Regulation of APO-2 ligand/trail expression in NK cells-involvement in NK cell-mediated cytotoxicity.

Apo-2L is a new member of the tumour necrosis factor (TNF) family shown to induce apoptosis in a number of tumour cell lines. Apo-2L mRNA is expressed by numerous human tissues. Here we report that Apo-2L is expressed and utilized by human Natural Killer (NK) cells. NK cells were shown to express surface Apo-2L in response to interleukin 2 (IL-2) activation, and this response was restricted to the CD3(-)population of the NK cells. Apo-2L mRNA and intracellular Apo-2L were present in both CD3(-)and CD3(+)NK cells; however, increased expression in response to IL-2 was only observed in CD3(-)CD56(+)cells. Also, IL-2-activated NK cells were shown to utilize membrane-bound Apo-2L in mediating lysis of Jurkat cells. Furthermore, Apo-2L-induced apoptosis of Jurkat cells was more rapid than FasL-induced apoptosis, indicating an important and distinct role for Apo-2L in apoptotic cell destruction. In conclusion, we report that NK cells express Apo-2L and that IL-2 activated CD3(-)NK cells utilize the Apo-2L pathway in mediating target cell lysis.     

Internal desynchronization of circadian rhythms and tolerance to shift work

Intolerance to shift work may result from individual susceptibility to an internal desynchronization. Some shift workers (SW) who show desynchronization of their circadian rhythms (e.g., sleep-wake, body temperature, and grip strength of both hands) exhibit symptoms of SW intolerance, such as sleep alteration, persistent fatigue, sleep medication dependence, and mood disturbances, including depression. Existing time series data previously collected from 48 male Caucasian French SW were reanalyzed specifically to test the hypothesis that internal synchronization of circadian rhythms is associated with SW intolerance and symptoms. The entry of the subjects into the study was randomized. Three groups were formed thereafter: SW with good tolerance (n=14); SW with poor tolerance, as evident by medical complaints for at least one year (n=19); and former SW (n=15) with very poor tolerance and who had been discharged from night work for at 1.5 yr span but who were symptom-free at the time of the study. Individual and longitudinal time series of selected variables (self-recorded sleep-wake data using a sleep log, self-measured grip strength of both hands using a Colin Gentile dynamometer, and oral temperature using a clinical thermometer) were gathered for at least 15 days, including during one or two night shifts. Measurements were performed 4-5 times/24 h. Power spectra used to quantify the prominent period (tau) and t-test, chi square, and correlation coefficient were used as statistical tools. The mean (+/-SEM) age of SW with good tolerance was greater than that of SW with poor tolerance (44.9+/-2.1 yrs vs. 40.1+/-2.6 yrs, p<.001) and of former SW discharged from night work (very poor tolerance; 33.4+/-1.7, p<.001). The shift-work duration (yrs) was longer in SW with good than poor tolerance (19.9+/-2.2 yrs vs. 15.7+/-2.2; p<0.002) and former SW (10.7+/-1.2; p<.0001). The correlation between subject age and shift-work duration was stronger in tolerant SW (r=0.97, p<.0001) than in non-tolerant SW (r=0.80, p<0.001) and greater than that of former SW (r=0.72, p<.01). The mean sleep-wake rhythm tau was 24 h for all 48 subjects. The number of desynchronized circadian rhythms (tau differing from 24 h) was greater in non-tolerant than in tolerant SW (chi square=38.9, p<.0001). In Former SW (i.e., 15 individuals assessed in follow-up studies done 1.5 to 20 yrs after return to day work), both symptoms of intolerance and internal desynchronization were reduced or absent. The results suggest that non-tolerant SW are particularly sensitive to the internal desynchronization of their circadian time organization.     

CIRCADIAN PATTERN OF SIMULATED FLIGHT PERFORMANCE OF PILOTS IS DERIVED FROM ULTRADIAN COMPONENTS *

Studies suggest some physiologic, cognitive, and behavioral 24h rhythms are generated by cyclic components that are shorter in period than circadian. The aim of this study was [1] to examine the hypothesis that 24h human performance rhythms arise from the integration of high-frequency endogenous components and [2] to quantify the contribution of each higher frequency component to the phenotype of the rhythm. We monitored the performance of 9 experienced pilots by employing an array of cognitive-based tests conducted in a flight simulator so that, over the 6-day experiment, data were obtained for each 2h interval of the 24h. The activity-rest schedule of the subjects, no matter the exact clock time schedule of sleep and activity, always consisted of 14h activity (when they carried out regular professional duties) and 10h rest, with at least 8h of sleep. The simulated combat scenarios consisted of simple and complex tasks associated with target interception, aircraft maneuvering, and target shooting and downing. The results yielded two indices: the number of prominent periodicities in the time series and the relative magnitude of the amplitude of each relative to the construction of the composite 24h waveform. Three cyclic components (8h, 12h, and 24h) composed the observed 24h performance pattern. The dominant period and acrophase (peak time) of the compound output rhythm were determined by the interplay between the amplitudes of the various individual ultradian components. Task complexity (workload) increases the expression of the ultradian entities in the 24h pattern. We constructed a model composed of the multiple ultradian components; the composite output defined a “time span” (of 2h-4h duration) as opposed to an exact “time point” of high and low performance, endowing elevated functional capability. (Chronobiology International, 18(6), 987-1003, 2001)     

Selective knockdown of the long variant of cellular FLICE inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis

Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIP(S)) and a long, 55-kDa form (c-FLIP(L)). The role of c-FLIP(S) as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIP(L) remains controversial. Although overexpression of transfected c-FLIP(L) inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIP(L) on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIP(L) in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIP(L) augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIP(L) functions primarily as an inhibitor of death receptor-mediated apoptosis.     

Euchronism,Allochronism, and Dyschronism: Is Internal Desynchronization of Human Circadian Rhythms a Sign of Illness?

The authors define a subject as euchronic when the circadian parameters—tau (τ=period), Ø (acrophse or peak time), A (amplitude), and M (MESOR=24 h rhythm‐adjusted mean)—of a set of circadian variables are within the confidence limits of appropriate reference values of healthy subjects (HS). We define internal desynchronization as a state in which the circadian τ of a set of rhythms differs from 24 h and when the τ of a given variable differs from that of other variables. Such a state was first observed in singly isolated HS without access to time cues and clues. Herein, data and analyses are presented demonstrating that internal desynchronization appears to be a rather common phenomenon in HS dwelling in their natural environment (i.e., in the presence of usual zeitgebers). This has been documented by longitudinal studies (n?15 days) of the circadian rhythm in sleep‐wakefulness, body temperature, right‐ and left‐hand‐grip strength, and reaction time involving a total of 246 HS and 134 shift workers (SW), with 45.5% showing good and 54.5% poor SW tolerance. The presence of internal desynchronization observed in SW was associated SW intolerance, with symptoms being sleep alteration/disturbances, sleeping‐pill dependence, persisting fatigue (asthenia), mood alteration, and digestive complaints. Internal desynchronization was also documented in groups of HS and tolerant SW, though it was almost the rule among the intolerant SW. The authors introduce two new terms: allochronism to describe the time organization of those SW who evidence internal desynchronization without detectable clinical symptoms, and dyschronism to describe the time organization of those SW who exhibit internal desynchrobization plus the symptoms of SW intolerance or medical illness. The condition of allochronism is not restricted only to SW tolerance, as it was detected in 112 HS without medical complains when exposed to various experimental conditions, including medications and placebos, sojourn in the high Arctic summer, intensive sport training, and task‐loaded cognitive performance testing. Dyschronism in SW who are sleep‐deprived is associated with persisting fatigue. An unpublished Gallup survey found that 47% of 2478 respondents experienced a state of asthenia during the previous 12 months, with symptoms mimicking those of SW intolerance. In one‐third of the cases, the origin of the asthenia was undetermined. Taking into account the high incidence of internal desynchronization found in past investigations and the clinical observation that sleep deprivation is a consequence of many acute and chronic medical conditions (nocturnal pain, nocturnal asthma, etc.), it is suggested that dyschronism may be responsible for the asthenia of unknown origin, at least for some persons. The interindividual (including sex‐related) variability in the propensity to exhibit an altered temporal organization, whether it be transient or persistent (i.e., reversible or non‐reversible) suggests the involvement of genetic factors. The Dian‐Circadian genetic model previously proposed by the authors seems pertinent to conceptualize and explain the various levels and output of internal desynchronization.     

Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL

Apo2L/TRAIL stimulates cancer cell death through the proapoptotic receptors DR4 and DR5, but the determinants of tumor susceptibility to this ligand are not fully defined. mRNA expression of the peptidyl O-glycosyltransferase GALNT14 correlated with Apo2L/TRAIL sensitivity in pancreatic carcinoma, non-small-cell lung carcinoma and melanoma cell lines, and up to 30% of samples from various human malignancies showed GALNT14 overexpression. RNA interference of GALNT14 reduced cellular Apo2L/TRAIL sensitivity, whereas overexpression increased responsiveness. Biochemical analysis of DR5 identified several ectodomain O-(N-acetyl galactosamine-galactose-sialic acid) structures. Sequence comparison predicted conserved extracellular DR4 and DR5 O-glycosylation sites; progressive mutation of the DR5 sites attenuated apoptotic signaling. O-glycosylation promoted ligand-stimulated clustering of DR4 and DR5, which mediated recruitment and activation of the apoptosis-initiating protease caspase-8. These results uncover a new link between death-receptor O-glycosylation and apoptotic signaling, providing potential predictive biomarkers for Apo2L/TRAIL-based cancer therapy.     

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.     

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.     

Intramolecular Interactions within the Human Immunodeficiency Virus-1 gp41 Loop Region and Their Involvement in Lipid Merging

The human immunodeficiency virus utilizes its gp41 fusion protein to mediate virus-cell membrane fusion. The conserved disulfide loop region in the gp41 hairpin conformation reverses the protein chain, such that the N-terminal heptad repeat and the C-terminal heptad repeat regions interact to form the six-helix bundle. Hence, it is conceivable that the sequential folded N- and C-terminal parts of the loop region also interact. We show that the N- and C-terminal parts of the loop preferably form disulfide-bonded heterodimers with slow oxidation kinetics. Furthermore, when the two parts were linked to a single polypeptide to form the full-length loop, only an intramolecular disulfide-bonded loop was formed. Fluorescence studies revealed that an interaction takes place between the N- and C-terminal parts of the loop in solution, which was sustained in membranes. Functionally, only a combination of the N- and C-loop parts induced lipid mixing of model liposomes, the level of which increased 8-fold when they were connected to a single polypeptide chain. In both cases, the activity was independent of the oxidation state of the cysteines. Overall, the data (i) provide evidence of a specific interaction between the N- and C-terminal parts of the loop, which can further stabilize gp41 hairpin conformation, and (ii) suggest that the interaction between the N- and C-terminal parts of the loop is sufficient to induce lipid merging without forming a disulfide bond.