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E R Podack

Publications and source records attributed to E R Podack.

At least 19 recordsLinked to original sources
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↗
PubMed1979

Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins.

Exposure of E. coli K12 W1485 to the cytolytic alternative pathway assembled from the 11 isolated pathway proteins resulted in killing of the bacteria, as evidenced by loss of viability. Lysis of the bacteria required introduction of lysozyme into the reaction mixture. The time-course and dose dependency of bacteriolysis in the isolated system were identical to those in C4-depleted serum. The bacteriolytic activity of the pathway was highly dependent on the concentration of the pathway proteins and became insignificant at 1:16 physiological concentration. Electron microscopic visualization of killed and of lysed bacteria revealed numerous complement membrane lesions and partial disintegration of the outer phospholipid membrane. Scanning electron microscopy showed that killed bacteria were enlarged, partially collapsed and exhibited irregular surface protrusions. Lysed bacteria were fragmented and appeared polymorphic. This study demonstrates that the alternative pathway, in absence of immunoglobulins, has the potential or eradicating gram-negative bacteria.

Blood Bactericidal Activity↗
PubMed1979

C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

Animals↗
PubMed1979

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Cell Membrane↗
PubMed1979

Membrane attack complex of complement: generation of high-affinity phospholipid binding sites by fusion of five hydrophilic plasma proteins.

The molecular basis of the membranolytic activity of the membrane attack complex (MAC) of complement was investigated. By using density gradient equilibrium ultracentrifugation, the binding of egg yolk lecithin to the isolated MAC and to its intermediate complexes and precursor proteins was measured. No stable phospholipid--protein complexes were formed with the MAC precursor components C5b--6, C7, C8, and C9. Stable complexes of phospholipid and protein were formed by C5b--7, C5b--8, C5b--9, and the MAC (C5b--9 dimer) and they exhibited densities of 1.2164, 1.184, 1.2055, and 1.2275 g/ml, respectively. The molar phospholipid/protein ratios for the four complexes were determined to be: C5b--7, 399:1, C5b--5, 841:1; C5b--9, 918:1; and C5b--9 dimer, 1460:1. Electron microscopy of the isolated phospholipid--protein complexes revealed no lipid bilayer structures. The magnitude of the phospholipid binding capacity of the MAC is consistent with the interpretation that the MAC forms phospholipid--protein mixed in micelles in lipid bilayers and biological membranes and thus causes formation of hydrophilic lipid channels.

Binding Sites↗
PubMed1979

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.

Cholesterol↗
PubMed1979

C1q: isolation from human serum in high yield by affinity chromatography and development of a highly sensitive hemolytic assay.

C1q, a subcomponent of the first component of complement, has been isolated from human serum in fully hemolytically active form by affinity column chromatography and gel filtration with Bio-Gel A-5M. The affinity column was prepared by covalent coupling of purified human IgG to CNBr-activated Sepharose 4B. Final yields of C1q ranged from 25 to 40% with 650- 890-fold purification based on recovery of hemolytic activity. The preparations were free of contaminating serum proteins as judged by SDS-polyacrylamide gel electrophoretic and immunochemical criteria. The final C1q preparations were also devoid of any demonstrable C1q-inhibitor activity. A C1q-depleted reagent (C1qD) was obtained from the nonabsorbed protein containing fractions of the human IgG-Sepharose 4B affinity column and utilized in conjunction with sensitized sheep erythrocytes (EA) for the detection and quantitation of C1q hemolytic activity. Employing optimal quantities of C1qD in the hemolytic assay mixture, the highly purified C1q preparations contained 0.5 to 1 x 10(13) effective molecules/mg and 0.5 to 1 x 10(12) effective C1q molecules/ml of human serum. This assay would therefore reproducibly detect less than 1 ng of C1q hemolytic activity.

Chromatography, Affinity↗
PubMed1979

Structural similarities between C6 and C7 of human complement.

A new method for the isolation of C6 and C7 by affinity chromatography of human serum with anti-C6 and anti-C7 coupled to Sepharose is described. C6 and C7 prepared by this method are hemolytically fully active, homogeneous proteins obtained in 25% yield. A comparison of the properties of isolated C6 and C7 gave the following results: The amino acid composition of the two proteins is very similar. The m.w. calculated from the amino acid content is 124,800 for C6 and 120,800 for C7. Both components are single chain glycoproteins migrating upon electrophoresis at pH 8.6 as beta 2-globulins, Both proteins are polymorphic as detected by isoelectrofocusing in polyacrylamide gels and range in their isoelectric points from pH 6.15 to 6.7. The UV spectra reveal only minor differences; the extinction coefficients are: EC6 = 1.71 cm2 X mg-1 and EC7 = 1.92 cm2 X mg-1. CD-spectra show 8% alpha-helix and 10% beta-structure for C6 and 10% alpha-helix and 14% beta-structure for C7. The structural similarities of C6 and C7 suggest their evolution from a common ancestral gene.

Chemical Phenomena↗