Russell H. Tomar

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells☆

Abstract We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10−8M and was found to be completely blocked by indomethacin. Adding back 10−8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.

Preferential homing of tumor-infiltrating lymphocytes in tumor-bearing mice

SummaryIn view of the current interest in the use of lymphoid cells in adoptive immunotherapy of patients with advanced cancer, we have studied the homing patterns of various lymphoid effector cells in mammary-tumor-bearing mice. Single-cell suspensions of total splenocytes, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells were prepared from the spleens of C3H/OuJ mice. Tumor-infiltrating lymphocytes (TIL) were isolated from mammary adenocarcinomas excised from retired breeder females of the same substrain. Effector cells were labeled with indium-111 and injected via a tail vein into female C3H/OuJ mice bearing one or more mammary tumors. Twenty-four hours after administration, total splenocytes, NK cells, and LAK cells distributed themselves evenly between normal mammary tissue and mammary adenocarcinomas. Only TIL had a higher concentration in tumors than in corresponding normal mammary tissue. The ability of the different lymphocyte preparations to lyse YAC-1 cells was determined by means of a 4-h 51Cr-release cytotoxicity assay. Cells harvested from LAK cell cultures and further enriched by centrifugation through a discontinuous Percoll gradient and interleukin-2 (IL-2)-stimulated TIL demonstrated the highest levels of cytotoxicity, while total splenocytes and fresh TIL were characterized by the lowest levels. Since IL-2-stimulated TIL were highly cytotoxic and exhibited better tumor localization than both NK cells and LAK cells in this system, they may be the lymphoid effectors of choice for adoptive immunotherapy of advanced cancer.

Response to and production of prostaglandin by murine thymus, spleen, bone marrow, and lymph node cells

Abstract Prostaglandins influence cellular immune responses of mice and men. We demonstrated that the proliferation of cells from the spleen, lymph node, bone marrow, and thymus of young adult C57BL/6 mice may be inhibited by exogenous and endogenous prostaglandin E 2 . Thymocytes produced very little prostaglandin in the presence or absence of mitogen; lymph node cells showed no increase in prostaglandin (PG) production with mitogen; bone marrow cells and splenocytes demonstrated fourfold and tenfold increases in production of prostaglandin in the presence of concanavalin A. Bone marrow and thymus cells were more sensitive to PG suppression. The sensitivity of the various cell populations, the quantities of supernate IPG, and augmentation by indomethacin suggest that PG may modulate cell function in the bone marrow, thymus, and spleen, but not in unchallenged lymph node.

Graves' disease, myasthenia gravis, and purpura.

Excerpt To the editor: A case of thyroiditis, myasthenia gravis, and idiopathic thrombocytopenic purpura was reported recently by Segal and Weintraub (Ann Intern Med85:761-762). We have seen a simi...

Decreased natural cytotoxicity in mice with high incidence of mammary adenocarcinoma

In an attempt to gather evidence relevant to the question of whether natural killer (NK) cells play a role in resisting the development of primary tumors, we compared natural cell-mediated cytotoxicity in two substrains of C3H mice. Animals of the C3H/OuJ substrain are at high risk for the formation of mammary adenocarcinomas, while C3Heb/FeJ mice have a low incidence of such tumors. Natural cytotoxicity of splenic mononuclear cells was lower in the high-risk substrain, suggesting that a lesion in NK cell activity may be involved in murine mammary tumorigenesis. This difference was observed in animals between 5 and 37 weeks of age. There was no significant difference in the number of splenic large granular lymphocytes between the substrains. A significant difference in the ability of splenic lymphocytes from the two substrains to bind to the target cells was noted. Since the binding capacity of lymphocytes was greater in mice with reduced NK cell activity, the lesion in cytotoxicity may exist at a postbinding step in the lytic sequence. It is felt that the C3H mouse may provide a useful model for studying the role of NK cells in controlling primary tumors.

Transfer factor: Hypoxanthine is a major component of a fraction with in vivo activity

Transfer factor was prepared from the leukocyte lysates of four donors with known skin test reactivity. After ultrafiltration and double-gel filtration on polyacrylamide gels, fraction IV of the preparation was found to have biologic activity. This fraction contained one major and occasionally one minor ultraviolet-absorbing and zero to one ninhydrin-detectable spots on thin-layer chromatography. The major ultraviolet spot was identified as hypoxanthine. Hypoxanthine was demonstrated to be responsible for the high 260 nm/280 nm ratio of preparations with biologic activity in vivo. It was not determined if hypoxanthine is required for transfer factor activity. In addition, an orcinol-negative preparation also had biologic activity.

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

SummaryWe have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h 51Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.

Delayed Skin Reactor from Streptokinase-Streptodornase: Stability Studies and Amino Acid Analysis

Delayed skin reactor (DSR), the material in streptokinase-streptodornase responsible for eliciting delayed hypersensitivity reactions in man, is stable as a lyophilized powder or when stored at -70 degrees C. Storage at a pH above or below 6.0 leads to a decrease in the ability of DSR to stimulate lymphocyte proliferation in vitro. Its molecular weight is 30,000 as estimated by sodium dodecyl sulfate gel electrophoresis and it is composed of approximately 260 amino acid residues without apparent sulfhydryl-bridged subunits. The extinction coefficient of DSR (A 1% 280 nm) is 2.6.

Cellular Requirements for Simulation by the Purified Streptococcal Antigen DSR

Delayed skin reactor (DSR) was prepared from streptokinase-streptodornase. Peripheral blood mononuclear cells, were collected from a Ficoll-Hypaque gradient and subsequently subdivided into T cell-, B cell-, or monocyte-depleted populations. Mononuclear cells with complement receptors (B cells) were removed by a nylon-wool column adsorption. Sheep erythrocyte rosette forming cells (T cells) were removed by centrifugation on a second gradient. Populations of cells which did not adhere to plastic and/or ingest iron filings were monocyte-depleted. While response to DSR as measured by incorporation of tritiated thymidine into DNA was not affected by removing B lymphocytes, it was significantly diminished by removing T lymphocytes. Monocyte-depleted cells incorporated less tritiated thymidine into DNA in both antigen stimulated and control cells, but the stimulation index was not significantly altered.

TRANSFER FACTOR - SOME BIOCHEMICAL OBSERVATIONS

Publisher Summary This chapter describes some biochemical observations of the transfer factors (TF). TF was made from healthy donors chosen for their skin test reactivity to streptokinase/streptodornase 100/25 units/ml., C . albicans , histoplasmin and PPD intermediate. All of initial donors had strong skin reactions to streptokinase-streptodornase and candida antigens. By thin-layer chromatography, one major UV spot was detected in all of TF-IV preparations. This migrated 43–44 mm the same place as hypoxanthine. In one preparation a second, faint, more rapidly migrating UV spot was observed and on two other preparations, a more rapid, also faint, ninhydrin spot was detected. It was observed that as hypoxanthine migrated identically to major UV component; aliquots of transfer factor were reacted with the enzyme, xanthine oxidase. Xanthine oxidase catalyzes the conversions of hypoxanthine to xanthine and xanthine to uric acid. It was found that one added xanthine oxidase to TF-IV preparations and to hypoxanthine of similar absorbance at 260 nm and observed the spectral changes with time.