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Biomedical subjects

G Berke

Publications and source records attributed to G Berke.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lytic reaction of in vivo primed peritoneal exudate CTL. Induction of high-conductance single channels in the target cell membrane.

CTL, primary effectors in immune responses, deliver a "lethal hit" signal to target cells, causing their destruction. The precise membrane events associated with the lethal hit remain elusive. We investigated the signal(s) mediating destruction of tumor target cells (EL4) by perforin-deficient peritoneal exudate CTL (PEL). We utilized patch clamp techniques to record electrophysiological events associated with the cytolytic interaction of PEL and EL4 in isolated conjugates. PEL-EL4 interaction resulted in induction in EL4 cells, of single channels (followed by EL4 destruction), with a mean conductance of 437 pS and a reversal potential of -1.0 mV, suggestive of nonselective pathways. Similar channels were induced in EL4 cells conjugated with perforin-rich PEL blasts (PEB), by perforin, postnuclear extract from PEL (pnPEL) and from other cytotoxic lymphocytes, but not from noncytolytic lymphocytes. As similar channels were induced by pnPEL in EL4 membrane patches, we propose that these channels result from a direct effect of PEL-derived channel-forming substance(s) on the target cell's membrane. Importantly, postnuclear extracts from perforin-devoid cytotoxic PEL-hybridomas induced similar channels, suggesting the presence of a nonperforin, channel-forming activity in PEL and PEL-hybridomas. Based on the present study, we conclude that the delivery of the lethal hit by cytolytic PEL and PEL-hybridoma is associated with induction in the target cell of high-conductance channels, which most likely mediate its destruction. We propose that these channels are related to the Fas pathway of lymphocytotoxicity.

Animals↗
PubMed1995

The CTL's kiss of death.

The potent and specific lytic activity of CTLs can occur by at least two distinct pathways. In the secretion and perforin-mediated pathway, the direct effect(s) on the target cell membrane of the pore-forming agent perforin, probably in conjunction with granzymes, also secreted from the CTLs, causes the target's demise. Intercytoplasmic transfer of granzymes is believed to be involved in inducing target apoptosis. In the Fas-mediated pathway, engagement of a CTL membrane ligand with an apoptosis-inducing target cell surface receptor, such as the FasL with Fas, triggers programmed disintegration of the CTL-bound target; secretion of granzymes and pore formation by perforin are not involved in this receptor-mediated mechanism. Despite the fundamental differences in their onset for both pathways, the downstream sequence of events that culminate in target cell apoptosis appears to be similar. Further studies will resolve this enigma.

Animals↗
PubMed1995

Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides.

The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8+ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.

Animals↗
PubMed1995

PELs and the perforin and granzyme independent mechanism of CTL-mediated lysis.

The central role of CTLs in immunopathology accounts for the increasing interest in deciphering the mechanism whereby they kill at the molecular level. Recent studies show that CTLs have two molecularly distinct lytic mechanisms at their disposal. The first involves the direct effect(s) of the pore-forming protein perforin, possibly in conjunction with granzymes. In recent years, experiments conducted in our laboratory led to an alternative pathway, of receptor-mediated mechanism for CTL killing, involving neither the secretion nor the lytic action of the pore-forming protein perforin or of granzymes. By this mechanism, engagement of a CTL membrane ligand and an apoptosis-inducing target cell surface receptor triggers the disintegration of the CTL-bound target cell. Cross-linking of apoptosis-inducing target cell surface molecules (e.g. Fas), induced upon binding of CTL ligands (e.g. Fas-L), may be required and sufficient to trigger target cell apoptosis. Intracellular lethal signals emanating from the cross-linked intracellular death domain of Fas are postulated.

Animals↗
PubMed1994

CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma.

Many mouse and human tumours express major histocompatibility complex (MHC) class I-associated antigens that constitute targets for syngeneic cytotoxic T lymphocytes (CTL). Genes encoding such antigens were isolated from a mouse mastocytoma and from human melanomas by genetic methods. Isolation and characterization of MHC class I-associated peptides has enabled specific anchor residues to be identified that are typical of peptides that bind to distinct class I molecules. Moreover, CTL specific to particular MHC-peptide combinations have been used to identify naturally occurring antigenic peptides in cell extracts and enabled them to be sequenced directly. Most known MHC ligands are of viral origin or are self peptides derived from normal proteins. Here we use total acid extraction and repeated fractionation to isolate and sequence Lewis lung carcinoma (3LL)-specific peptide(s), which shows sequence homology to the connexin 37 protein. Synthetic octamers based on these sequences bind to 'empty' H-2Kb molecules on RMA-S cells, sensitize RMA-S cells to lysis by specific anti-3LL CTL, and induce anti-tumour CTL. The tumour-associated peptide originates from mutated connexin 37 expressed in 3LL.

Amino Acid Sequence↗
PubMed1994

Mechanisms whereby cytotoxic T lymphocytes damage guinea-pig ventricular myocytes in vitro.

We studied possible mechanisms whereby cytotoxic T lymphocytes (CTL) damage the myocardium during the immunological rejection of the transplanted heart, by investigating the in vitro interaction between CTL and cardiac myocytes. We utilized the patch-clamp technique to record membrane currents and action potentials from concanavalin A-treated guinea-pig ventricular myocytes conjugated to mouse peritoneal exudate CTL (PEL). PEL-myocyte interaction reduced action potential duration at 50% repolarization (APD50) from 731.7 +/- 57.8 to 195.3 +/- 58.0 ms, action potential amplitude from 134.9 +/- 1.9 to 104.2 +/- 6.2 mV and resting membrane potential (Vm) from -80.9 +/- 0.5 to 72.5 +/- 1.5 mV. These changes were accompanied by generation of delayed afterdepolarizations, indicative of intracellular [Ca2+] overload. The electrophysiological alterations were associated with myocyte shortening (within 28.9 +/- 2.8 min) followed by complete cell destruction (within 43.5 +/- 4.3 min). To determine whether intracellular Ca2+ stores were involved in PEL-induced myocyte damage, the protective effects of ryanodine and caffeine were investigated. While ryanodine (10 microM) delayed the electrophysiological and morphological alterations, caffeine (5 mM) provided significant protection, suggesting that Ca2+ release from intracellular stores contributes to PEL-induced damage to the myocytes. Based on our findings, we suggest that the functional derangements seen in myocyte-lymphocyte conjugates can contribute to the overall decline in cardiac function during heart transplant rejection.

Action Potentials↗
PubMed1994

Effects of purified perforin and granzyme A from cytotoxic T lymphocytes on guinea pig ventricular myocytes.

OBJECTIVE: Involvement of cytotoxic T lymphocytes (CTL) in heart transplant rejection as well as in viral myocarditis is well established, but the precise mechanisms whereby infiltrating CTL damage the myocardium are unknown. The aim of the study was to investigate how CTL derived perforin, the serine protease granzyme A, and the combination of both, damage guinea pig ventricular myocytes. METHODS: Action potentials and membrane currents were recorded by means of the whole cell configuration from guinea pig ventricular myocytes. RESULTS: Resembling the effects of CTL derived lytic granules, perforin caused gradual myocyte shortening and contracture, leading to complete loss of the rod shaped morphology and to cell destruction. These changes were preceded by shortening of action potential duration and reduction of resting potential and action potential amplitude, followed by complete inexcitability. Granzyme A alone was ineffective, but accelerated the deleterious effects of perforin on the morphological and electrophysiological properties of myocytes. The effects of perforin were further evaluated by measuring membrane currents by means of the whole cell voltage clamp. Perforin induced discrete changes in membrane current, reminiscent of single ion channels, with large conductance and open time of up to several seconds. Linear regression analysis of the channel I-V relations resulted in a conductance of 890 pS and a reversal potential of -7.6 mV. These results suggest that perforin induces large non-selective channels, which can account for most of the observed adverse effects. CONCLUSIONS: As CTL participate in the immunological rejection of the transplanted heart, it is conceivable, but remains to be shown, that part of this damage is inflicted by perforin containing lytic granules.

Action Potentials↗
PubMed1994

The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects.

The characteristics of cytotoxic T lymphocyte (CTL) and natural killer (NK) cell recognition of and binding to target cells (conjugate formation), and the precise mechanism(s) by which the target cells are triggered to undergo apoptotic cell lysis are now being deciphered at the cellular and molecular levels. Involvement of a multitude of cell surface molecules, in addition to T cell receptor (TCR)-major histocompatibility (MHC)-peptide complexes, in the binding and signalling for lymphocyte-mediated lysis has been demonstrated. Two proposed mechanisms of lymphotoxicity currently appear to be valid: (i) a membranolytic one initiated by the formation of pores in target cell membranes by secreted molecules of lymphocyte origin, such as perforin and granzymes, and (ii) a nonsecretory one initiated by receptor-mediated triggering of apoptosis-inducing target cell surface molecules, but not involving the secretion of pore-forming agents and granzymes. Perforin and granzymes are probably involved in lymphocyte activation and are likely mediators of the membranolytic pathway of lymphotoxicity. Existence of the nonsecretory and receptor-triggered lytic mechanism was indicated by (i) the prelytic fragmentation of the target cell's DNA, which precedes release of intracellular (51Cr-labeled) components, (ii) the demonstration of cytolytic effector cells that are either devoid of or express background levels of lytic granules and perforin, and (iii) the observation that some CTL lyse target cells under conditions at which perforin and granzymes are neither secreted nor lytic, e.g. [Ca2+]o < 1 micromolar. These two mechanisms are not mutually exclusive and are probably used by different types of effector cells or by the same effector cells at different stages of differentiation. In fact, recent perforin gene knock-out experiments support the existence of both.

Animals↗
PubMed1994

Calcium channel blockers modify electrophysiological effects induced by lytic granules from cytotoxic T lymphocytes in guinea pig ventricular myocytes.

Damage to myocytes by infiltrating cytotoxic lymphocytes, containing lytic granules and the pore-forming protein, perforin, thereof, probably contribute to the immunological rejection of the transplanted heart, to autoimmune diseases and possibly to congestive heart failure associated with myocarditis. In the present study we investigated whether electrophysiological and morphological changes induced in guinea pig ventricular myocytes by lytic granules extracted from cytotoxic T lymphocytes, are modified by L-type Ca++ channel blockers. The organic blockers, verapamil (2 microM) and nisoldipine (500 microM) were unable to prevent or inhibit any of the deleterious effects of lytic granule on action potential and myocyte morphology, and the granule-induced increase in the membrane current measured at the end of 300-msec clamp pulse. In contrast, the inorganic blockers CoCl2 (3 mM) and NiCl2 (4 mM) provided considerable protection against the granule actions mentioned above, but an equally potent Ca++ blocker, CdCl2 (3 mM) was ineffective. The protective efficacy of CoCl2 (and probably that of NiCl2) was most likely due to its capacity to reduce or block the generation by lytic granules/perforin of large-conductance (approximately 1400 pS) channels responsible for inducing Ca++ overload and cell destruction. We consider these studies of importance because they direct further studies aimed at developing effective means for attenuating cytotoxic T lymphocyte-induced tissue damage, for example during transplant rejection or in autoimmune diseases.

Action Potentials↗
PubMed1993

Mechanisms whereby lytic granules from cytotoxic T lymphocytes damage guinea pig ventricular myocytes.

During immunological rejection of the transplanted heart, cytotoxic T lymphocytes (CTL) infiltrate the myocardium and by damaging the myocytes contribute to loss of function. To address one important aspect of heart transplant rejection, we investigated in guinea pig ventricular myocytes how CTL-derived lytic granules containing the pore-forming protein perforin reduce the membrane potential (VM) and cause cell damage. The reduction in VM was biphasic; within 8.4 +/- 1.9 min VM was reduced from a control value of -78.4 +/- 1.9 mV to -69.9 +/- 3.5 mV. Subsequently, within 6.7 +/- 2.1 min VM declined to -3.4 +/- 1.2 mV, associated with a progressive contracture. Under whole cell voltage clamp, in myocytes held at their resting potential (VM = -76.2 +/- 0.9 mV), granules induced discrete inward current steps (resembling 'single channel' activity), with a mean amplitude of -86.8 +/- 1.4 pA and open times lasting from seconds up to several minutes. The mean conductance and reversal potential calculated from the linear regression analysis of the I-V relations were 1390 pS and -6.8 mV, respectively. The probable non-selectivity of these 'channels' and the resultant loss of membrane K+ selectivity can account for the reduction in VM. At the same time, opening of these pores leads to Ca2+ overload, resulting in contracture and cell damage.

Animals↗
PubMed1993

Mechanism of lymphocyte-mediated cytolysis: functional cytolytic T cells lacking perforin and granzymes.

Involvement of the lytic protein perforin (c. 65,000 MW) and of granule proteases (granzymes) in cell lysis induced by cytolytic T lymphocytes (CTL) has been suggested, but is still controversial. For example, in vivo-primed peritoneal exudate CTL (PEL) have been found to express perforin and granzyme activity in amounts comparable to those found in non-lytic lymphocytes, although PEL are the most potent of all CTL. Exploiting several cloned CTL hybridomas developed in this laboratory and newly available molecular probes for detecting perforin, granzymes, protein and mRNA, we now directly demonstrate killer T lymphocytes which kill effectively and specifically, but are free from perforin, lytic granules and granzymes, all three of which have been postulated to be involved in lymphocyte-mediated killing. The CTL hybridomas are completely devoid of perforin and granzymes prior to, during, and after activation by antigen, mitogen or interleukin-2 (IL-2). The induction of lytic granules, perforin, and granzymes in the in vivo-primed PEL, but not in the cloned CTL hybridomas, upon cultivation in IL-2, further suggests the involvement of these constituents in antigen/lymphokine-induced CTL activation and differentiation rather than directly in their cytocidal activity. Together, these findings support a perforin- and granzyme-independent CTL lytic mechanism.

Animals↗
PubMed1992

Peritoneal exudate lymphocyte and mixed lymphocyte culture hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin.

We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.

Animals↗
PubMed1992

Immunological rejection of heart transplant: how lytic granules from cytotoxic T lymphocytes damage guinea pig ventricular myocytes.

We investigated the mechanism by which lytic granules extracted from cytotoxic T lymphocytes (CTL) damage guinea pig ventricular myocytes in order to determine whether their actions can be related to the overall immunological rejection of the transplanted heart. Granule-induced myocyte morphological changes and final destruction were preceded by shortening of action potential duration (APD) and reductions of the resting potential and the action potential amplitude. APD shortening was probably caused by a granule-induced increase in outward current (most likely non-specific). Ryanodine, which blocks Ca2+ release from the sarcoplasmic reticulum, did not interfere with the morphological and electrophysiological effects of lytic granules. Fura-2 imaging indicated that [Ca2+]i initially increased about 2-fold from 90.0 +/- 11.5 nM, while cell length decreased less than 5% from a mean value of 99.0 +/- 9.0 microns. A further increase in [Ca2+]i (greater than 10 fold) was associated with progressive contracture and destruction, suggesting that the structural damage inflicted by lytic granules is caused by [Ca2+]i overload. The results indicate that the cytocidal action of CTL-derived lytic granules may be involved in immunologically induced damage, even to the extent of rejection of the transplanted heart.

Action Potentials↗
PubMed1992

Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). I. Cell detachment and lysis induced by encephalitogenic CD4+ T lymphocytes.

Myelin basic protein (MBP) reactive CD4+ T lymphocytes, capable of inducing experimental autoimmune encephalomyelitis (EAE), were examined for their ability to damage target cells of central nervous system (CNS) origin. Damage was assessed by monitoring detachment of adherent astrocytes from substratum and astrocyte lysis. MBP-specific, but non-encephalitogenic CD4+ T cells mediated astrocyte detachment but not lysis. However, encephalitogenic CD4+ T cell lines were more efficient in causing astrocyte detachment and could also cause astrocyte lysis. The detachment and lytic activities of the MBP-reactive T cell lines tested were often independent of the presence of specific antigen, and were not restricted to syngeneic major histocompatibility (MHC) antigens. MBP often augmented the detaching and, if lytic, lytic activities of these T cells. The encephalitogenic CD4+ T cells also detached and lysed allogeneic 'bystander' fibroblasts in the presence of unlabelled syngeneic astrocytes, suggesting the involvement of a soluble mediator(s). Although MBP is essential for the initiation of EAE, the presence of MBP on cells of CNS origin, such as astrocytes and oligodendrocytes, does not appear to be necessary for their damage by MBP-specific CD4+ T cells. Immune CD4+ T cells, which penetrate the CNS, may disorganize brain tissue structure by lysing astrocytes directly and by damaging other brain cells indirectly by soluble mediators. Thus cellular detachment, in addition to cell lysis, mediated by MBP-specific CD4+ cells may contribute to EAE pathogenesis.

Animals↗
PubMed1992

Delineation of tissue damage mechanisms in experimental autoimmune encephalomyelitis (EAE). II. Characteristics of astrocyte detachment mediated by myelin basic protein (MBP) specific CD4+ T lymphocytes.

We have shown that encephalitogenic, myelin basic protein (MBP)-specific CD4+ T cells can cause astrocyte and oligodendrocyte detachment in vitro. Similar processes may damage the central nervous system (CNS) in vivo by causing disorganization and destruction of brain tissue structure. The finding that 'bystander' allogeneic fibrosarcoma cells were detached by MBP-specific CD4+ T cells only when syngeneic astrocytes were present, suggested that a soluble cell-detaching factor (CDF) is released during the specific astrocyte-CD4+ effector interaction. In this study, CDF activity was detected in the supernatants of MBP-reactive CD4+ T cells incubated with concanavalin A or astrocytes. Lymphocyte-induced astrocyte lysis, but not detachment, was inhibited by the protein synthesis inhibitors, cycloheximide and puromycin, indicating that de novo protein synthesis is required for this type of lysis, but not for detachment. Astrocyte detachment was not inhibited, but rather augmented, by the trypsin inhibitors, soybean trypsin inhibitor (SBTI) and alpha-1-antitrypsin (alpha 1), suggesting that the CDF activity is not due to tryptic serine proteases, although it may be protease susceptible. The heparanase inhibitor, heparin, inhibited CD4+ T cell-mediated astrocyte detachment at low doses, but augmented detachment at higher doses, indicating that detaching activity is not due to heparanases. The actin microfilament disrupting agent, cytochalasin B (CB), inhibited astrocyte detachment induced by MBP-specific CD4+ T cells. CB pretreatment of the target astrocytes, but not of the effector CD4+ T cells, inhibited astrocyte detachment, suggesting that the integrity of the target's, but not the effector's, cytoskeleton is required for astrocyte detachment. The results herein suggest that during astrocyte interaction with MBP-specific CD4+ T cells, soluble factors are released that trigger an intrinsic astrocyte detachment mechanism.

Animals↗
PubMed1991

T-cell-mediated cytotoxicity.

There are two competing theories to explain the mechanism(s) by which cytolytic T lymphocytes kill target cells: granule exocytosis of a pore-forming protein, and contact-induced internal disintegration. Accumulated evidence supports alternative pathways in lymphocytoxicity, possibly reflecting distinct effector functions expressed by different killer cells and cells at different stages of activation.

Animals↗