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Biomedical subjects

J L Rossio

Publications and source records attributed to J L Rossio.

At least 37 records · Page 2Linked to original sources
PubMed1986

Optimal strategies for developing human-human monoclonal antibodies.

Human monoclonal antibodies are desirable, especially as therapeutic agents, but the best means of producing them is still a matter of investigation. It is clear that human antibodies of predicted specificity from patients with autoimmune disease can be derived, and this may help unlock some of the mysteries of these illnesses. Human monoclonal antibodies against tumor-specific antigens for use in in vivo diagnosis and therapy remain desirable goals. Problems involved in their routine development include the lack of available, adequately immunized, and differentiated lymphocytes and the nature and paucity of the available human "myeloma" cell lines. These lines have been compared now by a number of authors who have reached similar conclusions. Our study directly compared the greatest number of cell lines and found UC729-6 and HF2 to be the best; on the other hand, our success in developing IgG-secreting hybridomas from U-266, using hyperimmunized lymphocytes, suggests that this line may only be capable of secretion with the more differentiated cell, the human equivalent of those hyperimmunized murine spleens. Hence both sides of the fusion equation must be made optimal. Two new approaches to circumvent this problem involve the use of either a human-murine myeloma chimera as the parental myeloma line or, more recently, genetic engineering techniques to substitute human constant regions for the murine while retaining the murine hypervariable region, preserving the binding specificity of the murine antibody.

Antibodies, Monoclonal↗
PubMed1986

Relationship of the clinical response and binding of recombinant interferon alpha in patients with lymphoproliferative diseases.

Patients with hairy cell leukemia (HCL) and chronic lymphocytic leukemia (CLL) were treated with recombinant interferon alpha A (rIFN-alpha A). The binding of iodinated recombinant interferon-alpha to baseline samples of peripheral blood mononuclear cells (PBMCs) from the leukemia patients was compared with clinical responsiveness to rIFN-alpha A. HCL patients (8/10) responded to rIFN-alpha A therapy, whereas none (0/10) of the CLL patients studied responded. The PBMCs from the eight responsive HCL patients bound approximately twice as much iodinated interferon as the PBMCs from nonresponsive CLL patients. This difference was due to more high-affinity receptors per cell with no difference in the affinity of the interferon-receptor interaction. However, because PBMCs from HCL patients were larger than PBMCs from CLL patients, the cell surface receptor density was similar. The leukemic cells from one of the two nonresponsive HCL patients bound iodinated interferon similarly to the cells from the responsive HCL patients, whereas the leukemic cells from the other nonresponsive HCL patient bound considerably less. The rapidity of response of the HCL patients did not correlate with the level of binding of iodinated interferon. Our results suggest that the absolute number of interferon receptors per cell may be only one of several important parameters in the response to rIFN-alpha A therapy, and that the responsiveness of a particular lymphoproliferative disease or a particular patient to rIFN-alpha A therapy cannot be predicted or explained solely by the degree of interaction between IFN and its cell surface receptor.

Cell Separation↗
PubMed1986

Ligand-specific calcium mobilization in IL 2 and IL 3 dependent cell lines.

Calcium mobilization is one of the earliest quantitative biochemical events associated with extracellular messenger stimulation of cellular proliferation. Therefore, purified recombinant lymphokines Interleukin 2 (IL 2) and Interleukin 3 (IL 3) were examined for ligand stimulation of calcium mobilization with highly specific interleukin-dependent cloned cell lines. Although IL 2 and IL 3 share neither amino acid structural homologies nor cellular tissue specificity, both recombinant polypeptides induced rapid influx of calcium into quiescent cells bearing specific receptors for the ligand. Neither ligand was capable of stimulating calcium mobilization in the non-homologous target cells which differed in tissue lineage. Stimulation of calcium mobilization demonstrated characteristic threshold dose-response kinetics and was observed under conditions where intracytoplasmic calcium levels were spontaneously depleted upon ligand deprivation. These data confirm that in addition to the activation of protein kinase C, growth regulatory polypeptides of mature T lymphocytes (IL 2) and hematopoietic stem cells (IL 3) share common biochemical features of the ligand-receptor signal transduction apparatus which initiates cellular proliferation.

Animals↗
PubMed1986

The BRMP IL-2 reference reagent.

The BRMP has established an interim IL 2 Reference Reagent. Unitage was assigned after a collaborative, 7 laboratory study. The standard has been distributed to over 350 investigators to date.

Indicators and Reagents↗
PubMed1986

Comparative evaluation of multiple lymphoid and recombinant human interleukin-2 preparations.

Six lymphoid human interleukin-2s (nIL-2s) [four from peripheral blood mononuclear cells (PBMC) and two from JURKAT cells] and six recombinant IL-2s (rIL-2s) were obtained for comparative evaluation. The main issues addressed were possible differences among the preparations in potency in T cell growth assays and other functional assays, and the possible presence of other cytokine activities or contaminants. Each preparation was assigned a standardized IL-2 activity in reference units (RU) by comparing its T cell growth promoting activity against the Biological Response Modifiers Program IL-2 (JURKAT) reference reagent. Relative to the IL-2 unitage indicated by the suppliers, the RU varied from 110-fold less to 38.5-fold more for the various preparations. Two nIL-2s and two rIL-2s contained significant levels of endotoxin. One nIL-2 contained low levels of both alpha and gamma interferon (IFN), and one nIL-2 had a high level of gamma IFN. All other IL-2s were negative for IFN activity. All IL-2 preparations significantly augmented human natural killer (NK) activity, although the amount of RU required varied from 0.1 to 50 RU. Four nIL-2s and three rIL-2s induced human PBMC to produce gamma IFN, whereas two nIL-2s and one rIL2 did not. All nIL-2s had substantial amounts of B cell growth factor activity, whereas none of the rIL-2s consistently displayed this activity. All IL-2s stimulated the tritiated thymidine [3H]TdR incorporation of human PBMC in the absence of other stimuli, in addition to augmenting the response to mitogen or alloantigens. Some nILs and IL-2s had effects on human monocytes such as inhibiting migration, inducing cytotoxic or growth inhibitory activity against tumor cells, and causing changes in cell surface markers. The IL-2s were also tested for activity in vitro and in vivo in mice. Although there was a 12-fold variation in activity among the preparations, all but one of the IL-2s showed augmentation of the mixed lymphocyte reaction activity and all IL-2s tested stimulated macrophage cytotoxicity in vitro. All IL-2s tested enhanced the mixed lymphocyte-allogeneic tumor cell reaction resulting in greater production of cytotoxic T cells. However, significant quantitative differences in potency were evident among the various IL-2 preparations, especially the nIL-2s. Only very high doses of IL-2 (intraperitoneal injection of 100,000 RU/animal) induced in vivo augmentation of splenic or peritoneal NK cells, although all IL-2s tested increased NK activity against tumor target cells in vitro with substantially lower doses (10-100 RU/ml).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

The generation of stable human T-cell hybridomas which constitutively produce interleukin-2 and chemotactic factor.

We report the successful generation of human T-cell hybridomas that constitutively secrete lymphokines. An acute lymphoblastic leukemia T-cell line, CCRF-H-SB2, free of reverse transcriptase and mycoplasma, was sensitized to hypoxanthine, aminopterin, and thymidine (HAT) by selecting out a mutant deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT) in 8-azaguanine. Peripheral blood T lymphocytes from normal donors were incubated in vitro with 10 micrograms/ml of concanavalin A for 48 h and subsequently fused with the CCRF-H-SB2 HAT-sensitive cell line. Following 5 weeks in culture, 38 of 440 wells (8.6%) demonstrated hybridoma growth. Supernatants of these cultures were screened for interleukin-2 (IL-2), chemotactic factor, interferon, migration inhibition factor, and macrophage-activating factor activities. Twelve (of 38) hybrids exhibited IL-2 activity, and eight of these were successfully cloned. The highest secreting clone was demonstrated to have mRNA to IL-2 while the parent CCRF-H-SB2 had no detectable mRNA to IL-2. Three hybrid cultures produced chemotactic factor; one was successfully cloned and grown in serum-free medium, where it continued to constitutively produce chemotactic factor as well as IL-2 activity. The chemotactic factor was determined to have the same molecular weight (12,500 daltons) as leukocyte-derived chemotactic factor. Constitutive IL-2 production remained stable for over 12 months. None of the hybridomas tested produced detectable levels of gamma interferon, migration inhibition factor, or macrophage activation factor. Because these T-cell hybridomas produce lymphokines constitutively and this phenotype is stable, they can be an important source of highly purified human lymphokines for clinical and laboratory investigations.

Antigens, Surface↗
PubMed1985

Biochemical characterization and separation of low molecular weight urinary thymocyte and fibroblast proliferative activities.

Normal human urine was found to contain two low molecular weight (ca. 4000 and 2000 molecular weight [mw]) fractions that exhibit IL-1-like properties. In vitro, both the 2K and 4K fractions demonstrated mouse thymocyte proliferative activity in the presence of phytohemagglutinin (PHA) (i.e., LAF-like), whereas only the 2K fraction also induced proliferation of human dermal fibroblasts. A third fraction (ca. 75,000 mw), which demonstrated both biologic activities, was shown to convert exclusively to the 2K and 4K activities after treatment with the nonionic detergent CHAPS (3-[3-cholamidopropyl)-dimethylammino]-1-propane sulfonate). Liquid isoelectric focusing of the 4K urinary LAF fraction revealed that the thymocyte costimulatory activity focused at pH 5.3 and 6.7. The thymocyte proliferative activity in the 2K pool focused at pH 7.5 and 8.7, as well as at pH 5.3 and 6.7. The 2K fibroblast activity, however, focused only at pH 7.5. Anion exchange chromatography also separated the fibroblast and thymocyte comitogenic proliferative activities. The fibroblast activity eluted as two sharp peaks between 0.5 and 0.7 M NaCl, whereas the 2K and 4K thymocyte activities eluted as a broader peak with 1 M NaCl. The origin of the 2K fibroblast proliferation factor remains to be established. However, since the urinary gel filtration fractions with low mw thymocyte costimulatory activity did not support the growth of IL-2-dependent cell lines, they are presumably fragments of IL-1 rather than related to IL-2. Results of this study further suggest that the lymphoproliferative activity and fibroblast proliferative activity of interleukin-1 are not necessarily coupled.

Adult↗
PubMed1984

Interleukin 1 activity in normal human urine.

Urine concentrates from normal individuals were shown to contain interleukin 1 (IL 1)-like activity when tested directly on human dermal fibroblasts and on C3H/HeJ mouse thymocytes in the presence of 1 microgram/ml phytohemagglutinin. Seventy-five percent of the urine samples tested, however, demonstrated the presence of a specific inhibitor of IL 1-promoted thymocyte proliferation. This inhibitor did not affect IL 2-promoted proliferation of mouse thymocytes or CT-6 cells or IL 1-promoted proliferation of human dermal fibroblasts. After gel filtration of the urine concentrates, even those samples that were inhibitory yielded fractions containing both thymocyte and fibroblast proliferative activity. The approximate m.w. of these activities were 75,000 and 15,000. In addition, two peaks of low m.w. thymocyte proliferative activity were noted at 4000 and 2000. The 2000 pool, but not the 4000 pool, also contained fibroblast proliferative activity.

Adult↗
PubMed1981

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice.

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.

Animals↗
PubMed1979

The chemistry and biology of thymosin. I. Isolation, characterization, and biological activities of thymosin alpha1 and polypeptide beta1 from calf thymus.

A partially purified extract from thymus tissue termed thymosin Fraction 5 has been shown to reconstitute immunological deficiencies resulting from the lack of thymic function in several animal models, as well as humans with primary and secondary immunodeficiency diseases. Thymosin Fraction 5 consists of a family of polypeptides with molecular weights ranging from 1,000 to 15,000. Several of these polypeptides contribute individually to the biological activity of the parent compound. Two polypeptide components of thymosin Fraction 5, termed thymosin alpha1 and polypeptide beta1, have been characterized chemically and biologically. Thymosin alpha1 is a highly acidic molecule composed of 28 amino acid residues. This polypeptide has potent biological activity and has been found to be 10 to 1,000 times as active as thymosin Fraction 5 in one in vivo and several in vitro bioassay systems designed to measure differentiation and function of thymus-dependent lymphocytes (T cells). Polypeptide beta1, in contrast, is inactive in our bioassay systems, suggesting that it is not involved in thymic hormone action. Sequence analysis and homology studies have indicated that polypeptide beta1, although present in Fraction 5, does not contribute to the biological activity of thymosin Fraction 5.

Animals↗
PubMed1979

Lymphokine-induced changes in macrophage transmembrane electrical potentials.

Intracellular electrical potentials have been measured in nonactivated and immunologically-activated macrophages obtained from the peritoneal cavities of mice. Normal macrophage potentials were established and found to become significantly more electronegative after in vitro exposure for 10 minutes to a lymphokine-containing supernatant which induced macrophage activation. This approach may reflect very early concomitants of such activation and is also useful in the study of other immunologic systems.

Animals↗
PubMed1978

Overview of thymosin activity.

Our current research program centers around the biologic and chemical characterization of the family of polypeptides present in thymosin fraction 5. A system of nomenclature has been developed and the peptides are being systematically isolated and chemically characterized. Thymosin fraction 5 and its component parts influence a variety of lymphocyte properties including cyclic nucleotide levels, migration inhibitory factor production, T-dependent antibody production, and expression of certain surface markers. Thymosin is being used in clinical trials to investigate its effects on immunodeficiency diseases, malignant diseases, and autoimmune diseases.

Animals↗
PubMed1977

Polyradiculoneuropathy accompanying procainamide-induced lupus erythematosus: evidence for drug-induced enhanced sensitization to peripheral nerve myelin.

Factors involved in the development of an insidious polyradiculoneuropathy in association with a procainamide-induced, lupuslike syndrome were explored. A 73-year-old man with this clinical syndrome had sural nerve changes consisting of loss of large myelinated fibers with evidence of remyelination and Schwann cell proliferation. The patient's lymphocytes showed marked incorporation of tritiated thymidine when cultured with either procainamide or extracts of human peripheral nerve myelin, and there was an enhanced response with the combination. We also found that procainamide-treated rats showed acceleration of lymphocyte sensitization to peripheral nerve myelin as judged by the early development of inhibition of macrophage migration and positive skin tests to extracts of peripheral nerve myelin. These studies suggest that procainamide can enhance lymphocyte sensitization to peripheral nerve myelin and may have predisposed this individual to development of a polyradiculoneuropathy.

Aged↗