Apoptotic Peptide Research Articles

Vaccination therapy of pancreatic carcinoma patients with autologous, tumor-lysate pulsed dendritic cells: Results of a phase II study

3069 Background: Vaccine therapy using dendritic cells can induce antitumor immunity. We report about the final results of a phase II-study using autologous, tumor-lysate pulsed dendritic cells (DC) for the treatment of patients with advanced pancreatic carcinoma. As all patients received concomitant chemotherapy, we investigated systematically the interplay between DC therapy and gemcitabine chemotherapy in a murine pancreatic carcinoma model, correlating the results of ex vivo immuno monitoring tools with clinical efficacy. Methods: Pancreatic carcinoma patients receiving abdominal surgery were recruited to the study. DC therapy was started after recurrence of pancreatic carcinoma or in a primarily palliative situation. Immune response was monitored by ELISPOT and proliferation assays. A pancreatic carcinoma vaccination model was established by transfecting murine pancreatic carcinoma cell line Panc02 with an OVA vector. Mice were vaccinated with bone marrow derived DC loaded with apoptotic Panc02OVA cells, OVA protein or SIINFEKL peptide. Intracellular IFN- γ staining and streptamer staining were used to examine the induction of antigen specific T cells. For tumor induction, Panc02OVA cells were orthotopically injected into C57BL/6 mice. Results: Twelve patients received DC vaccination. Four cases of at least intermittent partial remission or stable disease were seen. These patients had developed strong immunological antitumor responses as demonstrated by DTH skin testing, T cell proliferation assays and ELISPOT assays. Data in the murine model indicate that there is a synergistic clinical efficacy of gemcitabine and DC vaccination therapy despite reduction of specific cytotoxic T cells by concomitant gemcitabine therapy. Results of an in vitro cytotox assay using SIINFEKL-specific, transgene T-cells demonstrate that gemcitabine sensitizes Panc02OVA cells towards T cell mediated lysis. Conclusions: DC vaccination therapy can be of clinical benefit in the setting of advanced pancreatic carcinoma. DC therapy led to immunological responses despite concomitant chemotherapy. Data obtained in a murine model of pancreatic cancer support the concept of a combination of vaccination and chemotherapy. No significant financial relationships to disclose.

Ethanol Induces Apoptotic Death of β‐Endorphin Neurons in the Rat Hypothalamus by a TGF‐β1‐Dependent Mechanism

We have previously shown that developing beta-endorphin neurons, in the arcuate nucleus of the hypothalamus become increasingly apoptotic when exposed to ethanol. As in the previous study we have observed an involvement in transforming growth factor beta 1 (TGF-beta1) in mediation of the apoptotic process, the present study was conducted to determine the ethanol-induced changes in this apoptotic regulatory peptide signaling in the arcuate nucleus of the hypothalamus of neonatal rats. Pups were exposed to 11.34% ethanol in a milk-based diet or control diet on postnatal day (PND) 3 to PND7. Two hours after the last daily feeding, brains were collected and frozen in liquid nitrogen for analysis of various apoptosis regulatory proteins in the arcuate tissue by Western blots. Some animals were fixed in 4% paraformaldehyde and analyzed immunohistochemically. Ethanol exposure increased apoptotic death of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The cell death was associated with an increase in the tissue levels of TGF-beta1 in the mediobasal hypothalamus. This was correlated with a reduction in the arcuate level of retinoblastoma protein (Rb) phosphorylation. The reduced level of Rb phosphorylation was associated with an increased protein level of the cyclin dependent kinase inhibitor p27/kip but with a decreased protein level of cyclin dependent kinase 4 and cyclin D3. In addition, the apoptotic cell death was positively correlated with the level of Bclxs but negatively correlated with the level of the Bcl2. These results suggest that ethanol exposure increases TGF-beta1 signaling involving Bcl2 and Rb repression that may lead to apoptotic death of cells including beta-endorphin neurons in the arcuate nucleus of the hypothalamus.

The effect of obestatin on porcine ovarian granulosa cells

The aim of our in vitro experiments was to investigate the role of obestatin, a newly discovered metabolic hormone produced in the stomach and other tissues, in the direct control of ovarian cell proliferation, apoptosis and secretion. Porcine granulosa cells were cultured in the presence of obestatin (0, 1, 10 and 100 ng/ml medium). The expression of intracellular peptides associated with proliferation (PCNA, cyclin B1, MAP kinase), as well as markers of apoptosis (Bax, p53, Caspase 3), were detected using immunocytochemistry and Western immunoblotting. Secretion of progesterone (P 4), testosterone (T) and estradiol (E 2) was measured by EIA. Addition of obestatin (1–100 ng/ml) to the culture medium significantly stimulated the expression of PCNA and resulted in an increase in expression of cyclin B1 and MAPK. It also significantly increased the percentage of cells containing the apoptotic and anti-proliferating peptides p53, Caspase 3 and Bax. At 10 and 100 ng/ml, obestatin promoted the secretion of P 4, but not T or E 2. Our results are the first demonstration that obestatin directly controls porcine ovarian cell functions: it can stimulate proliferation (accumulation of rPCNA, cyclin B1 and MAPK), apoptosis (expression of p53, Caspase 3 and Bax) and the secretion of progesterone.

Zinc Irreversibly Damages Major Enzymes of Energy Production and Antioxidant Defense Prior to Mitochondrial Permeability Transition

Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, alpha-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 microm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.

A p53-derived apoptotic peptide derepresses p73 to cause tumor regression in vivo

The tumor suppressor p53 is a potent inducer of tumor cell death, and strategies exist to exploit p53 for therapeutic gain. However, because about half of human cancers contain mutant p53, application of these strategies is restricted. p53 family members, in particular p73, are in many ways functional paralogs of p53, but are rarely mutated in cancer. Methods for specific activation of p73, however, remain to be elucidated. We describe here a minimal p53-derived apoptotic peptide that induced death in multiple cell types regardless of p53 status. While unable to activate gene expression directly, this peptide retained the capacity to bind iASPP - a common negative regulator of p53 family members. Concordantly, in p53-null cells, this peptide derepressed p73, causing p73-mediated gene activation and death. Moreover, systemic nanoparticle delivery of a transgene expressing this peptide caused tumor regression in vivo via p73. This study therefore heralds what we believe to be the first strategy to directly and selectively activate p73 therapeutically and may lead to the development of broadly applicable agents for the treatment of malignant disease.

Peripheral CD8 T-Cell Responses to Apoptotic Cell Proteins and Peptides

The immune system must contend with the billions of cells that are turned over on a daily basis; many of these cells derived from the immune system itself. Recent evidence suggests that the apoptotic cells are processed and presented through classical pathways for MHC II presentation to CD4 T cells and also are cross-presented through a phagosome-cytosolic pathway or released into the cytosol for presentation to CD8 T cells. It appears that the continuous presentation of self-peptides is required for active maintenance of T-cell tolerance. Although for both CD4 and CD8 T cells, anergy is a dominant pathway for tolerance, presentation of self-peptides to CD8 and CD4 T may induce different responses. Phagocytosis of apoptotic cells leads to the production of TGF-beta and/or IL-10 by antigen-presenting cells and there is evidence to suggest that these cytokines favor the generation of CD4 T regulatory cells following encounter with self-antigen. In contrast, CD8 T cells undergo brief activation, an abortive proliferation ultimately leading to reduced long-term survival. The relative quantity and quality of all three signals of TCR engagement, costimulation versus coinhibition and quality of the cytokine response, distinguish T-cell responses to self-versus foreign antigens.

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1–28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1–18 and 1–5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P 4), testosterone (T), estradiol (E 2) and arginine–vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1–28, GHRP6 and ghrelin 1–18, but not by ghrelin 1–5. Expression of bcl-2 was suppressed by addition of ghrelin 1–28, GHRP6 and ghrelin 1–5, but promoted by ghrelin 1–18. The occurrence of PCNA was reduced by ghrelin 1–28, GHRP6, ghrelin 1–18 and ghrelin 1–5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1–28, GHRP6 and ghrelin 1–18, but not ghrelin 1–5. The accumulation of PKA decreased after treatment with ghrelin 1–28 and increased after treatment with GHRP6 and ghrelin 1–18 but not ghrelin 1–5. Secretion of P 4 by ovarian follicular fragments was decreased after addition of ghrelin 1–28 or ghrelin 1–5 but stimulated by GHRP6 and ghrelin 1–18. Testosterone secretion was inhibited by ghrelins 1–28 and 1–18, but not by GHRP6 or ghrelin 1–5. Estradiol secretion was reduced after treatment with ghrelin 1–28 but stimulated by ghrelins 1–18 and 1–5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1–28, GHRP6 and ghrelin 1–18, but inhibited by ghrelin 1–5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions—apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5–18 may be responsible for the major effects of ghrelin on the avian ovary.

Immunogenic and structural properties of the Asn-Gly-Arg (NGR) tumor neovasculature-homing motif

Tumor homing peptides containing the NGR motif, such as CNGRC and GNGRG, have been used for delivering cytokines, chemotherapeutic drugs, apoptotic peptides, and liposomes to a CD13 isoform expressed in tumor blood vessels. In view of the potential clinical applications of these drugs and considering the risk that NGR peptides could elicit blocking antibodies we have investigated the immunogenic properties of CNGRC and GNGRG in mice and rabbits, using various products containing these residues and different administration schedules. The results suggest that the immunogenicity of the NGR motif is very low, even when it is conjugated to tumor necrosis factor-α or to highly immunogenic carrier proteins. Molecular dynamics simulation experiments showed that both peptides have a strong propensity to populate a turn conformation. Superposition of predicted structures to the CTGNGRGEWKC loop of the 5th type I repeat of human fibronectin, a protein that contains four NGR motives, showed that the root mean square deviation of backbones was 0.7 Å for GNGRG and 0.5 Å for NGR. These results suggest that NGR peptides could mimic from an immunological point of view a “self” structure, likely the GNGRG loop of fibronectin, with important implications for the use of these targeting peptides in patients.

Expression of an anti apoptotic recombinant short peptide in mammalian cells

Understanding the mechanisms of the apoptotic and anti apoptotic processes may lead to a better way to control these cascades. Here we demonstrated for the first time the feasibility to express a short functional peptide in mammalian cells that abrogates the apoptosis cascade through interference with the proteolytic activity of the initiator caspase 9 and the executing caspase 3 enzymes. The expression of a short peptide that includes the pseudo-substrate motif of the apoptosis inhibitor protein P35 (Asp-Gln-Met-Asp) leads to the abrogation of cell death induced through either the mitochondrial or the death receptors pathways. Short open reading frames have been detected in several mammalian mRNAs, primarily upstream of the main long reading frame (uORFs), however, direct evidence for de-novo peptides translation has not been provided. Utilizing biochemical and imaging techniques we demonstrate here that the functional recombinant peptide was localized to the cytpoplasmic fraction of the cell. In conclusion, this work demonstrates that ribosomes recognize short ORFs to translate stable short recombinant peptides in mammalian cells. Expression of these intracellular peptides results in the knock down of apoptotic processes to generate apoptosis resistant stable cells.

Effects of HIV-1 Nef, a cytotoxic viral protein, on the growth of primary colorectal cancer

Colorectal cancer is the third leading cause of cancer deaths in American men and women. We describe the cytotoxic use of HIV-1 Nef protein and a cytotoxic peptide identified within the HIV-1 Nef structure in targeting human cancer cells in vitro and in vivo in a human xenograft model. A human colorectal tumor was implanted and propagated in the subcutaneous tissue of SCID mice. The mice were injected biweekly with the Nef apoptotic peptide. The tumor treated with Nef peptide underwent significant growth inhibition by as much as 300 percent when compared to the control (untreated) tumors. The Nef peptides were found to have an apoptotic effect on the human colon tumor similar to the effect seen on CD4 cells when the viral protein is secreted by the HIV-1 virus infected cells. The evidence from the xenograft mouse model suggests that the Nef peptides can be used to inhibit human colorectal cancer growth.

Biochemical Association of Poly(ADP‐ribose) Polymerase‐1 and Its Apoptotic Peptide Fragments with DNA Polymerase β

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.

Characterization of Nef-CXCR4 interactions important for apoptosis induction.

The HIV-1 Nef protein was analyzed for apoptotic structural motifs that interact with the CXCR4 receptor and induce apoptosis in CD4(+) lymphocytes. Two apoptotic motifs were identified. One centered on Nef amino acids (aa) 50 to 60, with the overlapping 20-mer peptides retaining about 82% of the activity of the full Nef protein. The second centered on aa 170 to 180, with the overlapping 20-mer peptides retaining about 30% of the activity of the full protein. Significant apoptotic abilities were observed for 11-mer motif peptides spanning aa 50 to 60 and aa 170 to 180, with a scrambled version of the 11-mer motif peptide corresponding to aa 50 to 60 showing no apoptotic ability. Hallmarks of apoptosis, such as the formation of DNA ladders and caspase activation, that were observed with the full-length protein were equally evident upon exposure of cells to these motif peptides. A CXCR4 antibody and the endogenous ligand SDF-1alpha were effective in blocking Nef peptide-induced apoptosis as well as the physical binding of a fluorescently tagged Nef protein, while CCR5 antibodies were ineffective. The CXCR4-negative cell line MDA-MB-468 was resistant to the apoptotic peptides and became sensitive to the apoptotic peptides upon transfection with a CXCR4-expressing vector. A fluorescently tagged motif peptide and Nef protein displayed physical binding to CXCR4-transfected MDA-MB-468 cells, but not to CCR5-transfected cells. The removal of the apoptotic motif sequences from the full-length protein completely eliminated the ability of Nef to induce apoptosis. However, these modified Nef proteins still retained the ability to enhance viral infectivity. Thus, specific sequences in the Nef protein appear to be necessary for Nef protein-induced apoptosis as well as for physical interaction with CXCR4 receptors.

Human dendritic cells induce tumor-specific apoptosis by soluble factors.

Dendritic cells (DCs) are the most potent antigen producing cells (APCs) for initiation of immune responses including anti-tumor immune responses. In previous reports, it has been shown that DCs efficiently take up and process apoptotic or necrotic bodies of tumor cells. It has also been shown that DCs pulsed with tumor cell apoptotic bodies, lysates or peptides generate potent anti-tumor immune responses. Direct interactions between DCs and viable tumor cells, however, have not been clearly elucidated. We report that monocyte-derived, CD1a+ immature DCs (iDCs) significantly inhibit the growth of breast tumor cells in coculture and transwell experiments in the presence of soluble CD40 ligand (sCD40L), LPS or both. The growth inhibition effects correlated with cell cycle arrest and apoptosis of breast tumor cells. The effects were associated with morphological changes of tumor cells from a round shape to a flat, spindle shape. In contrast, no inhibition of proliferation or morphological changes was observed on normal PBMC, K562 or breast fibroblasts. Interestingly, iDCs undergoing maturation induced by sCD40L+LPS induced a much stronger growth inhibitory effect than iDCs alone or mature DCs treated with sCD40L+LPS. Fractionation of supernatants showed the anti-tumor effects were mediated by a TNF-alpha-dependent and -independent mechanism. Soluble FasL and TRAIL were not involved. Our findings suggest that maturing DCs have the intrinsic ability to induce cell-cycle arrest and apoptosis of breast tumor cells through soluble factors, but not normal cells, in addition to their Ag presentation function.

Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach

Gene expression in haemocytes of the kuruma prawn (Penaeus japonicus) was investigated using an expressed sequence tag (EST) approach. Partial nucleotide sequences of cDNA library clones constructed from normal and white spot syndrome virus (WSSV)—infected P. japonicus haemocytes were determined. Of 635 clones obtained from the normal library, 284 (44·7%) significantly matched sequences in GenBank, and of 370 clones obtained from WSSV-infected library, 174 (47·0%) significantly matched sequences in the database. One hundred fifty-two deduced proteins were newly identified. Of these, 28 types were involved in biodefence. For the prophenoloxidase system, there are prophenoloxidase, coagulation factor G-β chain precursor, factor D, Masquarade-like protease, transglutaminase (TGase), clottable protein and eight types of protease inhibitors (two types of antileukoproteinase, alpha-2-macroglobulin, chelonianin, elastase inhibitor, two types of Kazal inhibitor and Kunitz-type inhibitor). For antibacterial peptides, there are bactinecin 11, penaeidin-2 precursor and lysozyme c type. The others defence-related proteins are basophil leukocyte interleukin-3-regulated protein, natural killer enhancing factor (NK-EF), integral membrane protein (CD34+), ESM-1, Notch homologue and Drac homologue. For the adhesion proteins, there are β-integrin, cell adhesion molecule (CAM) and three types of collagens. All ESTs representing protease inhibitors and tumour-related proteins were found only in the WSSV-infected library. Those encoding for apoptotic peptides were expressed at high levels in infected library. The putative defence proteins accounted for 2·7% of total ESTs in a normal shrimp library and 15·7% of the total ESTs in an infected library.

Transient vesicle leakage initiated by a synthetic apoptotic peptide derived from the death domain of neurotrophin receptor, p75NTR.

Peptides that induce apoptosis have potential as anticancer therapeutics. The design of safe, effective cancer therapeutic peptides requires characterization of the physical and chemical properties that influence activation of cell death in neoplastic cells. NTR365 is a synthetic pro-apoptotic peptide with an amino acid sequence derived from the death domain of p75(NTR). These studies were initiated to identify a potential mechanism for the apoptotic activity of NTR365 identified by Rabizadeh et al. We examined the interactions of this synthetic pro-apoptotic peptide with phospholipid vesicles. Fluorescence experiments demonstrate that the peptide induces leakage from large unilamellar vesicles. Leakage activity is transient and dependent on the presence of anionic lipid in the vesicles. Circular dichroism studies show that the NTR365 adopts a different conformation and may have altered vesicle affinity under conditions conducive to leakage. The active conformation of NTR365 differs from that of the NMR derived conformation. A related peptide with a single substitution is not apoptotically active, does not form a helical structure in the presence of vesicles and does not induce appreciable vesicle leakage under the same conditions as NTR365. These studies suggest that the demonstrated apoptotic activity of a closely related NTR364 peptide is linked to disruption of a membrane barrier and to the ability of the peptide to form a helical structure.

APAP, a sequence-pattern recognition approach identifies substance P as a potential apoptotic peptide

We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro-apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro-apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.

Viral, nonviral and peptide-mediated intra-articular transfer of genes and proteins

We have been examining the ability of different vectors to transfer therapeutic genes directly to joints following intra-articular injection. To date, we have examined the efficiency of intra-articular gene transfer to the rabbit knee mediated by adenoviral, adenoassociated virus (AAV), retrovirus and herpes simplex virus vectors as well as by over 100 different nonviral formulations. Moreover, we have examined the ability to repeat dose with the different types of viral vectors. A summary of our experiments to identify a clinically useful vector for intra-articular gene transfer will be presented. In an attempt to improve intra-articular gene transfer, we also have screened an M13 peptide phage display library for peptides able to facilitate internalization into synovial cells. We have identified a class of cationic peptides (PTD), similar to the cationic protein transduction domains found in Antennapedia (Antp) and HIV TAT peptide, as well as a class of more neutral and hydrophobic peptides (HAP). To determine the ability of the PTD and HAP peptides to facilitate internalization, the peptides were biotinylated and coupled to avidin-β-gal or streptavidin-Cy3. Although internalization mediated by the HAP peptides in culture and in vivo was not as efficient as the cationic peptides, they appeared to be more cell type specific. Several of the HAP and PTD peptides were fused to an antimicrobial peptide, KLAK, generating novel apoptotic peptides. These fusion peptides were able to impair rabbit and human synovial cell viability through disruption of mitochondria and induction of apoptosis. Intra-articular injection of these apoptotic peptides into arthritic rabbit joints resulted in extensive synovial cell apoptosis as well as reduction in leukocytic infiltration. An update of our progress using the different internalizing peptides for delivery of therapeutic proteins and genes will be presented.

Generation of an apoptotic intracellular peptide by gamma-secretase cleavage of Alzheimer's amyloid beta protein precursor.

The amyloid beta protein precursor (AbetaPP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical path leading to Abeta formation has been extensively studied since extracellular aggregates of amyloidogenic forms of Abeta peptide (Abeta42) are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of AbetaPP proteolysis is unknown. Although never previously described, cleavage of AbetaPP by gamma-secretase should release, together with Abeta, a COOH-terminal AbetaPP Intracellular Domain, herein termed AID. We have now identified AID-like peptides in brain tissue of normal control and patients with sporadic Alzheimer's disease and demonstrate that AID acts as a positive regulator of apoptosis. Thus, overproduction of AID may add to the toxic effect of Abeta42 aggregates and further accelerate neurodegeneration.

Dendritic cells: at the clinical crossroads.

Dendritic cells: at the clinical crossroads.

Vaccination against human cancers (review).

Classical and molecular immunological means of active tumor-specific immunization against human cancers yielded whole cell or tumor cell lysate vaccines of preventive value (reduced relapse rates) and dendritic cell-peptide or genetically engineered vaccines that may induce remissions even in metastatic disease. Active tumor-specific immunization was often successful in the past 50 years against experimental tumors maintained in the laboratory. During the epochs of classical and molecular immunology several vaccines were generated and used for the reduction of relapse rates of human cancer after surgical removal of the primary or metastatic tumors. Whole cell vaccines consist of X-irradiated autologous or allogeneic tumor cells coadministered with immunostimulants (BCG, Detox). Tumor cells haptenized biologically (as in viral oncolysates) or chemically were also used. Dendritic cell vaccines are prepared by transfection or transduction with tumor antigen-encoding DNA or by pulsing the cells with antigenic peptides in vitro; or collecting dendritic cells that engulfed apoptotic tumor cell DNA and/or peptide antigens in vivo for reinjection into the patient. Genetically engineered tumor cells are prepared in vitro to express MHC and peptides, costimulatory molecules (B7.1) and cyto- or lymphokines (interferons, interleukins, hematopoietic growth factors) for vaccination of patients. Antibody- and immune T cell-mediated immune reactions to autologous tumor cells are newly generated and/or quantitatively increased in immunized patients but do not always correlate with clinical response. Most vaccines are claimed to have reduced relapse rates presumably by inducing effective host immunity against micrometastases. Dendritic cell-peptide vaccines could induce partial or occasionally complete remissions in metastatic disease. The wrong antigenic presentation may result in tolerance induction toward the tumor; occasionally tumor enhancement may occur. Human tumor antigens when presented appropriately (with costimulatory molecules and with IL-2, IL-12) break the host's natural tolerance toward its tumor and induce rejection strength immune reactions even in patients with metastatic disease. Immune T cells thus generated could be collected for adoptive immunotherapy. For successful active specific immunization against human cancers the understanding of the immunoevasive maneuvers of the tumor cell (through FasL --> Fas; TRAIL; CD40L --> CD40; TGFbeta etc. systems) is essential.