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Biomedical subjects

E R Podack

Publications and source records attributed to E R Podack.

At least 91 records · Page 5Linked to original sources
PubMed1993

Interleukin-6 cDNA transfected Lewis lung carcinoma cells show unaltered net tumour growth rate but cause weight loss and shortened survival in syngeneic mice.

HuIL-6 cDNA, cloned into a neomycin resistant conferring expression vector, BMGNeo, was transfected into Lewis Lung Carcinoma (LLC) cells. LLC cells (5 x 10(6) ml-1) transfected with IL-6 cDNA (LLC-IL6) secreted IL-6 into the culture supernatant at a concentration of 9.9 ng ml-1 within 48 h. When 1,000,000 of untransfected LLC, BMGNeo vector transfected LLC (LLC-Neo) or LLC-IL6 cells were transplanted into C57BL/6 mice subcutaneously, the mean +/- s.d. of survival times of these mice were 33.3 +/- 9.7, 34.3 +/- 7.1 and 17.0 +/- 3.1 days, respectively. The survival time of LLC-IL6 cells transplanted mice was significantly shorter than that of LLC (P < 0.01) or LLC-Neo (P < 0.01) cells transplanted mice without a measurable difference of tumour size. Plasma concentration of IL-6 steadily increased in LLC-IL6 transplanted mice. Body weight and serum albumin were significantly lower in LLC-IL6 transplanted mice than in LLC transplanted mice. Mouse IL-1 alpha and mouse TNF-alpha were not detected in the plasma of LLC-IL6 transplanted mice. These data suggested that secretion of IL-6 from LLC cells was unable to alter net tumour growth rate but rather caused a state similar to cachexia without detectable increase of IL-1 alpha and TNF-alpha in the plasma. This state may be responsible for the shortened survival of LLC-IL6 tumour-bearing mice.

Animals↗
PubMed1992

Structure and function of the murine perforin promoter and upstream region. Reciprocal gene activation or silencing in perforin positive and negative cells.

Gene expression of the cytolytic protein perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine perforin promoter and upstream region. The murine perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in perforin-negative cell types the perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in perforin-negative cells, the transcriptional activity of the immediate proximal perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.

Animals↗
PubMed1992

Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.

Base Sequence↗
PubMed1992

Resistance and susceptibility of neural cells to lysis by cytotoxic lymphocytes and by cytolytic granules.

The susceptibility of neural cells to immune-mediated lysis by alloantigen-specific cytotoxic lymphocytes is important in understanding cell-mediated immune responses during rejection of transplanted neural tissues and in inflammatory responses of the central (CNS) and peripheral (PNS) nervous systems. In this study, we used 51Cr-release and granzyme A assays to examine whether primary cultures of astrocytes and CNS and PNS neurons could serve as targets for alloantigen-specific CTLs and granule-mediated lysis. The level of astrocyte killing by alloantigen-specific CTLs correlated with expression of the class I gene products of the major histocompatibility complex. Astrocytes cultured for 1-2 weeks did not express class I MHC antigens and were not susceptible to lysis by activated alloantigen-specific CTLs. Lengthening the astrocyte culture period to 3 weeks resulted in class I MHC antigen expression on the astrocyte surface and alloantigen-specific lysis. Astrocytes of all ages tested were susceptible to lysis by isolated cytolytic lymphocyte granules. PNS neurons of various ages tested also served as targets for CTLs and were lysed by isolated granules. In contrast, CNS neurons did not express class I MHC antigens and were highly resistant to killing by CTLs and lymphocyte granules. CNS neurons and astrocytes did not trigger specific granzyme A secretion from effector cells. In the presence of leucoagglutinin, CTLs-specific recognition of target cells is bypassed, and virtually any cell, regardless of its antigens, is killed nonspecifically. Although leucoagglutinin-treated CNS neurons and astrocytes triggered increased granzyme A secretion from effector cells, only astrocytes were lysed in an antigen-nonspecific manner, whereas CNS neurons remained strikingly resistant. These results suggest differences in the susceptibility of PNS and CNS neurons to T cell-mediated lysis. CNS neurons appear to possess protective mechanisms that render them refractory to CTL-mediated lysis and granule-mediated lysis, whereas PNS neurons and astrocytes are far more susceptible to both types of immune attack.

Animals↗
PubMed1992

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.

Antibodies, Monoclonal↗
PubMed1991

Perforin and granzyme B as markers for acute rejection in heart transplantation.

Histological analysis of endomyocardial biopsies (EMB) is regarded as the most satisfactory technique for monitoring crisis of rejection in heart transplanted patients. In this study, 42 biopsies from 14 patients who underwent heart transplantation were examined. Three patients did not present any rejection crisis at the date of the biopsy analysis, six were examined during an early rejection crisis (day 7-70 post-graft), and five were examined during a late rejection crisis (day 74-960 post-graft). Since granzyme B and perforin are proteins associated with cell lysis histological grading and cell phenotype analysis, in situ hybridization using granzyme B and perforin [35S]RNA probes was performed on 30 EMB to characterize the cytolytic activation of heart infiltrating cells. Our data suggest that granzyme B and perforin could be used as predictive markers for acute rejection in patients with early rejection crisis. Their detection might be an indication to administrate corticoids to resolve an acute rejection crisis. In contrast, their absence in patients with late rejection crisis appears as a good prognostic factor for the outcome of rejection and raises the question of the necessity to treat such patients with additional corticoid treatment.

Biomarkers↗
PubMed1991

A central role of perforin in cytolysis?

Studies on the gene structure, on the transcript, and on perforin protein are reviewed, including intracellular trafficking. Perforin transcription is tightly regulated and specific for CTL and NK. Two independent pathways for perforin induction exist, only one of them being IL-2 independent. Perforin expression in vitro and in vivo correlates with the functional expression of cytotoxicity in viral infection, transplant and tumor rejection, and in autoimmunity. Perforin together with granzymes is localized in cytolytic granules. However, the trafficking of those two proteins is quite different. Since the properties of perforin containing granules encompass the characteristics of secretory granules and of lyzosomes, the term granulosomes is used to describe this unique organelle. Evidence is reviewed to refute the concept that the homologous restriction factors of complement also restrict the lysis of homologous cells by perforin.

Animals↗
PubMed1989

Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences.

We have cloned the human perforin (P1) gene and sequenced 6.2-kb genomic DNA, containing 1.4-kb 5'-flanking region, the 5' untranslated region, the complete coding region and the beginning of the 3' untranslated region. The P1 gene including at least 95-bp 3' untranslated region is organized in only three exons: the first exon (97 bp) contains all but four nucleotides of the 5' untranslated region and was determined by primer extension and S1 nuclease mapping. This exon is separated by 1.7 kb from the second exon containing the remaining (4 bp) 5' untranslated region, the leader peptide and the N-terminal region of P1 up to--but not including--the C9 homologous region. The third exon is separated by a 1.2-kb intron and contains the remainder of the molecule, including at least 90 bp of the 3' untranslated region. This simple gene organization differs from that of the more complicated C9 gene. Because of the unusual intron in the 5' untranslated sequence the transcription initiation (cap) site is located almost 1.8 kb upstream of the ATG start signal. The more immediate 5' flanking sequence contains a CCAAT and GC box but lacks other known promoter elements. Instead, we find three different sequence repeats. One of them, a hexanucleotide sequence with the consensus GCCCTG of unknown significance occurs 19 times within a stretch of 240 bp. Further upstream we localized sequences homologous to the following enhancer and promoter elements: c-fos proto-oncogene, IFN-gamma and phorbol ester response elements, five cAMP response elements, and three motifs corresponding to general inducer elements. In addition, a sequence conserved in the 5'-flanking region of several T cell genes was identified. The 5' flanking regions of P1. CCP1 (granzyme B) and CCP2 (granzyme C) (kindly provided by Dr. Bleackley) contain as only significant homology cAMP response elements. These findings are consistent with a tight control and regulation of P1, which appears to be distinct from that of granzymes.

Amino Acid Sequence↗
PubMed1989

Perforin mRNA in primary peritoneal exudate cytotoxic T lymphocytes.

Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.

Animals↗
PubMed1989

Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis.

This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose-dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL-mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell-mediated cytolysis.

Aminoquinolines↗
PubMed1989

SC5b-7, SC5b-8 and SC5b-9 complexes of complement: ultrastructure and localization of the S-protein (vitronectin) within the macromolecules.

Purified terminal components of the complement system were used together with purified S-protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b-7, SC5b-8 and SC5b-9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50-70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double-diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S-protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b-7)n and (C5b-8)n, negatively stained SC5b-7 and SC5b-8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b-9 less than 27 S) appeared as wedge-shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl-S-protein and labeled with avidin-gold conjugates as electron-dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S-protein and the critical site for membrane binding of C5b-7 or C5b-8 and for initiation of C9 polymerization. These results support recent findings in which the function of S-protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin-binding domain.

Avidin↗
PubMed1989

Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus.

The analysis of gene expression in cytotoxic T cells by in situ hybridization of serial liver and brain sections from mice infected with lymphocytic choriomeningitis virus (LCMV) and immunostaining with T cell marker- and virus-specific antibodies revealed a close histological association of infiltrating lymphocytes expressing the perforin and granzyme A genes with virally infected cells. Maximal frequency of perforin and granzyme A mRNA-containing cells on liver sections preceded by about 2 days maximal LCMV-specific cytotoxicity of the lymphoid liver infiltrating cells. These results are most consistent with an involvement of perforin and granzyme A in cell-mediated cytotoxicity in vivo.

Animals↗
PubMed1989

Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9.

The nucleotide sequence coding for the cytotoxic T-lymphocyte (CTL) protein perforin 1 (P1) has been determined and the corresponding protein sequence has been derived. Murine CTL cDNA libraries contained in the vector lambda gt11 were screened by using a monospecific antiserum to purified P1. Three recombinant phages were isolated and their cDNA inserts were sequenced. The derived protein sequence contains 554 amino acids and displays, as expected, considerable homology with certain functional domains in the complement components C9, C8 alpha, C8 beta, and C7. The identity of P1 cDNA clones was verified by prokaryotic expression and the reactivities of antisera produced to the expressed proteins. In addition, antisera were produced to two synthetic peptides located in the center and C-terminal portions of P1. All antisera reacted with purified P1. In Northern blot analyses, P1 cDNA probes recognized a 2.9-kilobase mRNA only in CTL. Perforin mRNA was found in all cloned CTL and in all mixed lymphocyte reactions that gave rise to cytotoxic cells. Perforin mRNA was also detected in virus-specific CTL that had been generated in vivo and isolated from liver tissue of mice infected with lymphocytic choriomeningitis virus. The cell-specific expression of perforin is consistent with its postulated role in cytolysis.

Amino Acid Sequence↗