Response of Brain and Meningeal Metastases to Trastuzumab-Deruxtecan in a Patient with HER2-Low Breast Cancer: A Case Report.

Nervous system metastases (CNSm) are late events associated with poor outcomes in endocrine-sensitive HER2-negative breast cancer (BC) patients, especially in the presence of leptomeningeal disease (LMD). Effective treatments are extremely limited in this setting. The antibody-drug conjugate, trastuzumab-deruxtecan (T-DXd), which combines the anti-HER2 antibody trastuzumab with a topoisomerase type 1 inhibitor, showed high efficacy not only against HER2-positive but also HER2-low metastatic BCs, expressing HER2 at a lower level. To the best of our knowledge, this is the first report of a patient with metastatic endocrine-sensitive HER2-low BC suffering from BMs associated with LMD and sustained disease control when treated with T-DXd. Several recent case series have reported the activity of T-DXd in patients with HER2-positive disease and brain metastases or LMD, but none in HER2-low patients. This case is particularly relevant since more than 50% of BCs are HER2-low.

Both APRIL and antibody-fragment-based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) on multiple myeloma (MM) produces fast but not long-lasting responses. Reasons for treatment failure are poorly understood. CARs simultaneously targeting two antigens may represent an alternative. Here, we (1) designed and characterized novel A proliferation inducing ligand (APRIL) based dual-antigen targeting CARs, and (2) investigated mechanisms of resistance to CAR T cells with three different BCMA-binding moieties (APRIL, single-chain-variable-fragment, heavy-chain-only). METHODS: Three new APRIL-CARs were designed and characterized. Human APRIL-CAR T cells were evaluated for their cytotoxic function in vitro and in vivo, for their polyfunctionality, immune synapse formation, memory, exhaustion phenotype and tonic signaling activity. To investigate resistance mechanisms, we analyzed BCMA levels and cellular localization and quantified CAR T cell-target cell interactions by live microscopy. Impact on pathway activation and tumor cell proliferation was assessed in vitro and in vivo. RESULTS: APRIL-CAR T cells in a trimeric ligand binding conformation conferred fast but not sustained antitumor responses in vivo in mouse xenograft models. In vitro trimer-BBζ CAR T cells were more polyfunctional and formed stronger immune synapses than monomer-BBζ CAR T cells. After CAR T cell-myeloma cell contact, BCMA was rapidly downmodulated on target cells with all evaluated binding moieties. CAR T cells acquired BCMA by trogocytosis, and BCMA on MM cells was rapidly internalized. Since BCMA can be re-expressed during progression and persisting CAR T cells may not protect patients from relapse, we investigated whether non-functional CAR T cells play a role in tumor progression. While CAR T cell-MM cell interactions activated BCMA pathway, we did not find enhanced tumor growth in vitro or in vivo. CONCLUSION: Antitumor responses with APRIL-CAR T cells were fast but not sustained. Rapid BCMA downmodulation occurred independently of whether an APRIL or antibody-based binding moiety was used. BCMA internalization mostly contributed to this effect, but trogocytosis by CAR T cells was also observed. Our study sheds light on the mechanisms underlying CAR T cell failure in MM when targeting BCMA and can inform the development of improved treatment strategies.

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.

[2020 oncology update]. / Oncologie.

The COVID-19 pandemic that has swept around the world in early 2020 has changed our daily practice and habits. Fortunately, however, 2020 also brings its share of new approaches and therapeutic combinations as well as new therapies. These advances are improving the outcomes and quality of life of our patients across the spectrum of oncological diseases. This article summarises the latest oncological advances and novelties for 2020 in the following tumor entities : lung, breast, digestive, gynecological, urological and ENT.

La pandémie de Covid-19 survenue début 2020 dans le monde entier aura bouleversé notre pratique quotidienne et nos habitudes. Heureusement, sur le plan thérapeutique, l'année 2020 apporte également son lot de nouvelles approches et combinaisons thérapeutiques ainsi que l'introduction de nouvelles molécules, permettant d'améliorer le pronostic vital et la qualité de vie de nos patients, dans de nombreux domaines. Cet article résume les dernières avancées et nouveautés oncologiques de l'année 2020 dans les domaines suivants : poumon, sein, sphère digestive, gynécologique, urologique et ORL.

Guillain-Barré syndrome after adoptive cell therapy with tumor-infiltrating lymphocytes.

BACKGROUND: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) is a promising experimental immunotherapy that has shown high objective responses in patients with melanoma. Current protocols use a lymphodepletive chemotherapy before infusion of ex vivo expanded TILs, followed by high-dose interleukin-2 (IL-2). Treatment-related toxicities are mainly attributable to the chemotherapy regimen and to the high-dose IL-2 and are generally reversible. Neurological side effects have rarely been described. Nevertheless, due to improvements in cell production techniques and due to combinations with other immunomodulating molecules, side effects not previously described may be encountered. CASE PRESENTATION: We report the case of a 53-year-old heavily pretreated patient with melanoma who developed Guillain-Barré syndrome (GBS) 19 days after ACT using autologous TILs, given in the context of a phase I trial. He presented with dorsal back pain, unsteady gait and numbness in hands and feet. Lumbar puncture showed albuminocytological dissociation, and nerve conduction studies revealed prolonged distal motor latencies in median, ulnar, tibial and peroneal nerves, compatible with a GBS. The patient was treated with intravenous immunoglobulins and intensive neurological rehabilitation, with progressive and full recovery at 21 months post-TIL-ACT. Concomitant to the onset of GBS, a cytomegalovirus reactivation on immunosuppression was detected and considered as the most plausible cause of this neurological side effect. CONCLUSION: We describe for the first time a case of GBS occurring shortly after TIL-ACT for melanoma, even though we could not identify with certainty the triggering agent. The report of such rare cases is of extreme importance to build on the knowledge of immune cellular therapies and their specific spectrum of toxicities.

Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells.

Proper spindle positioning is crucial for spatial cell division control. Spindle positioning in human cells relies on a ternary complex comprising Gαi1-3, LGN and NuMA, which anchors dynein at the cell cortex, thus enabling pulling forces to be exerted on astral microtubules. We develop a live imaging siRNA-based screen using stereotyped fibronectin micropatterns to uncover components modulating spindle positioning in human cells, testing 1280 genes, including all kinases and phosphatases. We thus discover 16 components whose inactivation dramatically perturbs spindle positioning, including tyrosine receptor kinase 3 (TYRO3) and cyclin G associated kinase (GAK). TYRO3 depletion results in excess NuMA and dynein at the cortex during metaphase, similar to the effect of blocking the TYRO3 downstream target phosphatidylinositol 3-kinase (PI3K). Furthermore, depletion of GAK leads to impaired astral microtubules, similar to the effect of downregulating the GAK-interactor Clathrin. Overall, our work uncovers components and mechanisms governing spindle positioning in human cells.

Safety and Tolerability of Adoptive Cell Therapy in Cancer.

Adoptive T cell therapy (ACT) is a safe and effective personalized cancer immunotherapy that can comprise naturally occurring ex vivo expanded cells (e.g., tumor-infiltrating lymphocytes [TIL]) or T cells genetically engineered to confer antigen specificity (T-cell receptor [TCR] or chimeric antigen receptor [CAR] engineered T cells) to mediate cancer rejection. In recent years, some ACTs have produced unprecedented breakthrough responses: TIL therapy has moved from melanoma to solid tumor applications, TCR-engineered cells are developed for hematologic and solid tumors, and CAR-engineered T cells have received Food and Drug Administration (FDA) approval for the treatment of patients with certain B-cell malignancies. Although results are encouraging, to date, only a small percentage of patients with advanced malignancies can benefit from ACT. Besides ACT availability and accessibility, treatment-related toxicities represent a major hurdle in the widespread implementation of this therapeutic modality. The large variety of observed toxicities is caused by the infused cell product or as side effects of accompanying medication and chemotherapy. Toxicities can occur immediately or can be delayed. In order to render those highly promising therapeutic approaches safe enough for a wider pool of patients outside of clinical trials, an international consensus for toxicity management needs to be established.

ZYG-1 promotes limited centriole amplification in the C. elegans seam lineage.

Genome stability relies notably on the integrity of centrosomes and on the mitotic spindle they organize. Structural and numerical centrosome aberrations are frequently observed in human cancer, and there is increasing evidence that centrosome amplification can promote tumorigenesis. Here, we use C. elegans seam cells as a model system to analyze centrosome homeostasis in the context of a stereotyped stem like lineage. We found that overexpression of the Plk4-related kinase ZYG-1 leads to the formation of one supernumerary centriolar focus per parental centriole during the cell cycle that leads to the sole symmetric division in the seam lineage. In the following cell cycle, such supernumerary foci function as microtubule organizing centers, but do not cluster during mitosis, resulting in the formation of a multipolar spindle and then aneuploid daughter cells. Intriguingly, we found also that supernumerary centriolar foci do not assemble in the asymmetric cell divisions that precedes or that follows the symmetric seam cell division, despite the similar presence of GFP::ZYG-1. Furthermore, we established that supernumerary centrioles form earlier during development in animals depleted of the heterochronic gene lin-14, in which the symmetric division is precocious. Conversely, supernumerary centrioles are essentially not observed in animals depleted of lin-28, in which the symmetric division is lacking. These findings lead us to conclude that ZYG-1 promotes limited centriole amplification solely during the symmetric division in the C. elegans seam lineage.

TRACMIT: An effective pipeline for tracking and analyzing cells on micropatterns through mitosis.

The use of micropatterns has transformed investigations of dynamic biological processes by enabling the reproducible analysis of live cells using time-lapse fluorescence microscopy. With micropatterns, thousands of individual cells can be efficiently imaged in parallel, rendering the approach well suited for screening projects. Despite being powerful, such screens remain challenging in terms of data handling and analysis. Typically, only a fraction of micropatterns is occupied in a manner suitable to monitor a given phenotypic output. Moreover, the presence of dying or otherwise compromised cells complicates the analysis. Therefore, focusing strictly on relevant cells in such large time-lapse microscopy dataset poses interesting analysis challenges that are not readily met by existing software packages. This motivated us to develop an image analysis pipeline that handles all necessary image processing steps within one open-source platform to detect and analyze individual cells seeded on micropatterns through mitosis. We introduce a comprehensive image analysis pipeline running on Fiji termed TRACMIT (pipeline for TRACking and analyzing cells on micropatterns through MITosis). TRACMIT was developed to rapidly and accurately assess the orientation of the mitotic spindle during metaphase in time-lapse fluorescence microscopy of human cells expressing mCherry::histone 2B and plated on L-shaped micropatterns. This solution enables one to perform the entire analysis from the raw data, avoiding the need to save intermediate images, thereby decreasing data volume and thus reducing the data that needs to be processed. We first select micropatterns containing a single cell and then identify anaphase figures in the time-lapse recording. Next, TRACMIT tracks back in time until metaphase, when the angle of the mitotic spindle with respect to the micropattern is assessed. We designed the pipeline to allow for manual validation of selected cells with a simple user interface, and to enable analysis of cells plated on micropatterns of different shapes. For ease of use, the entire pipeline is provided as a series of Fiji/ImageJ macros, grouped into an ActionBar. In conclusion, the open source TRACMIT pipeline enables high-throughput analysis of single mitotic cells on micropatterns, thus accurately and efficiently allowing automatic determination of spindle positioning from time-lapse recordings.

Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning.

Zika virus (ZIKV) causes congenital microcephaly. Although ZIKV can impair cell cycle progression and provoke apoptosis, which probably contributes to disease aetiology through depletion of neural progenitor cells, additional cellular mechanisms may be important. Here, we investigated whether ZIKV infection alters centrosome number and spindle positioning, because such defects are thought to be at the root of inherited primary autosomal recessive microcephaly (MCPH). In addition to HeLa cells, in which centrosome number and spindle positioning can be well monitored, we analysed retinal epithelial cells (RPE-1), as well as brain-derived microglial (CHME-5) and neural progenitor (ReN) cells, using immunofluorescence. We established that ZIKV infection leads to supernumerary foci containing centriolar proteins that in some cases drive multipolar spindle assembly, as well as spindle positioning defects in HeLa, RPE-1 and CHME-5 cells, but not in ReN cells. We uncovered similar phenotypes in HeLa cells upon infection with dengue virus (DENV-2), another flavivirus that does not target brain cells and does not cause microcephaly. We conclude that infection with Flaviviridae can increase centrosome numbers and impair spindle positioning, thus potentially contributing to microcephaly in the case of Zika.

Inducing Differentiation of Premalignant Hepatic Cells as a Novel Therapeutic Strategy in Hepatocarcinoma.

Hepatocellular carcinoma (HCC) represents the second leading cause of cancer-related deaths and is reported to be resistant to chemotherapy caused by tumor-initiating cells. These tumor-initiating cells express stem cell markers. An accumulation of tumor-initiating cells can be found in 2% to 50% of all HCC and is correlated with a poor prognosis. Mechanisms that mediate chemoresistance include drug export, increased metabolism, and quiescence. Importantly, the mechanisms that regulate quiescence in tumor-initiating cells have not been analyzed in detail so far. In this research we have developed a single cell tracking method to follow up the fate of tumor-initiating cells during chemotherapy. Thereby, we were able to demonstrate that mCXCL1 exerts cellular state-specific effects regulating the resistance to chemotherapeutics. mCXCL1 is the mouse homolog of the human IL8, a chemokine that correlates with poor prognosis in HCC patients. We found that mCXCL1 blocks differentiation of premalignant cells and activates quiescence in tumor-initiating cells. This process depends on the activation of the mTORC1 kinase. Blocking of the mTORC1 kinase induces differentiation of tumor-initiating cells and allows their subsequent depletion using the chemotherapeutic drug doxorubicin. Our work deciphers the mCXCL1-mTORC1 pathway as crucial in liver cancer stem cell maintenance and highlights it as a novel target in combination with conventional chemotherapy. Cancer Res; 76(18); 5550-61. ©2016 AACR.

Gene expression profile of peripheral blood lymphocytes from renal cell carcinoma patients treated with IL-2, interferon-α and dendritic cell vaccine.

Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and T(REG)-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of T(REG)-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.

Regulatory T-cells and associated pathways in metastatic renal cell carcinoma (mRCC) patients undergoing DC-vaccination and cytokine-therapy.

PURPOSE: To evaluate CD4(+)CD25(+)FOXP3(+) T regulatory cells (T(REG)) and associated immune-regulatory pathways in peripheral blood lymphocytes (PBL) of metastatic renal cell carcinoma (mRCC) patients and healthy volunteers. We subsequently investigated the effects of immunotherapy on circulating T(REG) combining an extensive phenotype examination, DNA methylation analysis and global transcriptome analysis. DESIGN: Eighteen patients with mRCC and twelve volunteers (controls) were available for analysis. T(REG) phenotype was examined using flow cytometry (FCM). T(REG) were also quantified by analyzing the epigenetic status of the FOXP3 locus using methylation specific PCR. As a third approach, RNA of the PBL was hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays and the gene signatures were explored using pathway analysis. RESULTS: We observed higher numbers of T(REG) in pre-treatment PBL of mRCC patients compared to controls. A significant increase in T(REG) was detected in all mRCC patients after the two cycles of immunotherapy. The expansion of T(REG) was significantly higher in non-responders than in responding patients. Methylation specific PCR confirmed the FCM data and circumvented the variability and subjectivity of the FCM method. Gene Set Enrichment Analysis (GSEA) of the microarray data showed significant enrichment of FOXP3 target genes, CTLA-4 and TGF-ß associated pathways in the patient cohort. CONCLUSION: Immune monitoring of the peripheral blood and tumor tissue is important for a wide range of diseases and treatment strategies. Adoption of methodology for quantifying T(REG) with the least variability and subjectivity will enhance the ability to compare and interpret findings across studies.

The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells.

Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.

Indoleamine-2,3-dioxygenase enzyme expression and activity in polarized dendritic cells.

BACKGROUND AIMS: Polarized mature dendritic cells (DC) can activate cytolytic T-lymphocyte (CTL) responses and may be a more effective clinical strategy in DC-based cancer vaccines. A subset of mature DC can down-regulate the T-cell immune response through expression of indoleamine-2,3-dioxygenase (IDO). We determined whether polarizing DC ex vivo increased IDO expression and activity. METHODS: Peripheral blood monocytes from healthy volunteers were cultured ex vivo in polarizing and non-polarizing culture conditions. DC IDO expression was detected by Western blot. IDO enzyme activity was determined by high-performance liquid chromatography (HPLC) measurement of kynurenine (K) and tryptophan (T) concentrations in culture supernatants. RESULTS: IDO protein was markedly increased in DC after polarization (median 1222.4%, range 331.5-2113.3%) versus non-polarized DC (median 28.3%, range 3.7-119.8%; P=0.04). The median K/T ratio was significantly higher in polarized DC versus non-polarized DC (6.34, range 6.02-6.65, versus 0.047, range 0.004-0.541; P=0.04). IDO protein expression correlated with enzyme activity (r=0.80, P=0.002). CONCLUSIONS: DC polarizing culture conditions increased expression of IDO protein and IDO enzyme activity. DC culture and maturation methodologies may impact the effectiveness of adoptive DC therapy.

Clinical and immunologic effects of intranodal autologous tumor lysate-dendritic cell vaccine with Aldesleukin (Interleukin 2) and IFN-{alpha}2a therapy in metastatic renal cell carcinoma patients.

PURPOSE: To evaluate the clinical and immunologic outcomes of DC (dendritic cell) vaccine with interleukin (IL)-2 and IFN-alpha 2a in metastatic renal cell carcinoma patients. EXPERIMENTAL DESIGN: Eighteen consented and eligible patients were treated. Peripheral blood monocytes were cultured ex vivo into mature DCs and loaded with autologous tumor lysate. Treatment consisted of five cycles of intranodal vaccination of DCs (1 x 10(7) cells/1 mL Lactated Ringer's solution), 5-day continuous i.v. infusion of IL-2 (18MiU/m2), and three s.c. injections of IFN-alpha 2a (6MiU) every other day. Response Evaluation Criteria in Solid Tumors criteria were used for disease assessment. Correlative immunologic end points included peripheral blood lymphocyte cell phenotype and function as well as peripheral blood anti-renal cell carcinoma antibody and cytokine levels. RESULTS: All patients received between two and five treatment cycles. Toxicities consisted of known and expected cytokine side effects. Overall objective clinical response rate was 50% with three complete responses. Median time to progression for all patients was 8 months, and median survival has not been reached (median follow up of 37+ months). Treatment-related changes in correlative immunologic end points were noted and the level of circulating CD4(+) T regulatory cells had a strong association with outcome. Pre-IP-10 serum levels approached significance for predicting outcome. CONCLUSIONS: The clinical and immunologic responses observed in this trial suggest an interaction between DC vaccination and cytokine therapy. Our data support the hypothesis that modulation of inflammatory, regulatory, and angiogenic pathways are necessary to optimize therapeutic benefit in renal cell carcinoma patients. Further exploration of this approach is warranted.

Ex vivo expansion of tumor specific lymphocytes with IL-15 and IL-21 for adoptive immunotherapy in melanoma.

Although T central memory cells have been described as the most effective T-cell subtype against tumor growth, little is known about the requirements needed for their optimal ex vivo generation. Hence, our goal is to establish a protocol that will lead to consistent ex vivo generation of lymphocytes skewed toward a central memory phenotype. Antigen-specific T-cell lines were generated by ex vivo stimulation with Class-I and Class-II melanoma peptide pulsed dendritic cells in the presence of either IL-2 or IL-15 plus IL-21. Tumor specific lymphocytes of both central memory and effector characteristics were consistently generated from healthy donors and melanoma patients. IL15/IL21 cultures result in a cell population with a lower proportion of CD4(+)CD25(high)FoxP3(+) regulatory cells and higher number of CD8(+) and CD56(+) cells, and consequently render a higher yield of cells with a greater cytolytic activity and IFN-gamma production against melanoma cell lines.

Chronic angiotensin II receptor blockade induces cardioprotection during ischemia by increased PKC-epsilon expression in the mouse heart.

INTRODUCTION: This study was performed to investigate the role of chronic pretreatment with angiotensin II type 1 receptor antagonists (ARB) and angiotensin converting enzyme inhibitors (ACE-I) in myocardial infarction (MI) and ischemic preconditioning (iPC). Little is known about molecular mechanisms of MI and iPC, especially about protein kinase C (PKC) isozyme levels induced by chronic pharmacologic pretreatment with ARB and ACE-I. To address one of the most important signal molecules in iPC, the PKC system was investigated in an ischemia/reperfusion model using isolated mouse hearts. METHODS: C57/BL6 mice were treated orally with candesartan cilexetil or ramipril for 2 weeks. Isolated perfused hearts were subjected to 60 minutes of left anterior descending occlusion and 30 minutes of reperfusion. IPC was performed by 3 cycles of 5 minutes of ischemia prior to the infarct ischemia. Infarct size was measured using the propidium iodide method, and PKC isoenzymes were detected by immunoblotting in the membrane and cytosolic fraction. RESULTS: In the control group, iPC reduced infarct size from 59.8 +/- 4.2% to 24.5 +/- 1.7%. ARB pretreatment itself reduced the infarct size significantly (38.1 +/- 3.0%) in hearts without iPC. This protection could neither be enhanced by additional iPC (40.3 +/- 3.4%) nor blocked by the AT2-receptor antagonist PD123.319 (40.7 +/- 3.7%). The ARB-induced cardio protection, however, was abolished by chelerythrine (5 micromol/L) (71.7 +/- 6.6%, n = 11, P < 0.001). Furthermore, PKC-epsilon (PKC-epsilon) was significantly increased in the particulate fraction of ARB-pretreated mice. On the contrary, chronic treatment with ACE-I completely blocked iPC (57.7 +/- 3.9%, n = 12, P < 0.001) without any effect on infarct size itself (51.5 +/- 3.0%, n = 12). PKC-epsilon expression was significantly reduced. CONCLUSION: Chronic AT1-receptor antagonism is capable of protecting the heart against myocardial infarction in a PKC-epsilon-dependent way. Furthermore, chronic treatment with ACE-I is suggested to have suppressing effects on iPC, possibly caused by reduced PKC-epsilon expression.