The Association between Frailty and Short-Term Outcomes in an Intensive Care Unit Rehabilitation Trial: An Exploratory Analysis.

BACKGROUND: Physical therapy initiated early in an ICU stay may reduce functional deficits in critically ill patients; however, the association of frailty with outcomes in those receiving early in-ICU rehabilitation is unknown. OBJECTIVE: To estimate the association between frailty and 3 outcomes in patients enrolled in an ICU randomized clinical trial (RCT). DESIGN: Exploratory secondary analyses of the CYCLE pilot RCT (NCT02377830). SETTING: 7 Canadian ICUs. PARTICIPANTS: Previously ambulatory critically ill adults. INTERVENTION: Participants were randomized to early in-bed cycling plus routine physiotherapy versus early routine physiotherapy alone. MEASUREMENTS: Using regression analyses, we modelled the association between pre-hospital Clinical Frailty Scale (CFS) scores, Physical Function in ICU Test-scored (PFIT-s), muscle strength, and mortality at hospital discharge, adjusting for illness severity (APACHE II) and the randomized intervention. We explored the influence of imputing mean PFIT-s and strength scores for decedents, and with listwise deletion of decedents in a sensitivity analysis. RESULTS: Of 66 patients, 2 had missing data, 2 had incomplete data, and 21 died by hospital discharge. At hospital discharge for 66 patients, frailty was not associated with PFIT-s (mean difference (MD) [95% CI]=0.20, [-2.08, 2.74]) or muscle strength (1.96, [-12.6, 16.6]). A sensitivity analysis yielded consistent results. Frailty was also not associated with hospital mortality (odds ratio 0.91, [0.28 to 2.93]). CONCLUSION: We found no association between pre-hospital frailty, physical function, strength, or mortality at hospital discharge in critically ill patients enrolled in an early rehabilitation trial. Larger sample sizes are needed to further explore the association of frailty with these outcomes at hospital discharge.

Application of Moringa oleifera Lam. fractionated proteins for inactivation of Escherichia coli from water.

Chemical products traditionally used in the disinfection of water bodies often pose human health risks. For this reason, studies on natural coagulants such as Moringa oleifera Lam. represent an alternative for the inactivation of pathogenic microorganisms, among which is Escherichia coli. This study evaluated the effect of different concentrations of coagulants obtained from Moringa seed extracts and their protein fractions in the inactivation of E. coli during the coagulation/flocculation process. The coagulants studied were the aqueous extract, saline extract and protein fractions albumin and globulin, highlighting that the protein fractions were more effective on inactivating E. coli. The protein fraction globulin at a concentration of 10.0 mg L-1 showed bactericidal effects against E. coli within 18 min, whereas the albumin showed a bacteriostatic effect within 48 min because it isolated colonies in the sediment sample.

IPS-1 differentially induces TRAIL, BCL2, BIRC3 and PRKCE in type I interferons-dependent and -independent anticancer activity.

RIG-I-like receptors are the key cytosolic sensors for RNA viruses and induce the production of type I interferons (IFN) and pro-inflammatory cytokines through a sole adaptor IFN-ß promoter stimulator-1 (IPS-1) (also known as Cardif, MAVS and VISA) in antiviral innate immunity. These sensors also have a pivotal role in anticancer activity through induction of apoptosis. However, the mechanism for their anticancer activity is poorly understood. Here, we show that anticancer vaccine adjuvant, PolyIC (primarily sensed by MDA5) and the oncolytic virus, Newcastle disease virus (NDV) (sensed by RIG-I), induce anticancer activity. The ectopic expression of IPS-1 into type I IFN-responsive and non-responsive cancer cells induces anticancer activity. PolyIC transfection and NDV infection upregulate pro-apoptotic gene TRAIL and downregulate the anti-apoptotic genes BCL2, BIRC3 and PRKCE. Furthermore, stable knockdown of IPS-1, IRF3 or IRF7 in IFN-non-responsive cancer cells show reduced anticancer activity by suppressing apoptosis via TRAIL and anti-apoptotic genes. Collectively, our study shows that IPS-1 induces anticancer activity through upregulation of pro-apoptotic gene TRAIL and downregulation of the anti-apoptotic genes BCL2, BIRC3 and PRKCE via IRF3 and IRF7 in type I IFN-dependent and -independent manners.

Formation and reduction of streak artefacts in electron tomography.

We have analysed the formation of streak artefacts in the reconstruction based on the filtered back projection algorithm in electron tomography (ET) and accordingly applied an adaptive interpolation technique to artefact reduction. In the adaptive interpolation to recover the missing information, the edge positions in a projection curve were tracked to reduce the interpolation error. A simulation was used to demonstrate the effectiveness of the artefact reduction. Furthermore, image reconstruction of integrated circuit specimens in the ET experiments with the ultra-high voltage electron microscope show that the strong streak artefacts can be reduced effectively by our artefact reduction technique.

Involvement of IL-31 on scratching behavior in NC/Nga mice with atopic-like dermatitis.

Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen has not been identified. NC/Nga mice, spontaneously develop an eczematous AD-like skin lesion when kept under conventional conditions, but not under specific pathogen-free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL-31, may be involved in pruritus of AD, we examined the IL-31 expression in spontaneous dermatitis model which showed itch-associated long-lasting (over 1.5 s duration) scratching behavior and compared with that of hapten-induced contact dermatitis model without itch-associated long-lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long-lasting scratching counts were significantly increased. Yet in 2,4,6-trinitrochlorobenzene (TNCB)-sensitized and challenged mice (TNCB-applied NC/Nga mice), no significant increase in long-lasting scratching counts was observed. In conventional NC/Nga mice with long-lasting scratching behavior, expression of IL-31 mRNA was increased, while in TNCB-applied NC/Nga mice without long-lasting scratching behavior, the expression of IL-31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL-31 mRNA in conventional NC/Nga mice, but not so in TNCB-applied NC/Nga mice. These results suggest that IL-31 causes the itch-associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.

A critical role for mouse CXC chemokine(s) in pulmonary neutrophilia during Th type 1-dependent airway inflammation.

Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.

Constitutive IFN-alpha/beta signal for efficient IFN-alpha/beta gene induction by virus.

Efficient IFN-alpha/beta gene induction in virus-infected cells is an event central to innate immunity, in which the transcription factor IRF-7 plays a critical role together with IRF-3. Unlike IRF-3, IRF-7 is short-lived and its gene expression is dependent on IFN-alpha/beta signalling; hence, the signal-dependent enhancement of IRF-7 gene induction during viral infection is essential for positive-feedback regulation of IFN-alpha/beta gene induction. Here, we provide evidence that constitutive, IRF-3/IRF-7-independent production of IFN-alpha/beta in uninfected cells is critical for setting the IRF-7 expression levels, determining whether or not the positive-feedback mechanism will operate effectively upon viral infection. In fact, spleen cells are more dependent than fibroblasts on this mechanism; the IFN-alpha/beta gene induction is impaired more severely by blocking the IRF-7 induction pathway than by introducing an IRF-3 null mutation. Thus, the constitutive IFN-alpha/beta signal provides a foundation for the IRF-7-mediated enhancement of its own production in response to virus infection.

Cross talk of the interferon-alpha/beta signalling complex with gp130 for effective interleukin-6 signalling.

BACKGROUND: Signalling cross talk provides a molecular basis for modulating a given signalling pathway by another, and it is often critical for regulating cellular responses elicited by cytokines. Previously, we reported on the critical role of the IFN-alpha/beta signalling complex, generated by spontaneously produced IFN-alpha/beta, in efficient IFN-gamma signalling. RESULTS: In the present study, we have demonstrated that the IFN-alpha/beta signalling complex also contributes to efficient IL-6 signalling. In fact, IL-6-induced activation of the Stat1 and Stat3 transcription factors is markedly diminished in the absence of the IFN-alpha/beta signalling complex. The induction of several target genes for these factors is also diminished, both in vitro and in vivo. We provide evidence that the cytoplasmic tyrosine residues of IFNAR-1, which remains phosphorylated by a weak IFN-alpha/beta stimulation, provide docking sites for Stat1 and Stat3 to form homo- or heterodimers following IL-6 stimulation. Furthermore, a chemical cross-linking experiment revealed that IFNAR-1 and gp130, a common signal transducer for the IL-6 family of cytokines, exist in close proximity. CONCLUSIONS: The constitutive weak IFN-alpha/beta signal provides a foundation for strong cellular responses to IL-6, IFN-gamma, and possibly other cytokines. Our results also suggest the assembly of cytokine receptor subunits, which may represent a 'receptosome'-like structure, allowing the unique signalling cross talks to occur.

A weak signal for strong responses: interferon-alpha/beta revisited.

Biological systems have acquired adaptability and robustness against rapid environmental changes. A typical example is the immune system, which eradicates invading pathogens such as viruses. Interferons alpha and beta, which are produced in response to viral infection, are essential components of this system but are also produced at low levels in the absence of infection. What is the purpose of the constitutive weak interferon-alpha/beta signal?

Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of beta-catenin/T-cell factor 4-mediated gene transactivation.

Beta-catenin acts as a transcriptional coactivator by forming a complex with T-cell factor/lymphoid enhancer factor (TCF/LEF) DNA-binding proteins. Aberrant transactivation of a certain set of target genes by beta-catenin and TCF4 complexes has been implicated in familial and sporadic colorectal tumorigenesis. A colorectal cancer cell line, DLD-1, becomes irregularly multilayered, when maintained confluent for 2-3 weeks, and forms numerous dome-like polypoid foci piled-up over the surface of cell sheets. By the use of a strict tetracycline-regulation system, we found that the continuous suppression of beta-catenin/TCF4-mediated gene transactivation by dominant-negative TCF4B (deltaN30) reduced these piled-up foci and restored a simple monolayer of polarized columnar cells resembling normal intestinal epithelium. The restoration of epithelial cell polarity was evident in two ways: (a) the formation of microvilli over the apical surface; and (b) the distribution of a tight junction protein, ZO-1, to the lateral plasma membrane. Retroviral expression of stabilized beta-catenin (deltaN89) induced the formation of similar piled-up foci in untransformed IEC6 intestinal epithelial cells. Sulindac, a nonsteroidal antiinflammatory drug effective against colorectal tumorigenesis in familial adenomatous polyposis syndrome, suppressed the formation of foci. The loss of epithelial cell polarity may be a critical cellular event driving beta-catenin/TCF4-mediated intestinal tumorigenesis.

IRF family of transcription factors as regulators of host defense.

Interferon regulatory factors (IRFs) constitute a family of transcription factors that commonly possess a novel helix-turn-helix DNA-binding motif. Following the initial identification of two structurally related members, IRF-1 and IRF-2, seven additional members have now been reported. In addition, virally encoded IRFs, which may interfere with cellular IRFs, have also been identified. Thus far, intensive functional analyses have been done on IRF-1, revealing a remarkable functional diversity of this transcription factor in the regulation of cellular response in host defense. Indeed, IRF-1 selectively modulates different sets of genes, depending on the cell type and/or the nature of cellular stimuli, in order to evoke appropriate responses in each. More recently, much attention has also been focused on other IRF family members. Their functional roles, through interactions with their own or other members of the family of transcription factors, are becoming clearer in the regulation of host defense, such as innate and adaptive immune responses and oncogenesis.

T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma.

Bone resorption is regulated by the immune system, where T-cell expression of RANKL (receptor activator of nuclear factor (NF)-kappaB ligand), a member of the tumour-necrosis factor family that is essential for osteoclastogenesis, may contribute to pathological conditions, such as autoimmune arthritis. However, whether activated T cells maintain bone homeostasis by counterbalancing the action of RANKL remains unknown. Here we show that T-cell production of interferon (IFN)-gamma strongly suppresses osteoclastogenesis by interfering with the RANKL-RANK signalling pathway. IFN-gamma induces rapid degradation of the RANK adapter protein, TRAF6 (tumour necrosis factor receptor-associated factor 6), which results in strong inhibition of the RANKL-induced activation of the transcription factor NF-kappaB and JNK. This inhibition of osteoclastogenesis is rescued by overexpressing TRAF6 in precursor cells, which indicates that TRAF6 is the target critical for the IFN-gamma action. Furthermore, we provide evidence that the accelerated degradation of TRAF6 requires both its ubiquitination, which is initiated by RANKL, and IFN-gamma-induced activation of the ubiquitin-proteasome system. Our study shows that there is cross-talk between the tumour necrosis factor and IFN families of cytokines, through which IFN-gamma provides a negative link between T-cell activation and bone resorption. Our results may offer a therapeutic approach to treat the inflammation-induced tissue breakdown.

Cardioprotective effects of nicorandil in rabbits anaesthetized with halothane: potentiation of ischaemic preconditioning via KATP channels.

1. The roles of ATP-sensitive K+ channels (KATP channels) in ischaemic or pharmacological preconditioning in the rabbit heart remain unclear. Infarct limitation by ischaemic preconditioning was abolished by the KATP channel blocker glibenclamide under ketamine/xylazine anaesthesia, but not under anaesthesia induced by pentobarbital. Infarct limitation by the KATP channel opener pinacidil was detected under ketamine/xylazine anaesthesia, but not under pentobarbital anaesthesia. Thus, these effects appear to be anaesthetic dependent. 2. In the present study, we examined whether nicorandil (a KATP channel opener nitrate) exhibits cardioprotective actions under halothane anaesthesia, another commonly used volatile anaesthetic. Control animals were subjected to 40 min coronary occlusion and 120 min reperfusion. Before 40 min ischaemia, the nicorandil group received nicorandil (100 microg/kg per min, i.v., for 10 min), the 5' preconditioning (PC) group received 5 min ischaemia/20 min reperfusion, the 2.5'PC group received 2.5 min preconditioning ischaemia/20 min reperfusion, the nicorandil +2.5'PC group received both nicorandil and 2.5 min ischaemia/20 min reperfusion, the nicorandil +2.5'PC + 5-hydroxydecanoate (5HD) group received both nicorandil and 2.5 min ischaemia/20 min reperfusion in the presence of 5-hydroxydecanoate (5HD; a KATP blocker) and the 5HD group received 5 mg/kg, i.v., 5HD alone. Myocardial infarct size in control (n = 7), nicorandil (n = 5), 5'PC (n = 8), 2.5'PC (n = 5), nicorandil + 2.5'PC (n = 5), nicorandil + 2.5'PC + 5HD (n = 5) and 5HD (n = 4) groups averaged 44.4 +/- 3.6, 41.7 +/- 5.7, 17.8 +/- 3.2,* 34.1 +/- 4.8, 21.3 +/- 4.2,* 39.1 +/- 5.6 and 38.9 +/- 5.0% of the area at risk, respectively (*P <0.05 vs control). 3. Thus, nicorandil alone did not have an infarct size-limiting effect in halothane-anaesthetized rabbits. However, the results suggest that even when nicorandil alone does not demonstrate a direct cardioprotective effect, it may enhance ischaemic preconditioning via KATP channels. Key words: ATP-sensitive K+ (KATP) channel, ischaemic preconditioning, myocardial infarction, nicorandil, rabbit.

Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis.

The mutational inactivation of a tumor suppressor gene, adenomatous polyposis coli (APC), results in the accumulation of cytoplasmic beta-catenin protein and the activation of T-cell factor (TCF)/lymphoid enhancer factor transcriptional factors. A colorectal carcinoma cell line, DLD-1, was engineered to suppress transactivation by the TCF4/beta-catenin complex in a dominant-negative manner under the strict control of the tetracycline regulatory system. A large-scale comparison of the expression profiles, using two-color fluorescence hybridization of cDNA microarray, led to the identification of MDR1 as a target gene of the TCF4/beta-catenin complex. Luciferase reporter and gel retardation assays revealed the TCF4/beta-catenin responsive elements in the promoter of the human MDR1 gene. Corresponding to the accumulation of beta-catenin, expression of the MDR1 gene product was steadily up-regulated in adenomas and adenocarcinomas of 10 patients with familial adenomatous polyposis. In combination with cell proliferative activities of c-myc and cyclin D1, MDR1 may initiate colorectal tumorigenesis by suppressing cell death pathways programmed in intestinal epithelial cells.

Differential requirement of the cytoplasmic subregions of gamma c chain in T cell development and function.

The common cytokine receptor gamma chain (gammac), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of gammac consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its role in vivo remains totally unknown. In the present study, we expressed mutant forms of gammac, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on a gammac null background. We demonstrate that, unlike gammac or Jak3 null mice, expression of the latter, but not the former mutant, restores T lymphopoiesis in vivo, accompanied by strong expression of Bcl-2. On the other hand, the in vitro functions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the gammac membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of gammac in T cell development and function.

Cross talk between interferon-gamma and -alpha/beta signaling components in caveolar membrane domains.

Definition of cellular responses to cytokines often involves cross-communication through their respective receptors. Here, signaling by interferon-gamma (IFN-gamma) is shown to depend on the IFN-alpha/beta receptor components. Although these IFNs transmit signals through distinct receptor complexes, the IFN-alpha/beta receptor component, IFNAR1, facilitates efficient assembly of IFN-gamma-activated transcription factors. This cross talk is contingent on a constitutive subthreshold IFN-alpha/beta signaling and the association between the two nonligand-binding receptor components, IFNAR1 and IFNGR2, in the caveolar membrane domains. This aspect of signaling cross talk by IFNs may apply to other cytokines.

Simulation of the effect of magnification and image-rotation variations on three-dimensional reconstruction

Tilting a specimen may change the magnification and the image rotation for electron-microscopic images, because re-focusing with an objective lens is required to correct for the height variation of the tilted specimen. Computer simulation is performed to analyse the effect of variations in the magnification and the image rotation on the three-dimensional reconstruction in electron tomography.

International telemicroscopy with a 3 MV ultrahigh voltage electron microscope.

The ability for remote microscope operation via a network connection was added recently to the ultrahigh voltage electron microscope (UHVEM) in Osaka University, and used successfully for the observation of thick biological samples across the Pacific Ocean by researchers at the National Center for Microscopy and Imaging Research (NCMIR) at the University of California San Diego. High-quality images at video rate were transferred by a satellite link and control signals were transmitted by an ISDN connecting the workstations at both sites. Most microscope functions operated from the console of the UHVEM were replicated on the graphical user interface of the remote workstation. By clicking on icons or in boxes in the display window with a mouse, the researcher could operate the UHVEM from the remote-site. The total delay time for sending images and returning control signals was about 0.7 s, which did not interfere significantly with the smooth operation of the instrument. Researchers at the remote site were able to record images on film in the microscope which were later sent to San Diego.

[The application of the endotoxin test for globulin and other blood products].

The application of the endotoxin test for globulin and other blood products were investigated by two different limulus amebocyte lysate (LAL) test methods, colorimetric and kinetic turbidimetric methods, using two endotoxin-specific reagents. By the dilution of the blood products in 40 times or more, spiked endotoxin in the products was recovered accurately showing neither inhibition nor enhancement. The definite difference was not shown between the results obtained by the two LAL test methods. According to the method of the endotoxin test described under General Tests of The Japanese Pharmacopeia (thirteenth edition), JPXIII, the maximum valid dilution (MVD) for these products will be calculated to be 40 or more, so it is capable to measure the endotoxin limit for each product. This study indicates that the endotoxin test is applicable to measure the endotoxin content in globulin and other blood products as an alternative method for the rabbit pyrogen test.