Proteomic identification of synaptic caspase substrates.

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states.

Snake venom toxin from Vipera lebetina turanica induces apoptosis of colon cancer cells via upregulation of ROS- and JNK-mediated death receptor expression.

BACKGROUND: Abundant research suggested that the cancer cells avoid destruction by the immune system through down-regulation or mutation of death receptors. Therefore, it is very important that finding the agents that increase the death receptors of cancer cells. In this study, we demonstrated that the snake venom toxin from Vipera lebetina turanica induce the apoptosis of colon cancer cells through reactive oxygen species (ROS) and c-Jun N-terminal kinases (JNK) dependent death receptor (DR4 and DR5) expression. METHODS: We used cell viability assays, DAPI/TUNEL assays, as well as western blot for detection of apoptosis related proteins and DRs to demonstrate that snake venom toxin-induced apoptosis is DR4 and DR5 dependent. We carried out transient siRNA knockdowns of DR4 and DR5 in colon cancer cells. RESULTS: We showed that snake venom toxin inhibited growth of colon cancer cells through induction of apoptosis. We also showed that the expression of DR4 and DR5 was increased by treatment of snake venom toxin. Moreover, knockdown of DR4 or DR5 reversed the effect of snake venom toxin. Snake venom toxin also induced JNK phosphorylation and ROS generation, however, pretreatment of JNK inhibitor and ROS scavenger reversed the inhibitory effect of snake venom toxin on cancer cell proliferation, and reduced the snake venom toxin-induced upregulation of DR4 and DR5 expression. CONCLUSIONS: Our results indicated that snake venom toxin could inhibit human colon cancer cell growth, and these effects may be related to ROS and JNK mediated activation of death receptor (DR4 and DR5) signals.

Pain behavior in the formalin test persists after ablation of the great majority of C-fiber nociceptors.

Although the formalin test is a widely used model of persistent pain, the primary afferent fiber types that underlie the cellular and behavioral responses to formalin injection are largely unknown. Here we used a combined genetic and pharmacological approach to investigate the effect of ablating subsets of primary afferent nociceptors on formalin-induced nocifensive behaviors and spinal cord Fos protein expression. Intrathecal capsaicin-induced ablation of the central terminals of TRPV1+neurons greatly reduced the behavioral responses and Fos elicited by low-dose (0.5%) formalin. In contrast, genetic ablation of the MrgprD-expressing subset of non-peptidergic unmyelinated afferents, which constitute a largely non-overlapping population, altered neither the behavior nor the Fos induced by low-dose formalin. Remarkably, nocifensive behavior following high-dose (2%) formalin was unchanged in mice lacking either afferent population, or even in mice lacking both populations, which together make up the great majority of C-fiber nociceptors. Thus, at high doses, which are routinely used in the formalin test, formalin-induced "pain" behavior persists in the absence of the vast majority of C-fiber nociceptors, which points to a contribution of a large spectrum of afferents secondary to non-specific formalin-induced tissue and nerve damage.

A signaling polypeptide derived from an innate immune adaptor molecule can be harnessed as a new class of vaccine adjuvant.

Modulation of intracellular signaling using cell-permeable polypeptides is a promising technology for future clinical applications. To develop a novel approach to activate innate immune signaling by synthetic polypeptides, we characterized several different polypeptides derived from the caspase recruitment domain (CARD) of IFN-beta promoter stimulator 1, each of which localizes to a different subcellular compartment. Of particular interest was, N'-CARD, which consisted of the nuclear localization signal of histone H2B and the IFN-beta promoter stimulator 1CARD and which localized to the nucleus. This polypeptide led to a strong production of type I IFNs and molecular and genetic analyses showed that nuclear DNA helicase II is critically involved in this response. N'-CARD polypeptide fused to a protein transduction domain (N'-CARD-PTD) readily transmigrated from the outside to the inside of the cell and triggered innate immune signaling. Administration of N'-CARD-PTD polypeptide elicited production of type I IFNs, maturation of bone marrow-derived dendritic cells, and promotion of vaccine immunogenicity by enhancing Ag-specific Th1-type immune responses, thereby protecting mice from lethal influenza infection and from outgrowth of transplanted tumors in vivo. Thus, our results indicate that the N'-CARD-PTD polypeptide belongs to a new class of vaccine adjuvant that directly triggers intracellular signal transduction by a distinct mechanism from those engaged by conventional vaccine adjuvants, such as TLR ligands.

Expresión de bcl-2, ki-67 y caspasa-3 en lesiones cancerosas de la mucosa oral. Resultados preliminares / Bcl-2, ki-67 and caspasa-3 expression in oral cancer lesions. Preliminary results

El cáncer oral representa del 2 al 4% de todos los cánceres diagnosticados, siendo el carcinoma epidermoide el tumor más común encontrado a nivel histológico. En varios estudios se ha constatado que el uso de la clasificación TNM del cáncer oral no resulta de utilidad a la hora de establecer el pronóstico tumoral. Nos encontramos en la era de los marcadores tumorales que actualmente pueden ser detectados y medidos con las más modernas técnicas de inmunohistoquímica. Material y método: aplicación de la técnica inmunohistoquímica peroxidasa- antiperoxidasa para la detección de las moléculas bcl-2, caspasa-3 y Ki-67 en lesiones cancerosas. Resultados preliminares y discusión: la detección molecular del marcador antiapoptótico bcl-2, el marcador proapoptótico caspasa 3 y el marcador de proliferación celular Ki-67 nos informará sobre la situación más o menos grave del paciente respecto de su proceso canceroso (AU)

Oral cancer represents 2%-4% of all types of cancer that are diagnosed and epidermal carcinoma is the most common tumour found in oral cavity. Some experimental studies have shown that TNM classification is not useful when you want to know tumour prognosis. Now we are in the modern inmunohistochemical period with new techniques for cancer diagnose. Material and method: use the peroxidase-antiperoxidase inmunohistochemical technique to detect bcl-2, caspasa-3 and Ki-67 molecules in cancerous lesions. Preliminary results and discussion: molecular detection of the antiapoptotic bcl-2 molecule, the proapoptotic caspasa-3 molecule and the cellular proliferation indicator Ki-67 will show us about the gravity of the patient situation (AU)

Liquen plano oral (II). Mecanismos apoptóticos y posible malignización / Oral Lichen planus (II), apoptolic mechanism and possible malignization

Se presenta un estudio histopatológico realizado con una muestra de 52 pacientes con liquen plano oral evaluados en la Universidad Complutense de Madrid con el objetivo de valorar los fenómenos apoptóticos y su posible vinculación con la malignización de las lesiones (AU)

A histopathological study is carried out on 52 patients with oral lichen planus at the Faculty of Dentistry of Madrid, with the aim to assess apoptotic phenomena and their possible relationship with the malignization of the lesions (AU)

A DNA vaccine co-expressing antigen and an anti-apoptotic molecule further enhances the antigen-specific CD8+ T-cell immune response.

We have shown that DNA encoding the anti-apoptotic protein Bcl-xL enhances E7-specific CD8+ T-cell responses and DNA encoding pro-apoptotic protein caspase-3 suppresses E7-specific CD8+ T-cell responses when co-administered intradermally via gene gun with DNA encoding human papillomavirus type 16 (HPV-16) E7 linked to the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1). E7 and LAMP-1 are linked to form the chimeric Sig/E7/LAMP-1 (SEL). Because co-administration does not ensure delivery of both constructs to a single cell, we used pVITRO, a mammalian expression vector with double promoters, to ensure expression of both molecules in the same cell. We vaccinated C57BL/6 mice with pVITRO-SEL-Bcl-xL, pVITRO-SEL-mtBcl-xL, pVITRO-SEL, or pVITRO-SEL-caspase-3 intradermally via gene gun and intramuscularly via injection. We demonstrated that vaccination with pVITRO achieved similar results to a co-administration strategy: that Bcl-xL enhanced the E7-specific CTL response and caspase-3 suppressed the E7-specific CTL response. In addition, we found intradermal vaccination elicited significantly higher numbers of E7-specific CD8+ T cells compared to intramuscular vaccination. Thus, intradermal vaccination with a pVITRO vector combining an anti-apoptotic strategy (Bcl-xL) and an intracellular targeting strategy (SEL) further enhances the E7-specific CD8+ T-cell response and guarantees co-expression of both encoded molecules in transfected cells.

More efficient mobilisation of peripheral blood stem cells with HiDAC+AMSA+G-CSF than with mini-ICE+G-CSF in patients with AML.

We have compared the efficacy of two PBSC mobilisation regimens, mini-ICE+filgrastim (second consolidation) and HiDAC+AMSA+filgrastim (third consolidation), in two consecutive cohorts of patients with AML CR1 receiving treatment according to a joint protocol. Group A: 18 patients, aged 41 (21-65) years, were mobilised with mini-ICE (idarubicin 8 mg/m(2)+cytarabine 800 mg/m(2)+etoposide 150 mg/m(2) days 1-3) followed by filgrastim 300-480 microg once daily s.c. from day 11 after start of chemotherapy. Only four patients reached >5 CD34+ cells/microl blood (B-CD34+) and were able to undergo leukaphereses. Two out of 18 (11%) reached the defined target of >/=2.0 x 10(6) CD34+ cells/kg after 1-3 leukaphereses. Group B: 20 patients, aged 50 (29-67) years, received HiDAC+AMSA (cytarabine 3 g/m(2) b.i.d. days 1, 3, 5+amsacrine 150 mg/m(2) q.d. days 2, 4) followed by filgrastim at a similar dose starting on day 7. A total of 18 patients reached B-CD34+ >5/microl and underwent PBSC harvesting, starting on day 23 (14-29) and yielding 4.0 (0.9-21) x 10(6) CD34+ cells/kg. Of 20 patients, 17 (85%) reached the defined target of >/=2.0 x 10(6) CD34+ cells/kg after 1-3 leukaphereses. We conclude that HiDAC+AMSA+G-CSF - in contrast to mini-ICE+G-CSF - is an efficient regimen for mobilising PBSC in patients with AML CR1.

Caspase-3-dependent apoptosis in vascular smooth muscle cell by proteasome inhibition.

The effects of a number of substances on neointima formation following angioplasty have been investigated in animal models. It was suggested that delivering of proteasome inhibitor to the site of vascular injury would be a potential therapeutic approach in prevention of vascular restenosis. But the mechanisms underlying biologic activities of proteasome inhibition in vascular smooth muscle cells (VSMCs) are largely unknown. We have investigated effects of proteasome inhibition on VSMCs using proteasome inhibitor MG115. MG115 induced apoptotic death in VSMCs as determined by viability, morphology, and DNA fragmentation. Proteasome inhibition was accompanied by up-regulation of p53, p21, and p27. In contrast, there were no appreciable alterations in the levels of Bcl-2 and Bax. Proteasome inhibition was followed by activation of caspase-3 but not of -8. The induction of apoptosis was suppressed by treatment with a selective inhibitor of the caspase-3 family, z-DEVD-fmk but not by NG-monomethyl-L-arginine. These results indicate that proteasome inhibition induces apoptosis in VSMCs by activation of caspase-3.

Glucocorticoid cotreatment induces apoptosis resistance toward cancer therapy in carcinomas.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Glucocorticoids (GCs) are frequently used as cotreatment because they may have potent proapoptotic properties and reduce nausea, hyperemesis, and acute toxicity on normal tissue. In contrast to the proapoptotic effect of GCs in lymphoid cells, resistance toward cancer therapy-mediated apoptosis was induced in solid tumors of human cervix and lung carcinomas. Filter hybridization, expression data, as well as functional assays identified multiple core apoptosis molecules, which are regulated by GCs in a pro- or antiapoptotic manner. Both antiapoptotic genes such as FLIP and members of the Bcl-2 and IAP family as well as proapoptotic elements of the death receptor and mitochondrial apoptosis pathways were down-regulated in carcinomas resulting in a decreased activity of caspase-8, caspase-9, and caspase-3. In contrast, death receptor and mitochondrial apoptosis signaling as well as caspase activity was enhanced by dexamethasone in lymphoid cells. To restore apoptosis sensitivity in dexamethasone-treated carcinomas, caspase-8 and caspase-9 were transfected. This resensitized tumor cells in vitro and xenografts in vivo to cisplatin induced cell death. These data therefore raise concern about the widespread combined use of GCs with antineoplastic drugs or agents in the clinical management of cancer patients.

A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.

Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers.

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Blocking caspase-activated apoptosis improves contractility in failing myocardium.

Cardiac myocyte apoptosis has been demonstrated in end-stage failing human hearts. The therapeutic utility of blocking apoptosis in congestive heart failure (CHF) has not been elucidated. This study investigated the role of caspase activation in cardiac contractility and sarcomere organization in the development of CHF. In a rabbit model of heart failure obtained by rapid ventricular pacing, we demonstrate, using in vivo transcoronary adenovirus-mediated gene delivery of the potent caspase inhibitor p35, that caspase activation is associated with a reduction in contractile force of failing myocytes by destroying sarcomeric structure. In this animal model gene transfer of p35 prevented the rise in caspase 3 activity and DNA-histone formation. Genetically manipulated hearts expressing p35 had a significant improvement in left ventricular pressure rise (+dp/dt), decreased end-diastolic chamber pressure (LVEDP), and the development of heart failure was delayed. To better understand this benefit, we examined the effects of caspase 3 on cardiomyocyte dysfunction in vitro. Microinjection of activated caspase 3 into the cytoplasm of intact myocytes induced sarcomeric disorganization and reduced contractility of the cells. These results demonstrate a direct impact of caspases on cardiac function and may lead to novel therapeutic strategies via antiapoptotic regimens.

Autologous hematopoietic stem cell transplantation in chronic myeloid leukemia with different clinical stages.

Seven patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) were treated with an ICE-based regimen plus G-CSF with the aim of mobilizing and collecting Ph-negative peripheral stem cells in the setting of an autologous transplant program. Five patients had CML in the first chronic phase and 2 in the accelerated phase. All patients had been previously treated with interferon-alpha. Median value and ranges for harvested mononuclear cells, CD34+ cells and CFU-GM, respectively: 5.65 x 10(8)/kg (2.61-11.38); 1.48 x 10(6)/kg (0.216-3.5), and 3.43 x 10(4)/kg (0.243-11.6). FISH was the only useful method for detection of minimal residual disease on apheresis product showing <5% t(9;22) positive cells in 2 cases and <10% positive cells in 4 other cases. Four of seven autologous grafts have been transplanted to date. Busulfan conditioning was used in 1 case and TBI/Cy conditioning in 3 other cases. All patients are alive and well following transplantation and are on interferon-alpha therapy.

17-beta-estradiol induces an inhibitor of active caspases.

We have shown previously that caspase-6 activity is lethal to human neurons (LeBlanc et al., 1999; Zhang et al., 2000). Here we find that 17-beta-estradiol but not 17-alpha-estradiol prevents caspase-6-mediated neuronal cell death. 17-beta-estradiol-treated neuronal extracts directly inhibit recombinant active caspase-6, caspase-3, caspase-7, and caspase-8 in vitro. We conclude that 17-beta-estradiol induces a caspase inhibitory factor (CIF) that is preventing neuronal apoptosis. The induction of CIF occurs within 10 min of 17-beta-estradiol exposure to neurons, does not require de novo protein synthesis, and involves mitogen-activated protein kinase activation. The effect is antagonized by the estrogen receptor antagonist tamoxifen. In contrast, 17-beta-estradiol does not induce CIF or prevent caspase-mediated cell death in cultured astrocytes. CIF does not act through oxidation of the caspase active site. CIF activity copurifies with proteins of between 12 and 14 kDa in size. Our results indicate that 17-beta-estradiol induces an inhibitor of active caspases through a receptor-mediated nongenomic pathway and provide an additional mechanism for the neuroprotective action of 17-beta-estradiol that is likely highly relevant to the understanding of the role of estrogen against Alzheimer's disease.

The MCS + revision A2 for highly efficient PBSC collection.

Autologous PBSC transplantation is an integral component of the management of hemato-oncology patients. In order to reduce the number of sessions needed to collect the desired number of repopulating cells there has been significant research activity in developing progressively more and more effective technologies and techniques. Recently our group has been involved in the rejuvenation of the MCS + apparatus for both platelet and PBSC collection. The so called "version A2 protocol" is aimed at collecting PBSC in a very efficient way. This protocol is characterized by high blood flow rates both in the collection and reinfusion (80 ml/min) recirculation (56 ml/min) and collection phases (30 ml/min). Only one recirculation is carried out every 5 cycles and only from 5 to 7 are carried out for a single procedure. Twenty-seven collections were carried out of which 25 were evaluable in terms of PBSC efficiency. These averaged 68.8% in an average procedure time of 3.5-5 h for processing an average of 7,052 ml of blood. The RBC contamination was reduced to approximately 5.02 g of hemoglobin and the average volume of the product to 177 ml. If these results are confirmed, the gap between CFC and DFC PBSC is progressively closing.

Selective and protracted apoptosis in human primary neurons microinjected with active caspase-3, -6, -7, and -8.

We have shown previously that caspase-6 is activated in serum deprivation-mediated human neuronal cell death and correlates with increased production of Alzheimer's disease (AD) amyloid beta peptide (Abeta). Here, we show by direct microinjection of recombinant active enzymes that caspase-6 (>0.5 pg/cell) induces a protracted course of apoptosis in neurons in a caspase-specific, dose- and time-dependent manner in the presence of serum. Only transient activation of caspase-6 is required to initiate apoptosis. Caspase-6 induces apoptosis directly without the activation of other caspase effectors. Doses of caspase-6 of <0.25 pg/cell induce only 20% cell death within 16 d but render neurons vulnerable to oxidative stress, indicating that caspase activation affects neurons despite the absence of cell death. Caspase-3 induces neuronal apoptosis in 20% of the cells, whereas caspase-7 or -8 do not induce apoptosis. In contrast, astrocytes undergo apoptosis within 24 hr when microinjected with caspase-3 but not caspase-6, -7, or -8. These results show cell type-specific vulnerability to caspases in the CNS. The results suggest that activation of caspases in human neurons does not lead to an immediate and rapid process of cell death but provokes a protracted form of apoptosis. Activation of caspases in human neurons may participate in the long-term overproduction of Abeta and other potential toxic fragments resulting from caspase-mediated proteolysis. These results are consistent with the protracted and age-dependent nature of AD.

Early total white blood cell recovery is a predictor of low number of apheresis and good CD34(+) cell yield.

OBJECTIVE: We analysed peripheral blood progenitor cell (PBPC) mobilisation and collection in order to assess the main factors related to CD34(+) cell yields in patients affected by haematological malignancies. PATIENTS AND METHODS: The features of CD34(+) cell mobilisation of patients with haematological malignancies that underwent autologous bone marrow transplantation were examined. Mobilisation chemotherapy consisted mainly of cyclophosphamide (CY) 4 or 7 g/m(2) followed by growth factors. Leukapheresis was started when the WBC counts reached 1.0x10(9)/l with the aim to collect at least 5x10(6) CD34(+) cells/kg body weight. The aphereses were performed on continuous-flow blood cell separators. The analysed variables were: age, diagnosis, CT mobilisation regimen, type of growth factor, number of previous CT lines, prior radiotherapy, days for WBC recovery and number of aphereses procedures to achieve the target of CD34(+) cells. RESULTS: There were 41 consecutive patients (26 M/15 F): 21 non-Hodgkin's lymphoma (NHL), 15 Hodgkin's disease (HD), two chronic myeloid leukaemia (CML) and three multiple myeloma (MM). Eleven patients could not collect the proposed threshold of CD34(+) cells. CY 4 mobilised patients recovered WBC counts in less days (P=0.03). By ANOVA, the days to WBC recovery had a linear function of the predictors "number of aphereses" and "type of mobilisation CT" (coefficients: 0.86 and 0.95, respectively). For the number of aphereses and WBC recovery after CT mobilisation, we obtained a correlation coefficient of 0.36 (P=0.02). CONCLUSION: This study shows that it is feasible to mobilise and collect PBPC in patients previously treated with CT with or without RT. There was a linear correlation between the days for WBC recovery and the number of aphereses needed to collect the target number of CD34(+) cells. The study suggests that early WBC recovery, using mainly CY 4 mobilisation chemotherapy, is an important predictor of a low number of aphereses to achieve a good CD34(+) yield.

Caspase inhibitor z-VAD-FMK inhibits keratocyte apoptosis, but promotes keratocyte necrosis, after corneal epithelial scrape.

The purpose of this study was to determine whether the caspase inhibitor z-VAD-FMK could be applied topically prior to epithelial scrape injury to inhibit keratocyte apoptosis. Rabbit corneas were treated with z-VAD-FMK or vehicle alone prior to epithelial scrape injury. Cell fate was analysed at 4 hr after epithelial scrape using quantitative TUNEL assay, propidium iodide staining, and transmission electron microscopy. Less stained anterior stromal keratocytes were detected with the quantitative TUNEL assay in corneas pre-treated with z-VAD-FMK than in corneas pretreated with vehicle at 4 hr after epithelial scrape. This difference appeared to be confirmed by propidium iodide staining of keratocyte nuclei. It was observed that fewer nuclei were stained with propidium iodide in the DMSO vehicle treated corneas compared to the z-VAD-FMK treated corneas. Analysis of corneas with transmission electron microscopy, however, indicated that many anterior stromal keratocytes in corneas pretreated with z-VAD-FMK, but not vehicle, had cell morphologic changes more consistent with necrosis. Although pretreatment of corneas with the caspase inhibitor z-VAD-FMK inhibited keratocyte apoptosis detected with the TUNEL assay, transmission electron microscopy revealed that many anterior stromal keratocytes in z-VAD-FMK-treated corneas instead died by necrosis. Thus, z-VAD-FMK is unlikely to be useful to modulate corneal would healing through inhibition of keratocyte apoptosis induced by epithelial injury. The TUNEL assay should not be used to monitor cell fate without confirmation using analyses that also detect necrosis.