mL, or a 10-mL saline control solution on separate occasions. Antinociception (tested via a point pressure stimulation device), arterial blood pressure, heart rate, cardiac output, pulmonary capillary wedge pressure, and arterial and mixed venous blood gases were measured every 30 min for 8 h. Guanfacine produced a longer duration of antinociception (guanfacine = 8 h vs clonidine = 5.5 h). Increases in PaCO2 were more pronounced in the clonidine group. There were no marked hemodynamic differences between the two drugs. Pretreatment with epidural idazoxan, an alpha 2-antagonist, blocked the antinociceptive effects of guanfacine. Because of a longer duration of action and less respiratory depression, epidural guanfacine may be superior for postoperative analgesia and chronic pain syndromes.

Lower antibody response to tetanus toxoid associated with higher auto-anti-idiotypic antibody in old compared with young humans.

The production of anti-tetanus toxoid (TT) antibody and anti-anti-TT (auto-anti-Id) antibody has been measured in three young and three old persons. Both antibodies were measured using ELISA. We found that: (i) the anti-TT response of old subjects was lower than that of young subjects, (ii) serum auto-anti-Id antibody concentration was higher in old compared with young humans before boosting with TT, and (iii) anti-TT antibodies from different humans shared Id cross-reactivity when tested with a rabbit anti-Id antibody. Thus, the serum anti-TT antibody response to boosting was inversely correlated with the serum level of auto-anti-Id. This conclusion is consistent with the view that the higher level of auto-anti-antibody in older subjects contributes to their impaired antibody response to TT.