OBJECTIVE

To evaluate how optical coherence tomography (OCT) angiography depicts clinical fundus findings in patients with diabetic retinopathy (DR).

METHODS

Prospective study evaluating imaging technology.

METHODS

Forty-seven eyes of 25 patients with DR were scanned using a high-speed 840-nm-wavelength spectral-domain optical coherence tomography instrument (RTVue XR Avanti; Optovue, Inc, Fremont, California, USA). Blood flow was detected using the split-spectrum amplitude-decorrelation angiography algorithm. Fluorescein angiography (FA) images were also obtained in all eyes and the ability to visualize microaneurysms, retinal nonperfused areas, and neovascularization was compared with that of the en face OCT angiograms.

RESULTS

In 42 eyes, microaneurysms detected by FA near the macula appeared as focally dilated saccular or fusiform capillaries on OCT angiograms of the superficial and/or deep capillary plexus. Retinal nonperfused areas visualized by FA appeared as lesions with no or sparse capillaries on OCT angiograms. Area measurement of retinal nonperfusion near the macula in 7 eyes revealed a difference between the extent of nonperfused areas in superficial and deep plexuses. In 4 eyes, the vascular structures of neovascularization at the optic disc were clearly visualized on OCT angiograms. Decreases and re-increases of flow in new vessels were quantified in an eye treated with anti-vascular endothelial growth factor.

CONCLUSIONS

OCT angiography can clearly visualize microaneurysms and retinal nonperfused areas and enables closer observation of each layer of the retinal capillaries. Quantitative information on new vessels can also be obtained. OCT angiography may be clinically useful to evaluate the microvascular status and therapeutic effect of treatments for DR.

Diffusion tensor magnetic resonance imaging (DT-MRI) was applied for in vivo quantification of myelin loss and regeneration. A transgenic mouse line (Oligo-TTK) expressing a truncated form of the herpes simplex virus 1 thymidine kinase gene (hsv1-tk) in oligodendrocytes was studied along with two induced phenotypes of myelin pathology. Myelin loss and axonal abnormalities differentially affect values of DT-MRI parameters in the brain of transgenic mice. Changes in the anisotropy of the white matter were assessed by calculating and mapping the radial (D perpendicular) and axial (D parallel) water diffusion to axonal tracts and fractional anisotropy (FA). A significant increase in D perpendicular attributed to the lack of myelin was observed in all selected brain white matter tracts in dysmyelinated mice. Lower D parallel values were consistent with the histological observation of axonal modifications, including reduced axonal caliber and overexpression of neurofilaments and III beta-tubulin. We show clearly that myelination and axonal changes play a role in the degree of diffusion anisotropy, because FA was significantly decreased in dysmyelinated brain. Importantly, myelin reparation during brain postnatal development induced a decrease in the magnitude of D( perpendicular) and an increase in FA compared with the same brain before recovery. The progressive increase in D parallel values was attributed to the gain in normal axonal morphology. This regeneration was confirmed by the detection of enlarged oligodendrocyte population, newly formed myelin sheaths around additional axons, and a gradual increase in axonal caliber.

Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

A number of DNA viruses carry apoptosis-inhibiting genes which enable the virus to escape from the host response. The adenovirus E1B 19K protein can inhibit apoptosis induced by E1A, tumour-necrosis factor-alpha, FAS antigen and nerve growth factor deprivation. The molecular basis of this inhibition remains poorly understood, but the fact that protection is seen in the absence of other viral proteins suggests that E1B 19K targets cellular proteins. We report here the identification of three cellular proteins that bind E1B 19K. One of these is a new member of the bcl-2 family, which we have called bak (for bcl-2 homologous antagonist/killer). This protein, which is expressed in a wide variety of cell types, binds to E1B 19K and to the Bcl-2 homologue Bcl-XL (ref. 17) in yeast. In addition, overexpression of bak in sympathetic neurons deprived of nerve growth factor accelerates apoptosis and blocks the protective effect of co-injected E1B 19K.

The death receptors tumour-necrosis factor receptor-1 (TNFR1) and CD95 (also known as FAS and APO-1) transduce signals that promote cell death by apoptosis. However, these receptors are also capable of inducing anti-apoptotic signals through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) or through activation of the proliferative mitogen-activated protein kinase (MAPK) cascade. Recent findings reveal a role for receptor internalization and endosomal trafficking in selectively transmitting the signals that lead either to apoptosis or to the survival of the cell.

Mathematical modeling is required for understanding the complex behavior of large signal transduction networks. Previous attempts to model signal transduction pathways were often limited to small systems or based on qualitative data only. Here, we developed a mathematical modeling framework for understanding the complex signaling behavior of CD95(APO-1/Fas)-mediated apoptosis. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, and neurodegeneration. During the last decade many of the molecular mechanisms of apoptosis signaling have been examined and elucidated. A systemic understanding of apoptosis is, however, still missing. To address the complexity of apoptotic signaling we subdivided this system into subsystems of different information qualities. A new approach for sensitivity analysis within the mathematical model was key for the identification of critical system parameters and two essential system properties: modularity and robustness. Our model describes the regulation of apoptosis on a systems level and resolves the important question of a threshold mechanism for the regulation of apoptosis.

Fanconi anemia (FA) is a heritable human cancer-susceptibility disorder, delineating a genetically heterogenous pathway for the repair of replication-blocking lesions such as interstrand DNA cross-links. Here we demonstrate that one component of this pathway, FANCJ, is a structure-specific DNA helicase that dissociates guanine quadruplex DNA (G4 DNA) in vitro. Moreover, in contrast with previously identified G4 DNA helicases, such as the Bloom's helicase (BLM), FANCJ unwinds G4 substrates with 5'-3' polarity. In the FA-J human patient cell line EUFAFANCJ G4 unwinding function correlates with the accumulation of large genomic deletions in the vicinity of sequences, which match the G4 DNA signature. Together these findings support a role for FANCJ in the maintenance of potentially unstable genomic G/C tracts during replication.

Diffusion tensor imaging (DTI) is used to study tissue composition and architecture in vivo. To increase the signal to noise ratio (SNR) of DTI contrasts, studies typically use more than the minimum of 6 diffusion weighting (DW) directions or acquire repeated observations of the same set of DW directions. Simulation-based studies have sought to optimize DTI acquisitions and suggest that increasing the directional resolution of a DTI dataset (i.e., the number of distinct directions) is preferable to repeating observations, in an equal scan time comparison. However, it is not always clear how to translate these recommendations into practice when considering physiological noise and scanner stability. Furthermore, the effect of different DW schemes on in vivo DTI findings is not fully understood. This study characterizes how the makeup of a DW scheme, in terms of the number of directions, impacts the precision and accuracy of in vivo fractional anisotropy (FA), mean diffusivity (MD), and principal eigenvector (PEV) findings. Orientation dependence of DTI reliability is demonstrated in vivo and a principled theoretical framework is provided to support and interpret findings with simulation results. As long as sampling orientations are well balanced, differences in DTI contrasts due to different DW schemes are shown to be small relative to intra-session variability. These differences are accentuated at low SNR, while minimized at high SNR. This result suggests that typical clinical studies, which use similar protocols but different well-balanced DW schemes, are readily comparable within the experimental precision.

Re-programming of metabolic pathways is a hallmark of physiological changes in cancer cells. The expression of certain genes that directly control the rate of key metabolic pathways including glycolysis, lipogenesis and nucleotide synthesis are drastically altered at different stages of tumor progression. These alterations are generally considered as an adaptation of tumor cells; however, they also contribute to the progression of tumor cells to become more aggressive phenotypes. This review summarizes the recent information about the mechanistic link of these genes to oncogenesis and their potential utility as diagnostic markers as well as for therapeutic targets. We particularly focus on three groups of genes; GLUT1, G6PD, TKTL1 and PGI/AMF in glycolytic pathway, ACLY, ACC1 and FAS in lipogenesis and RRM2, p53R2 and TYMS for nucleotide synthesis. All these genes are highly up-regulated in a variety of tumor cells in cancer patients, and they play active roles in tumor progression rather than expressing merely as a consequence of phenotypic change of the cancer cells. Molecular dissection of their orchestrated networks and understanding the exact mechanism of their expression will provide a window of opportunity to target these genes for specific cancer therapy. We also reviewed existing database of gene microarray to validate the utility of these genes for cancer diagnosis.

Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals previously implicated in hyperglycemia-induced vascular damage and inactivated 2 important antiatherogenic enzymes, prostacyclin synthase and eNOS. In 2 nondiabetic rodent models--insulin-resistant, obese Zucker (fa/fa) rats and high-fat diet-induced insulin-resistant mice--inactivation of prostacyclin synthase and eNOS was prevented by inhibition of FFA release from adipose tissue; by inhibition of the rate-limiting enzyme for fatty acid oxidation in mitochondria, carnitine palmitoyltransferase I; and by reduction of superoxide levels. These studies identify what we believe to be a novel mechanism contributing to the accelerated atherogenesis and increased cardiovascular disease risk occurring in people with insulin resistance.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.

We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular administration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune diseases.

Diffusion tensor imaging (DTI) measures have shown to be sensitive to white matter (WM) damage in multiple sclerosis (MS), not only inside focal lesions but also in user-defined regions in the so-called normal-appearing white matter (NAWM). New analysis techniques for DTI measures are now available that allow for hypothesis-free localization of damage. We performed DTI measurements of 30 MS patients selected for low focal lesion loads, and of 31 age-matched healthy controls and analyzed these using tract-based spatial statistics (TBSS). Patients were found to have a lower fractional anisotropy (FA) compared to controls in a number of brain regions, including the fornices, the left corona radiata, the inferior longitudinal fasciculus in both hemispheres, both optic radiations, and parts of the corpus callosum. In the regions of reduced FA, an increase in radial diffusivity and a less pronounced increase of axial diffusivity were found. Neurocognitive assessment showed that patients had normal visuospatial memory performance, just-normal attention, and impaired processing speed; the latter was associated with abnormal FA in the corpus callosum, an area which was relatively devoid of lesions visible on proton density-weighted images in our patients. TBSS can be useful in future studies with other MS patient samples to provide an unbiased localization of damage and generate location-specific hypotheses.

Cognitive abilities such as working memory (WM) capacity decrease with age. To determine the neurophysiological correlates of age-related reduction in working memory capacity, we studied 10 young subjects (<35 years of age; mean age=29) and twelve older subjects (>55 years of age; mean age=59) with whole brain blood oxygenation-level dependent (BOLD) fMRI on a 1.5 T GE MR scanner using a SPIRAL FLASH pulse sequence (TE=24 ms, TR=56 ms, FA=60 degrees , voxel dimensions=3.75 mm(3)). Subjects performed a modified version of the "n" back working memory task at different levels of increasing working memory load (1-Back, 2-Back and 3-Back). Older subjects performed as well as the younger subjects at 1-Back (p=0.4), but performed worse than the younger subjects at 2-Back (p<0.01) and 3-Back (p=0.06). Older subjects had significantly longer reaction time (RT) than younger subjects (p<0.04) at all levels of task difficulty. Image analysis using SPM 99 revealed a similar distribution of cortical activity between younger and older subjects at all task levels. However, an analysis of variance revealed a significant group x task interaction in the prefrontal cortex bilaterally; within working memory capacity, as in 1-Back when the older subjects performed as well as the younger subjects, they showed greater prefrontal cortical (BA 9) activity bilaterally. At higher working memory loads, however, when they performed worse then the younger subjects, the older subjects showed relatively reduced activity in these prefrontal regions. These data suggest that, within capacity, compensatory mechanisms such as additional prefrontal cortical activity are called upon to maintain proficiency in task performance. As cognitive demand increases, however, they are pushed past a threshold beyond which physiological compensation cannot be made and, a decline in performance occurs.

Memory and attentional control impairments are the two most common forms of dysfunction following mild traumatic brain injury (TBI) and lead to significant morbidity in patients, yet these functions are thought to be supported by different brain networks. This 3 T magnetic resonance diffusion tensor imaging (DTI) study investigates whether microstructural integrity of white matter, as measured by fractional anisotropy (FA) within a small set of individually localized regions of interest (ROIs), is associated with these cognitive domains in normal adults and adults with mild TBI. Results in a sample of 23 normal controls reveal a significant correlation between attentional control and FA within a ROI in the left hemisphere anterior corona radiata. Furthermore, the controls demonstrate a correlation between memory performance and FA in a ROI placed in the uncinate fasciculus. Next, to examine whether these relationships are found in the pathological ranges of attention, memory and microstructural white matter integrity associated with mild TBI, these analyses were applied to a group of 43 mild TBI patients. Results, which generally demonstrated a wider range of attention, memory and FA scores, replicated the correlation between attentional control and FA in left hemisphere anterior corona radiata, as well as the correlation between memory performance and FA in the uncinate fasciculus. These two sets of brain-behaviour relationships were highly specific, as shown by a lack of correlation between attention and uncinate fasciculus FA and the lack of correlation between memory performance and anterior corona radiata FA. Furthermore, a 'correlational double dissociation' was demonstrated to exist between two distinct frontal structures independently associated with attention and memory, respectively, via a series of multiple regression analyses in both normal controls and adults with mild TBI. The results of the multiple regression analyses provide direct evidence that tract-specific variation in microstructural white matter integrity among normal controls and among mild TBI patients can account for much of the variation in performance in specific cognitive domains. More generally, such findings suggest that diffusion anisotropy measurement can be used as a quantitative biomarker for neurocognitive function and dysfunction.

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.

Postmortem histological studies have demonstrated that myelination in human brain white matter (WM) continues throughout adolescence and well into adulthood. We used in vivo diffusion-weighted magnetic resonance imaging to test for age-related WM changes in 42 adolescents and 20 young adults. Tract-Based Spatial Statistics (TBSS) analysis of the adolescent data identified widespread age-related increases in fractional anisotropy (FA) that were most significant in clusters including the body of the corpus callosum and right superior corona radiata. These changes were driven by changes in perpendicular, rather than parallel, diffusivity. These WM clusters were used as seeds for probabilistic tractography, allowing us to identify the regions as belonging to callosal, corticospinal, and prefrontal tracts. We also performed voxel-based morphometry-style analysis of conventional T1-weighted images to test for age-related changes in grey matter (GM). We identified a cluster including right middle frontal and precentral gyri that showed an age-related decrease in GM density through adolescence and connected with the tracts showing age-related WM FA increases. The GM density decrease was highly significantly correlated with the WM FA increase in the connected cluster. Age-related changes in FA were much less prominent in the young adult group, but we did find a significant age-related increase in FA in the right superior longitudinal fascicle, suggesting that structural development of this pathway continues into adulthood. Our results suggest that significant microstructural changes in WM continue throughout adolescence and are associated with corresponding age-related changes in cortical GM regions.

Body fluids of cancer patients contain TEXs (tumour-derived exosomes). Tumours release large quantities of TEXs, and the protein content of exosome or MV (microvesicle) fractions isolated from patients' sera is high. TEXs down-regulate functions of immune cells, thus promoting tumour progression. We isolated TEXs from tumour cell supernatants and sera of patients with solid tumours or AML (acute myelogenous leukaemia). The molecular profile of TEXs was distinct from that of circulating exosomes derived from normal cells. TEXs were co-incubated with activated T-cells, conventional CD4(+) CD25(neg) T-cells or CD56(+) CD16(+) NK (natural killer) cells respectively. TEXs down-regulated CD3ζ and JAK3 (Janus kinase 3) expression in primary activated T-cells and mediated Fas/FasL (Fas ligand)-driven apoptosis of CD8(+) T-cells. TEXs promoted CD4(+) CD25(neg) T-cell proliferation and their conversion into CD4(+) CD25(hi)FOXP3+ (FOXP3 is forkhead box P3) Treg cells (regulatory T-cells), which also expressed IL-10 (interleukin 10), TGFβ1 (transforming growth factor β1), CTLA-4 (cytotoxic T-lymphocyte antigen 4), GrB (granzyme B)/perforin and effectively mediated suppression. Neutralizing antibodies specific for TGFβ1 and/or IL-10 inhibited the ability of TEXs to expand Treg cells. TEXs obtained at diagnosis from AML patients' sera were positive for blast-associated markers CD33, CD34, CD117 and TGFβ1, and they decreased cytotoxic activity of NK cells isolated from NC (normal control) donors, induced Smad phosphorylation and down-regulated NKG2D receptor expression. Correlations between the TEX molecular profile or TEX protein levels and clinical data in cancer patients suggest that TEX-mediated effects on immune cells are prognostically important. In contrast with exosomes released by normal cells, TEXs have immunosuppressive properties and are involved in regulating peripheral tolerance in patients with cancer.

Magnetic resonance diffusion tensor imaging (DTI) enables the discrimination of white matter pathways before myelination is evident histologically or on conventional MRI. In this investigation, 14 premature neonates with no evidence of white matter abnormalities by conventional MRI were studied with DTI. A custom MR-compatible incubator with a novel high sensitivity neonatal head coil and improved acquisition and processing techniques were employed to increase image quality and spatial resolution. The technical improvements enabled tract-specific quantitative characterization of maturing white matter, including several association tracts and subcortical projection tracts not previously investigated in neonates by MR. Significant differences were identified between white matter pathways, with earlier maturing commissural tracts of the corpus callosum, and deep projection tracts of the cerebral peduncle and internal capsule exhibiting lower mean diffusivity (Dav) and higher fractional anisotropy (FA) than later maturing subcortical projection and association pathways. Maturational changes in white matter tracts included reductions in Dav and increases in FA with age due primarily to decreases in the two minor diffusion eigenvalues (lambda2 and lambda3). This work contributes to the understanding of normal white matter development in the preterm neonatal brain, an important step toward the use of DTI for the improved evaluation and treatment of white matter injury of prematurity.

Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.

When expressed in heterologous cells, the viral FLIP protein (vFLIP) of Kaposi's-sarcoma-associated herpesvirus (KSHV) has been reported both to block Fas-mediated apoptosis and to activate the NF-kappaB activation pathway by interaction with IkappaB kinase (IKK). In a yeast-two-hybrid screen, we identified IKKgamma as an interacting partner of vFLIP. We expressed fragments of IKKgamma in mammalian cells and bacteria, and identified the central CCR3/4 (amino acids 150-272) as the vFLIP binding region. To investigate the proteins interacting with vFLIP in a KSHV-infected primary effusion lymphoma (PEL) cell line, we immunoprecipitated vFLIP and identified four associated proteins by mass spectrometry: IKK components IKKalpha, beta and gamma, and the chaperone, Hsp90. Using gel filtration chromatography, we demonstrated that a single population of vFLIP in the cytoplasm of PEL cells co-eluted and co-precipitated with an activated IKK complex. An inhibitor of Hsp90, geldanamycin, inhibited IKK's kinase activity induced by vFLIP and killed PEL cells, suggesting that vFLIP activation of IKK contributes to PEL cell survival.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.