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Biomedical subjects

G Berke

Publications and source records attributed to G Berke.

At least 19 recordsLinked to original sources
PubMed2001

Electrophysiologic perturbations and arrhythmogenic activity caused by activation of the Fas receptor in murine ventricular myocytes: role of the inositol trisphosphate pathway.

INTRODUCTION: Experimental evidence suggests a major role for Fas receptor activation in a wide range of myocardial pathologies. Because clinical situations, which are likely to be associated with Fas activation, are accompanied by a variety of ventricular arrhythmias, the major goal of this study was to investigate the ionic mechanisms responsible for these phenomena. METHODS AND RESULTS: To delineate the origin of Fas-mediated electrophysiologic perturbations, the transient outward K+ current I(to) and the L-type Ca2+ current I(Ca,L) were studied in murine ventricular myocytes treated with the Fas-activating monoclonal antibody Jo2. Jo2 decreased I(to) (4.36 +/- 1.2 pA/pF vs 17.48 +/- 2.36 pA/pF in control, V(M) = +50 mV; P < 0.001) and increased I(Ca,L) (-13.17 +/- 1.38 pA/pF vs -3.94 +/- 0.78 pA/pF in control, V(M) = 0 mV; P < 0.001). Pretreatment of ventricular myocytes with ryanodine or thapsigargin prevented the electrophysiologic effects of Jo2, suggesting that [Ca2+]i elevation is important for Fas-mediated action. In agreement with our previous studies demonstrating dependence of Fas-based myocyte dysfunction on an intact inositol trisphosphate (1,4,5-IP3) pathway, the effects of Jo2 on I(to) and I(Ca,L) were prevented by the phospholipase C (generates 1,4,5-IP3) blocker U73122, and by xestospongin C (tested with I(to)), a specific blocker of IP3-operated sarcoplasmic reticulum Ca2+ release channels. Furthermore, intracellular perfusion with 1,4,5-IP3, but not with 1,3,4-IP3, caused electrophysiologic effects resembling those of Jo2. CONCLUSION: Decreased I(to) and increased I(Ca,L) underlie Fas-induced action potential alterations and arrhythmias in murine ventricular myocytes, effects that appear to be mediated by 1,4,5-IP3-induced intracellular calcium release.

Animals↗
PubMed2000

Tumor immunity in perforin-deficient mice: a role for CD95 (Fas/APO-1).

CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39. 5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF-, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.

Animals↗
PubMed2000

Vocal cord augmentation with cultured autologous fibroblasts.

Bovine collagen is an acceptable agent for vocal cord medialization; however, it produces only a temporary effect. As a foreign protein bovine collagen is susceptible to host collagenase and can induce immune response. Autologous collagen has become recently available, but it is less effective as a medialization agent. The study examines human skin fibroblasts growing in culture. Human skin bioptates were taken from the retroauricular area. Fibroblasts in culture were tested for scar contractility and ability to produce type I collagen (by flow cytometry with labeled antibodies). After five passages in culture the cells produced normal type I collagen, exhibited normal contractility, and did not induce no tumors in nude mice.

Animals↗
PubMed1999

Cardiac dysfunction in murine autoimmune myocarditis.

We have investigated the pathophysiological basis of cardiac dysfunction in autoimmune myocarditis and in the resulting dilated cardiomyopathy. To this end we utilized the myosin-induced autoimmune myocarditis model in BALB/c mice. Myocarditis has been found to be associated with massive ventricular lymphocyte infiltration and a 50% reduction in tail artery blood flow, reflecting the depressed cardiac function in myocarditis. Action potential characteristics of control and diseased isolated ventricular myocytes were (mean+/-SEM): resting potential: -68.1+/-1. 1,-68.3+/-0.7 mV; action potential amplitude: 96.5+/-10.4, 92.3+/-4. 4 mV; action potential duration at 80% repolarization (APD80) 38+/-5, 116+/-24* ms; * P<0.05. We utilized the whole cell voltage clamp technique to explore ion currents involved in APD prolongation and arrhythmogenic activity, and found that in diseased myocytes the transient outward current (Ito) was markedly attenuated. At a membrane potential of +40 mV, in control and in diseased myocytes, I(to) current density was 14.7+/-1.5 and 6.5+/-1.4 pA/pF, respectively, P<0.005. In contrast, the L-type Ca2+current (ICa,L) remained unchanged. To further explore the basis for cardiac impairment, we simultaneously measured [Ca2+]i transient and contraction in isolated normal and diseased myocytes. The major findings indicated that both the relaxation kinetics of [Ca2+]i transients and myocyte contraction were significantly faster in the diseased myocytes. In conclusion, substantial, potentially reversible, electrophysiological and mechanical perturbations in ventricular myocytes from mice with myosin-induced autoimmune myocarditis appear to contribute to disease-related cardiac dysfunction.

Action Potentials↗
PubMed1998

The regulation of CD95 ligand expression and function in CTL.

Previous studies with CTL lines and CTL hybridomas have suggested that functional CD95 (APO-1/Fas)-ligand (CD95L) expression on effector CTLs is a consequence of specific CTL-target recognition and TCR triggering of newly transcribed CD95L. Such a control on the expression of CD95L could provide a double safeguard for killing only cognate target cells. Here the regulation of CD95L expression and function was tested in in vivo primed, alloreactive peritoneal exudate CTL (PEL) from perforin-deficient (P0) mice. CD95L-based, PEL-mediated cytotoxicity was blocked by brefeldin A, an inhibitor of intracellular protein transport, but not by the protein synthesis inhibitor emetine, the immunosuppressive drug cyclosporin A, or the DNA transcription inhibitor actinomycin D. CD95L mRNA transcripts in freshly isolated PEL were shown by RT-PCR; CD95L surface expression was evident by staining with Fas-Fc as well as CD95L Abs. Undiminished CD95L expression and cytocidal activity were found in PEL incubated for 48 h in culture, without adding Ag, mitogen, or cytokines. PEL expressed functional CD95L, yet exhibited target cell-specific killing, except when encountering high CD95-expressing cells. The results indicate that PEL use CD95L probably expressed in the Golgi and/or on the cell surface and do not require newly transcribed CD95L upon target cell conjugation. Hence the TCR-triggered recruitment of preformed CD95L, rather than its biosynthesis, controls CD95L-based specific lysis induced by CTL.

Animals↗
PubMed1998

Fas (CD95/Apo-1)-mediated damage to ventricular myocytes induced by cytotoxic T lymphocytes from perforin-deficient mice: a major role for inositol 1,4,5-trisphosphate.

Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene-knockout (P-/-) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca2+]i transients, and contractions of myocytes conjugated to P-/- PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P-/- PELs were, respectively, as follows: Vm, -73.2+/-1.5 and -53.6+/-6.4 mV (mean+/-SEM); action potential amplitude, 117.9+/-3.9 and 74.3+/-21.2 mV; and action potential duration at 80% repolarization, 17+/-6 and 42+/-13 milliseconds (all P<.05). P-/- PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca2+]i increased during the cytocidal interaction with P-/- PELs, from a fluorescence ratio of 0.82+/-0.05 (n=7) to 1.98+/-0.09 (n=13) (P<.05). All of the effects caused by P-/- PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 microg/mL), an antagonist of inositol trisphosphate (IP3)-operated sarcoplasmic reticulum Ca2+ channels, or U-73122 (2 micromol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP3 analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P-/- PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP3. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.

Action Potentials↗
PubMed1998

Activation of influenza-specific memory cytotoxic T lymphocytes by Concanavalin A stimulation.

Traditionally, the in vitro activation of virus-specific memory cytotoxic T lymphocytes (CTLs) has been achieved by stimulating the CTLs with antigen-presenting cells (APCs) infected with an appropriate virus or pulsed with virus-specific antigenic peptides. Here, we describe the utilization of the polyclonal activator Concanavalin A (ConA) for in vitro restimulation of memory CTLs from virus-primed mice. Using this simple method, the activation of splenocytes with ConA for 3 days (i) eliminates the need to stimulate with virus-pulsed APCs and (ii) generates CD8+ CTLs that exhibit virus specificity and MHC-restricted lytic activity similar to CTLs obtained by conventional viral restimulation. In vitro ConA stimulation of splenocytes from BALB/c mice primed with the A/Texas/77 or A/Japanese/57 strain of influenza virus and from C57L/J mice infected with the A/Texas strain, generated CTLs with specific lytic activity. Hence reactivation of memory CTLs by this method is a general phenomenon rather than a mouse or viral strain-specific one. The ConA stimulation method used here had a recall of long-term (1 year) memory CTLs that effectively lysed virally infected targets. Further ConA-stimulated effector lymphocytes from virally primed animals have been shown to recognize and subsequently lyse target cells pulsed with virus or virus-derived peptides. The ConA reactivation of specific anti-viral CTLs may facilitate (i) studying anti-viral CTL responses and (ii) identifying of viral epitopes when unknown or when appropriate viral stimulation is impossible.

Animals↗
PubMed1997

Channel formation and [Ca2+]i accumulation induced by perforin N-terminus peptides: comparison with purified perforin and whole lytic granules.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells express the pore forming protein perforin, which contributes to lymphocytotoxicity. The hallmark of perforin action is opening high-conductance transmembrane channels that enable massive influx of Ca2+ ions (deleterious to many cell types), as well as granzymes, which may trigger the apoptotic pathway. To explore the functional domains in the perforin molecule, we investigated in PN71 lymphocytes, the ability of perforin N-terminus synthetic peptides (compared to purified perforin and perforin-containing lytic granules), to cause intracellular Ca2+ ([Ca2+]i) accumulation and open transmembrane channels. To this end, we used the whole cell recording technique and Indo 1 fluorescence to measure membrane currents and [Ca2+]i, respectively. We have demonstrated that the N-terminus peptide Hu-34 (amino acids 1-34) closely resembled perforin action, reflected by [Ca2+]i accumulation and channel activity, while shorter peptides (e.g., Hu-16) generated mostly short-lived channels but no [Ca2+]i elevation. Hence, the first 34 amino acids of the perforin N-terminus sequence are sufficient for the perforin action.

Amino Acid Sequence↗
PubMed1997

Fluorescence depolarization as an early measure of T lymphocyte stimulation.

We have used the Cellscan, an apparatus capable of measuring optical properties of individual cells, to study changes in fluorescence polarization associated with T cell stimulation. We show that the fluorescence polarization of human peripheral blood lymphocytes (PBL) labeled with fluorescein diacetate (FDA) is markedly reduced upon exposure to the mitogenic lectins phytohemagglutinin (PHA), concanavalin A (ConA), or to phorbol esters. Methyl alpha-D-mannopyranoside (alphaMM) is able to reverse the depolarizing effect induced by ConA as long as the cells are not committed to proliferate. H7 and staurosporin, both inhibitors of protein kinase C (PKC), inhibit the depolarization induced by PHA. The mitogen-induced depolarization is dependent on metabolic energy. The results support the use of fluorescence depolarization of FDA-labeled PBL, monitored by the Cellscan, as a sensitive means of measuring early lymphocyte stimulation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Involvement of the IP3 cascade in the damage to guinea-pig ventricular myocytes induced by cytotoxic T lymphocytes.

We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca2+] ([Ca2+]i). Since these deleterious effects cannot be accounted for by increased L-type Ca2+ current (ICa,L), we hypothesize that [Ca2+]i overload due to Ca2+ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP3) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca2+]i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from -80.9 +/- 0.7 to -59.9 +/- 2.5 mV, action potential amplitude from 139.5 +/- 1.4 to 80.6 +/- 1.7 mV and action potential duration to 50% repolarization (APD50) from 797 +/- 97 to 52 +/- 12 ms. The ratio of fluorescence at 340 and 380 nm (R340/380) increased from a control value (in nonconjugated myocytes) of 0.71 +/- 0.02 to 2.07 +/- 0.03, 30 min, after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 micrograms/ml), an antagonist of IP3-induced Ca2+ release from SR channels, or U-73122 (2 microM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in [Ca2+]i; after 60 min of PEL-myocyte interaction, R340/380 was 1.15 +/- 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP3-induced Ca2+ release from intracellular stores. Pharmacological targeting of IP3 in heart transplant rejection is thus suggested.

Animals↗
PubMed1997

Killing mechanisms of cytotoxic lymphocytes.

Killer lymphocytes are primary immune effectors of virus, certain bacteria, and tumor immunity and play a role in autoimmunity and transplant rejection. This article reviews progress in deciphering the mechanisms by which they kill target cells through induction of apoptosis by either the secretory, perforin/granzyme-based pathway or the nonsecretory pathway, (i.e., by triggering the cell-surface death receptor Fas (CD95) by the membrane-bound Fas ligand of the killer).

Animals↗
PubMed1996

Cell-mediated cytotoxicity results from, but may not be critical for, primary allograft rejection.

Tumor cells insensitive to lysis through the Fas and TNF pathways were injected either subcutaneously or into the peritoneal cavities of allogeneic perforin-less (P0) and perforin wild-type (P2) mice. In three of four cases, the tumors were rejected equally rapidly in both strains of mice. Rejection was accompanied by vigorous in vitro cytotoxicity in P2, but not in P0 mice. The rapid clearance of allografted cells in mice where all three known cytolytic pathways are seriously compromised raises important questions about the involvement of cell-mediated cytotoxicity, as defined by current assay techniques, in primary allograft rejection.

Animals↗
PubMed1996

Abnormal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachexia.

Excessive urea excretion associated with a negative nitrogen balance and massive loss of skeletal muscle mass (cachexia) is a frequent life threatening complication in malignancies and HIV infection. As these patients have often elevated interleukin-6 (IL-6) and abnormally low cystine levels, we have now determined the intracellular levels of glutathione and other cysteine derivatives in the liver and muscle tissue of IL-6-treated or tumor-bearing C57BL/6 mice. IL-6 treatment or inoculation of the MCA-105 fibrosarcoma caused a significant increase in hepatic gamma-glutamyl-cysteine synthetase activity and a decrease in the sulfate level, glutamine/urea ratio, and glutamine/glutamate ratio, suggesting that a decrease of the proton generating cysteine catabolism in the liver may increase carbamoyl-phosphate synthesis and urea formation at the expense of net glutamine synthesis. Treatment with cysteine, conversely, caused an increase in sulfate, glutamine/urea ratios, and glutamine/glutamate ratios and may thus be a useful therapeutic tool in clinical medicine. In contrast to the liver, muscle tissue of tumor-bearing mice showed decreased glutathione and increased sulfate levels, suggesting that the cysteine pool may be drained by an increased cysteine catabolism in this tissue. The findings indicate that tumor cachexia is triggered initially by IL-6 and is later sustained by processes driven by an abnormal cysteine metabolism in different organs.-Hack, V., Gross, A., Kinscherf, R., Bockstette, M., Fiers, W., Berke, G., and Dröge, W. Abnormal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachexia.

Animals↗
PubMed1996

Interaction of cytotoxic T lymphocytes and guinea pig ventricular myocytes. Pharmacological modulation by blocking K+ currents in cytotoxic T lymphocytes.

Infiltrating cytotoxic T lymphocytes (CTLs) are important immune effectors that damage the myocardium during heart transplant rejection as well as in cardiomyopathy and Chagas' heart disease. We have previously shown that in an in vitro model of murine-derived peritoneal exudate CTL (PEL)-guinea pig ventricular myocyte interaction, PEL induced in conjugated myocytes reduction of resting membrane potential and action potential (AP) amplitude, shortening of AP duration, delayed afterdepolarizations (DADs), and myocyte contracture and destruction. Since these findings indicated that cytotoxicity was largely caused by [Ca2+]i overload, in the present study we tested the hypothesis that blocking the L-type Ca2+ current (ICa,L) in the myocyte will eliminate the trigger for Ca2+ release from intracellular stores and will reduce [Ca2+]i overload and subsequent myocyte deterioration. CoCl2 (3 mmol/L) prevented PEL-induced AP changes, induction of DADs, and myocyte destruction. Since verapamil (2 mumol/L) was ineffective, indicating that the CoCl2 protection was not due to block of ICa,L, we tested whether the different action of these Ca2+ channel blockers was due to their differential effect on the PEL's K+ current (IK), previously shown to participate in lymphocyte activation and cytotoxicity. In agreement with their protective efficacy, CoCl2 but not verapamil blocked IK in PELs, suggesting that this is the mechanism for the protection provided by CoCl2. To support this notion, we tested the effect of the scorpion-derived peptide margatoxin (10 nmol/L), a specific K+ channel blocker in lymphocytes, on PEL-myocyte interaction and on PEL's IK; margatoxin prevented PEL-induced cytotoxicity and also blocked IK in PEL. Based on these findings, an alternative modality for attenuating CTL-induced lymphocytotoxicity is proposed.

Animals↗