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Biomedical subjects

E R Podack

Publications and source records attributed to E R Podack.

At least 73 records · Page 4Linked to original sources
PubMed1995

Deoxyribonuclease induction in apoptotic cytotoxic T lymphocytes.

IL-2-dependent CTLL2 cells, upon IL-2 deprivation, die by apoptosis, which is accompanied by the fragmentation of genomic DNA. Two major deoxyribonuclease activities were detected in the extract of IL-2-deprived CTLL2 cells in a zymographic assay. They were designated nuc-58 and nuc-40, based on their apparent molecular mass of 58 and 40 kDa. The activity of both DNases was greatly induced in CTLL2 cells deprived of IL-2 or treated with the kinase inhibitor staurosporine. Deregulated expression of bcl-2 cDNA suppressed the induction of both nuclease activities. Nuc-58 was dependent on both Ca2+ and Mg2+ ions alone. Nuc-40 showed a preferential nuclear localization over that of nuc-58, which was found primarily in the cytoplasm. Optimal activity of both DNases required neutral pH and was inhibited by zinc ions. The physicochemical characteristics of the nucleases indicate that they are novel DNases associated with apoptosis in CTLL2 cells.

Animals↗
PubMed1995

Perforin expression in endometrium during the menstrual cycle.

Perforin is a marker of functionally active cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We examined perforin expression in endometrium throughout the menstrual cycle. In addition, perforin expression was studied in endometrial leukocytes in women with anovulatory cycles with or without progesterone therapy and in postmenopausal women. In the endometrium, perforin-positive endometrial lymphocytes increased in numbers in the middle through late secretory phases. In women with anovulatory cycles, the proliferative endometrium, before estrogen withdrawal endometrial breakdown, contained less perforin-positive lymphocytes compared with the premenstrual endometrium of the normal menstrual cycle. The progestin-induced endometrial decidualization was accompanied by an extensive recruitment of perforin-positive cells. In contrast, the postmenopausal atrophic endometrium showed complete absence of perforin-positive cells. The lymphocytes isolated from secretory endometrium were CD3-, CD56+ (approximately 80%) or CD3+, CD8+ T-cell receptor (TCR)-gamma delta- (approximately 20%). Almost 90% +/- 5.5 of the CD3-, CD56+, and up to 25% +/- 7% of CD8+ T lymphocytes contained perforin. Perforin expression was further confirmed in endometrial lymphocytes by Northern blot analysis. In vitro, isolated endometrial lymphocytes exhibited NK-like cytotoxicity. After cyclic hormone withdrawal during normal menstrual cycle, these perforin-positive cytotoxic cells may be involved in endometrial stromal breakdown during menstruation.

Blotting, Northern↗
PubMed1995

Perforin expression in human cell-mediated luteolysis.

After ovulation and in the absence of fertilization, the human corpus luteum regresses in an orderly sequence of morphological changes. This study demonstrated that luteal regression involved progressive infiltration of lymphocytes and macrophages. The inflammatory infiltrate began in the theca externa and gradually invaded granulosa cells, with maximum accumulation of lymphocytes and macrophages at the time of menstruation. Immunoperoxidase staining showed that the majority of lymphocytes were CD2+, CD3+, CD8+ T lymphocytes, and 15% of these T cells expressed perforin, a cytolytic protein implicated as a mediator of cytotoxicity. The remaining mononuclear infiltrate showed strong reactivity with monocyte/macrophage markers. These findings indicate that (a) a physiologic cell-mediated inflammatory process in the regressing human corpus luteum is mediated mainly by CD8+ T lymphocytes and cells of monocyte/macrophage lineage and (b) perforin expression in T lymphocytes supports a possible role for cytolytic T cells during the physiologic inflammatory response in human luteolysis.

Corpus Luteum↗
PubMed1995

Characteristics of perforin expressing lymphocytes within the first trimester decidua of human pregnancy.

PROBLEM: The number of perforin (P)-positive cells in decidua of pregnancy is larger than that observed in any other pathological condition. The aim was to investigate the distribution and the phenotype of P+ cells. METHOD: Decidual tissue was obtained from the first trimester vaginal termination of pregnancy. Tissue distribution of P+ cells was analyzed by immunohistochemistry. The method for simultaneous measurement of P and cell surface is presented. RESULTS: There is no difference in number and distribution of P+ cells between decidua basalis (DB) and decidua parietalis (DP). The percentage of P+ decidual lymphocytes (DL) is two times higher than in peripheral blood lymphocytes (PBL) (55% vs. 27%), and the prevalent phenotype is CD3- CD4- CD8- CD2+ (95%) CD11c+ (68%) and CD56+ (82%). CD56bright+ DL are also Pbright+ and this is the largest DL subpopulation (42.4% DL). Two different subpopulations of CD8+ DL exist: 1) CD8bright+, which are CD3+ CD56- P- and 2) CD8dim+, which are CD3- CD56+ P+. CONCLUSION: P expressing DL are prevalently nonclassical NK cells (CD16-) with low cytolytic activity but fully equipped with potent cytolytic machinery (Pbright+). There are no classical cytotoxic lymphocytes (CTL) (CD3+ CD8+ P+) in the decidua, and all CD8+ P+ cells are CD3- CD56+. The number of P+ cells is even higher in DP in the vicinity of noninvasive trophoblast, than in DB.

Biomarkers↗
PubMed1995

Analysis of perforin expression in human peripheral blood lymphocytes, CD56+ natural killer cell subsets and its induction by interleukin-2.

In this study new evidence is obtained by the use of an anti human perforin monoclonal antibody (mAb), anti P1, concerning the number of perforin positive cells in human peripheral blood lymphocytes (PBL). It is shown that about 23% of PBL is perforin positive and that this percent increases by the treatment in RPMI 1640 medium alone to 33% and with 1000 U r hIL-2 to 46%. Assessment of the cytotoxicity potential of NK cells from PBL, freshly isolated and treated, against tumor cell line K562 by the standard NK cell 4-hr 51-chromium release assay, indicates a significant enhancement in their cytotoxicity. By FACStar sorting and analysis of the CD56+ NK cell population new evidence is obtained which shows that about 25-30% of this population represents the CD56bright+ subset, while 70-75% represents the CD56dim+ subset. As the two subsets were shown to differ functionally they were stained with anti P1 for the evaluation of perforin content and it was found that both of them are positive for perforin from 97-99%, suggesting that the functional difference is not due to perforin content. In this sense, as NK cells are constitutively positive for perforin, the increase in the cytotoxicity of NK cells induced by IL-2 is most likely due to the synthesis and expression of various adhesion molecules on NK cells which increase their cytotoxic potential, as well as, that the detected increase in the number of perforin positive cells by this lymphokine does not belong to NK, but to the T lymphocyte population. The data obtained in this study indicate the possibility of perforin detection in human lymphocytes by an anti human perforin mAb and the change in the number of perforin positive cells after stimulation with interleukin-2.

Antibodies, Monoclonal↗
PubMed1995

Perforin- and Fas-mediated cytotoxic pathways are not required for allogeneic resistance to bone marrow grafts in mice.

Failure to engraft is a major complication in allogeneic bone marrow transplantation (BMT) using T cell-depleted donor marrow. Previous work has demonstrated that radioresistant host natural killer (NK) cells, CD8+ T cells, and T cells with NK markers participate in the active rejection of donor hematopoietic stem cells in murine models of allogeneic BMT. However, the precise role of cell-mediated cytotoxic mechanisms in marrow allograft rejection remains controversial. To determine the role of perforin- and Fas-mediated cytotoxicity in allogeneic resistance, we transplanted T cell-depleted allogeneic bone marrow into perforin-deficient (perforin 0/0), Fas-ligand-defective (gld/gld), and normal mice. Short-term resistance was measured using a sensitive in vitro assay for colony formation by spleen cells from BMT recipients. The findings we report here demonstrate that strong allogeneic resistance remains largely intact in perforin-deficient and Fas-ligand-defective recipient mice. Thus, perforin- and Fas-mediated cytotoxic pathways are not required for resistance to bone marrow allografts in mice. We conclude that alternative pathways of cytotoxicity and/or soluble factors can mediate resistance to allogeneic BM.

Animals↗
PubMed1994

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.

AIDS Vaccines↗
PubMed1994

Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.

Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

Amino Acid Sequence↗
PubMed1994

Soluble and membrane-bound TNF-alpha are involved in the cytotoxic activity of B cells from tumor-bearing mice against tumor targets.

Splenic B cells from BALB/c mice bearing mammary adenocarcinomas are capable of performing Ab-dependent cellular cytotoxicity. Effector-target conjugation after 18 h results in minimal cytoplasmic damage, whereas extensive nuclear disintegration is observed. To determine whether splenic B cells from tumor-bearing mice can effect direct cytotoxicity against tumor cells, L929 and WEHI 164 cells were used as targets. B lymphocytes from tumor-bearing mice, but not from normal animals, were capable of lysing these two types of tumor cells. However, only a low level of cytotoxicity could be detected when the nontumorigenic 3T3 cells were used as targets. To elucidate the mechanism of cytotoxicity of these killer B cells, RNase protection assays were performed using perforin, granzyme A, TNF-alpha, and lymphotoxin probes. No perforin, granzyme A, or lymphotoxin RNA could be detected in purified preparations of B cells from normal and tumor-bearing mice. B cells from normal mice did not have TNF-alpha RNA. In contrast, B cells from tumor bearers expressed TNF-alpha RNA. TNF-alpha could be detected in supernatants from both unstimulated and stimulated tumor bearers' splenic B cells, as measured by ELISA, and its lytic activity was neutralized by anti-TNF-alpha Ab. Western blots revealed the presence of TNF-alpha on the surface of the killer B cells. Paraformaldehyde-fixed B cells from tumor-bearing mice but not from normal animals were able to lyse TNF-alpha-sensitive tumor targets. This cytotoxicity was neutralized by anti-TNF-alpha Ab. These results suggest that TNF-alpha in soluble and membrane-bound forms may be involved in the mechanism of cytotoxicity exerted by B cells from tumor-bearing mice.

Animals↗
PubMed1994

In vitro and in vivo growth of B16F10 melanoma cells transfected with interleukin-4 cDNA and gene therapy with the transfectant.

In an attempt to develop the most effective cytokine gene therapy, we transfected mouse interleukin(IL)-2, mouse IL-4, and human IL-6 cDNAs into mouse melanoma cells, B16F10. Transfection with IL-4 cDNA decreased the tumorigenicity of B16F10 most strongly. We investigated whether gene therapy with IL-4-transfected B16F10 cells was possible. Flow-cytometric analysis showed that major histocompatibility complex class I and II expression in B16F10 and IL-4-cDNA-transfected B16F10 (B16F10-IL4) cells did not differ. Doubling times of B16F10 and B16F10-IL4 were 20.1 and 21.1 h respectively. The growth of B16F10 cells was retarded if C57BL/6 mice were inoculated with B16F10-IL4 at the contralateral sides. When 5 x 10(5) B16F10 cells were transplanted subcutaneously into the flanks of C57BL/6 mice, they all developed a tumor mass, whereas no tumor masses formed in those transplanted with B16F10-IL4 cells within 60 days. No nude, severe combined immunodeficient or beige mice were able to reject parental B16F10 or B16F10-IL4 cells, although, B16F10-IL4 tumor growth in all these immunodeficient mice was slower than that of B16F10. Therefore, we concluded that T and natural killer cells are necessary for rejection of B16F10-IL4 tumor cells.

Animals↗
PubMed1994

Perforin expression by thyroid-infiltrating T cells in autoimmune thyroid disease.

Infiltration of the thyroid gland by lymphocytes is a hall-mark of autoimmune thyroid disease; it is particularly evident in Hashimoto's thyroiditis but is also seen in most patients with Graves' disease. Infiltrating cells are comprised primarily of T lymphocytes, of which only a minority appears to be activated. Their precise pathogenic role is largely unknown. Since perforin has been a marker for functionally activated cytotoxic T cells in situ we elected to assess the presence of perforin-containing cells in thyroid-infiltrating lymphocytes and establish their phenotype. Cells were isolated from seven subtotal thyroidectomy specimens, five from patients with Graves' disease and two with Hashimoto's thyroiditis. The novel findings were as follows: CD4+ perforin-containing T cells occurred only in Hashimoto's glands, suggesting a class II-restricted component of cytotoxicity; in Graves' disease, and to a lesser extent in Hashimoto's, perforin-expressing cells were primarily T cell receptor alpha beta- CD4-CD8- (double negative); double negative perforin-containing cells in peripheral blood of normal individuals were largely gamma delta + T cells. In Hashimoto's samples, the predominant population of T cells expressing perforin was CD8+. By comparison, in studies of the synovial fluid of knee joints from patients with rheumatoid arthritis only a minor population of the perforin-containing cells was double-negative. The data suggest significant differences in cytotoxic autoimmune mechanisms between the two autoimmune thyroid diseases. Functional characterization of double-negative T cells is necessary to define their role in autoimmunity.

Arthritis, Rheumatoid↗
PubMed1994

Gene therapy for Lewis lung carcinoma with tumor necrosis factor and interleukin 2 cDNAs co-transfected subline.

Gene therapy with cytokine cDNA will provide a new tool for cancer treatment. We have already reported that immunization with interleukin-2 (IL2) cDNA transfected Lewis lung carcinoma (LLC) cells induced anti-tumor immunity, which, however, was not strong enough to eradicate an established tumor. In an attempt to develop more effective gene therapy methods, we have used tumor cells co-transfected with IL-2 and tumor necrosis factor (TNF) cDNAs. These cDNAs were introduced into pBMG-Neo and pcDV-X819 vectors, respectively, and then co-transfected into LLC cells. The co-transfectants were selected by incubating them in a medium containing G418 followed by the limiting dilution method twice to obtain IL2 and TNF cDNA co-transfected LLC (LLC-TNF-IL2) cells. When 5 x 10(5)/ml LLC-TNF-IL2 cells were incubated for 48 h, they secreted 7.56 U/ml TNF and 527.0 U/ml IL2 into the culture supernatant. When C57BL/6 mice were transplanted with 1 x 10(6) LLC-TNF-IL2 cells, all the tumors were rejected. The growth of transplanted LLC, but not B16F10 melanoma cells, was retarded in mice inoculated with LLC-TNF-IL2 on their contralateral sides, which suggests specific immunity was induced. The immunization effect by the co-transfectant was superior to that of the IL2- and TNF-transfectants alone.

Animals↗
PubMed1993

Functional effects of CD30 on a large granular lymphoma cell line, YT. Inhibition of cytotoxicity, regulation of CD28 and IL-2R, and induction of homotypic aggregation.

Studies are described revealing novel regulatory functions for the lymphocyte activation Ag CD30. A new mAb, C10, reactive with YT cells binds to CD30 and induces inhibition of the cytotoxicity of YT for Raji cells. C10 inhibition of cytotoxicity requires several hours preincubation of YT with C10; the antibody has no effect if added directly to YT cytotoxicity assays. CD30 stimulation by C10 down-regulates CD28 expression on YT by > 80% within 48 h. Because CD28 is required for YT cytotoxicity toward Raji cells and other B7/BB1 bearing targets, it is suggested that inhibition of cytotoxicity of YT is mediated by control of CD28 expression and/or signaling via CD30. Accordingly, conjugation of YT with Raji is only slightly affected by CD30-mediated down-regulation of CD28, and perforin mRNA steady state levels are not changed at all. C10 treatment of YT cells additionally down-regulates the expression CD45 and up-regulates IL-2R p55. Moreover, CD30 stimulation by C10 causes homotypic aggregation of YT. Homotypic aggregation is slow, requiring gene transcription, translation, metabolic energy at elevated temperature (37 degrees C), magnesium ions, and an intact cytoskeleton. These studies offer insights into the function of CD30 as a complex regulator of T cells.

Amino Acid Sequence↗
PubMed1993

Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2.

To determine whether the apoptotic cell death induced by anti-cancer agents could be inhibited by bcl-2, we established a bcl-2-transfected human small cell lung cancer cell line, SBC-3/Bcl2. SBC-3/Bcl2 showed higher resistance to ADM, CPT-11 and MMC compared with the parental line SBC-3, with relative resistance values of 3.4, 7.6 and 5.7, respectively. However, there was no difference in sensitivity to CDDP, VP-16, ACNU, MTX and taxol between SBC-3 and SBC-3/Bcl2. Agarose gel electrophoresis showed typical DNA fragmentation of SBC-3 following treatment with CPT-11 or MMC, in a concentration-dependent manner. In contrast, the same concentration of the drugs did not induce DNA fragmentation in SBC-3/Bcl2. Treatment with CDDP resulted in the same degree of DNA fragmentation in SBC-3 and SBC-3/Bcl2. These studies indicate that bcl-2 can modulate the cytotoxicity of some anti-cancer agents by inhibiting the process of apoptosis.

Animals↗
PubMed1993

Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2.

Absence of interleukin 2 (IL-2) from IL-2-dependent cells, such as the cytotoxic T-cell line CTLL2, causes DNA fragmentation and programmed cell death (apoptosis). We found that, upon initiation, DNA degradation proceeds rapidly. IL-2-deprived CTLL2 cells can be rescued from apoptosis by the addition of IL-2 2 h prior to the onset of detectable DNA breakdown. Addition of inhibitors of transcription with IL-2 abolished the IL-2-mediated rescue of CTLL2 cells. Thus it appears that IL-2-mediated gene transcription is necessary for survival. Deregulated expression of a protooncogene, bcl-2, inhibits apoptosis of cells dependent on other hematopoietic growth factors. To determine whether bcl-2 was active in CTLL2 cells, we transfected CTLL2 cells with a plasmid containing bcl-2 cDNA expressed under the metallothionein promoter and observed prolonged survival of the transfected cells upon IL-2 deprivation. Cell growth, however, was arrested in the G0/G1 or G2/M phases of the cell cycle. The prolonged survival of bcl-2 transfectants allowed the analysis of endogenous bcl-2 mRNA levels by Northern blot analysis. The expression of endogenous bcl-2 was down-regulated within 8 h of IL-2 withdrawal and was not detected after 3 days. Addition of IL-2 induced endogenous bcl-2 expression within 8 h. Full recovery of bcl-2 expression was achieved by 24 h after IL-2 addition. We conclude that the survival of death-prone CTLL2 cells may be viewed as IL-2-dependent suppression of suicide, probably by the IL-2-induced expression of the cellular bcl-2 gene.

Actins↗
PubMed1993

Combination effect of vaccination with IL2 and IL4 cDNA transfected cells on the induction of a therapeutic immune response against Lewis lung carcinoma cells.

In order to develop a more effective method of immunotherapy we have transfected mouse interleukin-2 (IL2) or mouse interleukin-4 (IL4) cDNA into a spontaneous non-immunogenic murine lung cancer. Lewis lung carcinoma (LLC). IL2 cDNA transfection more strongly decreases tumorigenicity of LLC than IL4 cDNA transfection. Recombinant-human-IL2 treatment of mice that were transplanted with untransfected LLC could not prolong their survival. In contrast, vaccination with IL2-cDNA-transfected LLC (LLC-IL2) and LLC-IL2 mixed with IL4-cDNA-transfected LLC (LLC-IL4) could significantly suppress tumor growth of LLC in a tumor-specific manner. The vaccination with LLC-IL2 mixed with the same number of LLC-IL4 cells was more suppressive to the growth of LLC than that with LLC-IL2 cells alone, while LLC-IL4 vaccination alone was ineffective. Nude, severe-combined-immune-deficient (SCID) and beige mice were unable to reject LLC-IL2 cells. However, immunodeficient mice responded to LLC-IL2, but not to LLC, since their survival times after transplantation with LLC-IL2 cells were significantly longer than the survival time of normal or immunodeficient mice transplanted with untransfected LLC cells. We conclude that vaccination with IL2-producing tumors and, with more pronounced effect, in combination with IL4-producing tumors, is able to induce an immune response to this normally non-immunogenic tumor. Tumor rejection appears to be achieved by the combined activity of CTL and NK cells. This strategy has potential for new immunotherapeutic interventions in cancer patients.

Animals↗
PubMed1993

Regulation of perforin gene expression in a T cell hybrid with inducible cytolytic activity.

A mouse x rat T cell hybrid (PC60) that does not require interleukin (IL)-2 for proliferation, was used as a model to study regulation of perforin gene expression. Perforin mRNA is barely detectable in non-induced PC60 cells; however, a 30-fold induction is observed after stimulation with IL-1 alone. Peak perforin mRNA levels were reached after 10 h of induction with IL-1, and these levels were maintained for as long as the stimulus was present. IL-2 by itself has no detectable effect. However, in combination with IL-1 it shows the same induction kinetics as IL-1 alone for the first 10 h, subsequently there is synergism (100-fold induction) between IL-1 and IL-2. The induction response was mainly due to increased transcriptional rates of the perforin gene, and require newly synthesized proteins. The half-life of perforin mRNA in this system is about 5 h. In addition, we confirm the existence of two types of mouse perforin mRNA that differ in their 5' untranslated regions, and show evidence that both mRNA are translated in vivo with similar efficiencies.

Animals↗