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Biomedical subjects

F W Ruscetti

Publications and source records attributed to F W Ruscetti.

212 records · Page 12Linked to original sources
PubMed1978

Long-term culture of human antigen-specific cytotoxic T-cell lines.

Long-term cultures of human cytotoxic T-cell lines (H-CTLL) were established. H-CTLL cells were strictly dependent on growth upon a T-cell growth factor (TCGF) produced by phytohemagglutinin-stimulated normal human peripheral blood lymphocytes. H-CTLL cells were maintained in TCGF-dependent exponential proliferative culture for over 4 mo during which time they continued to mediate stimulator antigen-specific cytotoxicity as measured by a 4-h 51Cr-release assay. H-CTLL cells recovered from cryopreserved stocks and re-established in long-term culture demonstrated similar high levels of antigen-specific cytotoxicity. H-CTLL cells were 95--100% E-rosette positive and expressed normal T and Ia-like cell surface markers. The ability to sustain differentiated antigen-specific T-effector cells in long-term culture may provide a new means for the study of both the mechanism and regulation of T-cell-mediated immunity.

Antigens↗
PubMed1978

Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds.

A human leukemic cell line (designated HL-60) has recently been established from the peripheral blood leukocytes of a patient with acute promyelocytic leukemia. This cell line displays distinct morphological and histochemical commitment towards myeloid differentiation. The cultured cells are predominantly promyelocytes, but the addition of dimethyl sulfoxide to the culture induces them to differentiate into myelocytes, metamyelocytes, and banded and segmented neutrophils. All 150 clones developed from the HL-60 culture show similar morphological differentiation in the presence of dimethyl sulfoxide. Unlike the morphologically immature promyelocytes, the dimethyl sulfoxide-induced mature cells exhibit functional maturity as exemplified by phagocytic activity. A number of other compounds previously shown to induce erythroid differentiation of mouse erythroleukemia (Friend) cells can induce analogous maturation of the myeloid HL-60 cells. The marked similarity in behavior of HL-60 cells and Friend cells in the presence of these inducing agents suggests that similar molecular mechanisms are involved in the induction of differentiation of these human myeloid and murine erythroid leukemic cells.

Butyrates↗
PubMed1977

Functional and morphologic characterization of human T cells continuously grown in vitro.

Long-term growth (now over 13 months) of thymus-derived lymphocytes from numerous normal human bone marrow and peripheral blood cell samples was accomplished by using a factor present in media obtained from mitogen-stimulated human peripheral blood lymphocytes. This long-term growth could neither be initiated nor maintained by mitogens alone. All cell cultures were greater than 90% E rosette-positive, whereas the tests for B cell markers, surface IgG and IgM, and EAC rosette were routinely negative. There was no evidence for the presence of granulocytes, monocytes, and their precursors in these cultures. The E rosette-positive cells were then tested to see if they had T cell functions. PHA, Con A, and pokeweed mitogens stimulated lymphproliferative responses in these cultures comparable to those of fresh peripheral blood cells. These proliferating cells were also able to release cell mediators, such as interferon and colony-stimulating activity. Further evidence for the T lymphocyte nature of these cultured cells was obtained from one-way mixed leukocyte cultures in which these cells responded to but were unable to stimulate allogeneic cells. The functional and morphologic characteristics of these cultured cells show that these cells are T cells that grow continuously in vitro.

Cells, Cultured↗
PubMed1976

Selective in vitro growth of T lymphocytes from normal human bone marrows.

Selective growth of T lymphocytes occurred when unfractionated normal human bone marrow cells were cultured with conditioned medium obtained from phytohemagglutinin-stimulated normal human lymphocytes (Ly-CM). Cultures of up to 90 percent T cells have been maintained for more than 9 months. The T cells exhibited a strict growth dependence upon Ly-CM and were consistently negative for Epstein-Barr viral information.

Bone Marrow Cells↗
PubMed1976

Specific release of neutrophillic- and eosinophilic-stimulating factors from sensitized lymphocytes.

Mitogen-stimulated lymphocytes recently have been shown to release colony-stimulating activity (CSA), a stimulator of granulopoiesis in vitro. Whether CSA release is a result of specific antigen-lymphocyte interactions has been investigated by determining the ability of spleen lymphocytes isolated from Trichinella spiralis and Bacille Calmette Guerin (BCG) infected mice to release CSA. Lymphocytes were incubated with different antigens to obtain conditioned media (CM). Conditioned media prepared from Trichinella-sensitized lymphocytes incubated with Trichinella antigen (CM-1) resulted in a fourfold increase in the number of colonies from murine marrow cultured in methylcellulose as compared to controls. The addition of Trichinella antigen to either nonsensitized or BCG-sensitized lymphocytes did not stimulate CSA release. Similar results were obtained with lymphocytes from BCG-infected mice. Liquid cultures stimulated with CM resulted in an increase in 3h-thymidine incorporation and cell numbers. Using CM-1 to stimulate marrow from Trichinella-infected mice resulted in a three- to fourfold increase in the number of eosinophils present after 4 days. All other CMs tested stimulated only neutrophil production...

Animals↗
PubMed1975

Release of colony-stimulating activity from thymus-derived lymphocytes.

Colony-stimulating activity (CSA) is essential for in vitro differentiation of bone marrow cells into colonies of granulocytes and mononuclear cells. While blood monocytes and macrophages are a major source of CSA, recent studies have indicated that CSA may be produced by lymphocytes responding to immunologic stimulation. Lymphocytes, purified from spleens and thymuses of mice by glass wool columns, were incubated in CMRL-1066 medium with fetal calf serum in vitro. Lymphocytes from the thumus and spleen released CSA when cultured in vitro, with peak levels of CSA observed after 7 days of incubation. Stimulation of cultures with phytohemagglutinin, concanavalin A, or pokeweed mitogen resulted in a 2-5-fold increase in CSA release, with peak levels of CSA released after 4 days of incubation. Thymus-dependent lymphocytes were responsible for the release of CSA from unstimulated and mitogen-stimulated cultures, since the incubation of these cultures with rabbit anti-mouse T cell sera abolished their ability to release CSA. Anti-mouse B cell sera had no effect on the ability of lymphocyte cultures to release CSA. These studies suggest that thymocytes and thymus-derived lymphocytes can release CSA in vitro and may be responsible for the increase in CSA observed in certain immunologic reactions.

Animals↗
PubMed1975

Regulation of the release of colony-stimulating activity from mitogen-stimulated lymphocytes.

Lymphocytes stimulated by Concanavalin A (Con A) as well as other mitogens release colony stimulating activity (CSA) which stimulated granulocyte and mononuclear cell formation from hematopoietic progenitor cells in vitro. Removal of Con A from lymphocyte cultures by the competitive inhibitor, alpha methyl mannoside, resulted in the cessation of CSA release regardless of the time the inhibitor was added to the culture. However, removal of Con A after 6 hr of incubation had no effect on cellular DNA synthesis and lymphocyte transformation. In addition, vinblastine and cytosine arabinoside completely inhibited DNA synthesis but had no effect on CSA release. CSA release is dependent on an active energy process since it was completely and reversibly inhibited by the inhibitors of protein synthesis puromycin and cycloheximide. These results suggest that CSA release from stimulated lymphocytes requires continuous binding of mitogen and active protein synthesis but is not dependent on DNA synthesis and cell proliferation.

Animals↗
PubMed1972

Accumulation of 70S monoribosomes in Escherichia coli after energy source shift-down.

When Escherichia coli is shifted from glucose-minimal to succinate-minimal medium, a transient inhibition of protein synthesis and a time-dependent redistribution of ribosomes from polysomes to 70S monosomes occurs. These processes are reversed by a shift-up with glucose. In a lysate made from a mixture of log-phase and down-shifted cells, the 70S monosomes are derived solely from the down-shifted cells and are therefore not produced by polysome breakage during preparation. This conclusion is supported by the absence of nascent proteins from the 70S peak. The monosomes are not dissociated by NaCl or by a crude ribosome dissociation factor, so they behave as "complexed" rather than "free" particles. When down-shifted cells are incubated with rifampin to block ribonucleic acid (RNA) synthesis, the 70S monosomes disappear with a half-life of 15 min. When glucose is also added this half-life decreases to 3 min. The 70S particles are stable in the presence of rifampin when chloramphenicol is added to block protein synthesis. We interpret these data to mean that the existence of the 70S monosomes depends on the continued synthesis of messenger RNA and their conversion to free ribosomes (which dissociate under our conditions) is a result of their participation in protein synthesis. Finally, a significant fraction of the RNA labeled during a brief pulse of (3)H-uracil is found associated with the 70S peak. These results are consistent with the hypothesis that the 70S monosomes are initiation complexes of single ribosomes and messenger RNA, which do not initiate polypeptide synthesis during a shift-down.

Bacterial Proteins↗
PubMedDate not supplied

Sequence diversity of SIV(Mne) Nef in vivo and in vitro.

We have compared nef gene sequences isolated by PCR from peripheral blood lymphocyte DNA of macaques which had been inoculated with either biologically or molecularly cloned SIV(Mne). Two samples from each animal obtained either early after infection (week 2-8) or after significant CD4+ depletion (week 21-137) were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two more in all but one. Two of the common exchanges are located about 40 residues apart in the Nef core sequence, but are in proximity on the tertiary structure as judged by computer modelling using the structure of the HIV Nef core protein as a guide. Most recurring in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIVsm/HIV-2 groups. Animals inoculated with virus already containing the "late version" nef gene developed a more aggressive disease. The macaque adapted (MA)nef conferred a threefold higher infectivity to the cloned virus, but had no effects on CD4 downregulation. Propagation of virus with MAnef in tissue culture resulted in the rapid emergence of variants with newly attenuated nef. These findings suggest that the selective pressure on nef in vivo and in vitro are different.

Amino Acid Sequence↗
PubMedDate not supplied

Hematopoietic switch from lymphoid to granulocytic development in 3LL tumor-bearing mice.

A significant splenomegaly and lymphadenopathy develops during the progressive growth of Lewis Lung (3LL) tumors in mice. Enlarged spleen and lymph nodes occur because of a pronounced increase in granulocytes in these organs. This granulocytosis in spleen and lymph node was not simply due to recruitment of granulocytes from peripheral blood to spleen and lymph nodes, but also a result of development and/or differentiation of granulocytes from the bone marrow. There was a marked increase in development of myeloid lineage cells, whereas lymphoid populations including T cells and B cells, were dramatically decreased in bone marrow and peripheral blood of 3LL tumor-bearing mice. These data demonstrate that host hematopoiesis shifts from lymphoid to granulocytic development in the 3LL tumor-bearing mice. Interestingly, a somatic mutation of N-Ras gene was found in 3LL tumor cells at codon 61, suggesting that mutated N-Ras may contribute to induction of granulocytosis in 3LL tumor-bearing mice.

Animals↗
PubMedDate not supplied

Phytohemagglutinin-induced human T-lymphocyte colonies: cellular and T-cell growth factor requirements are the same as for suspension culture growth.

Both T-cell colony formation and T-cell growth in liquid culture requires phytohemagglutinin, either adherent cells or conditioned medium from mononuclear cells, and perhaps other cooperating cells. Despite these similarities, there are some data that the two systems may differ. While it is clear that a source of T-cell growth factor (TCGF) is an absolute requirement for the growth of T cells in liquid culture, less is known of the factors influencing T-cell colony formation. In these studies, production of TCGF during T-cell colony formation was determined. High levels of TCGF can be extracted from methylcellulose when colony formation is rapidly increasing. When colony size is at its maximum, measurable levels of TCGF decline and after 2 weeks little or no TCGF is detectable, suggesting that the factor is used by colony-forming cells. The only requirement for these colony-forming cells to form secondary colonies in replating experiments is exogenous TCGF- The need for adherent cells in primary colony formation can be substantially replaced by interleukin 1, a factor which is produced by adherent cells and functions by amplifying TCGF production. Other promoters of T-cell colony formation may also amplify TCGF production. Addition of dexamethasone to the assay decreases the number and size of the T-cell colonies formed by 75-90%, and colony formation can be completely restored by the addition of purified TCGF. The addition of antigen, lectin, 12-O-tetradecanoyl-phorbol-13-acetate or interleukin 1 cannot overcome the specific dexamethasone-mediated inhibition of colony formation. Although there may be additional requirements for colony formation depending upon the T-cell subset and state of differentiation of the cells, it appears that TCGF is the ultimate proliferative signal in both liquid and colony T-cell growth.

Cell Division↗