Codominant Role of Interferon-γ- and Interleukin-17-Producing T Cells During Rejection in Full Facial Transplant Recipients.

Facial transplantation is a life-changing procedure for patients with severe composite facial defects. However, skin is the most immunogenic of all transplants, and better understanding of the immunological processes after facial transplantation is of paramount importance. Here, we describe six patients who underwent full facial transplantation at our institution, with a mean follow-up of 2.7 years. Seum, peripheral blood mononuclear cells, and skin biopsy specimens were collected prospectively, and a detailed characterization of their immune response (51 time points) was performed, defining 47 immune cell subsets, 24 serum cytokines, anti-HLA antibodies, and donor alloreactivity on each sample, producing 4269 data points. In a nonrejecting state, patients had a predominant T helper 2 cell phenotype in the blood. All patients developed at least one episode of acute cellular rejection, which was characterized by increases in interferon-γ/interleukin-17-producing cells in peripheral blood and in the allograft's skin. Serum monocyte chemotactic protein-1 level was significantly increased during rejection compared with prerejection time points. None of the patients developed de novo donor-specific antibodies, despite a fourfold expansion in T follicular helper cells at 1 year posttransplantation. In sum, facial transplantation is frequently complicated by a codominant interferon-γ/interleukin-17-mediated acute cellular rejection process. Despite that, medium-term outcomes are promising with no evidence of de novo donor-specific antibody development.

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis.

GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type 'GRASP' domains. Fas/CD95 contains a C-terminal leucine-valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65-Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-X(L) as a candidate apoptotic binding partner for GRASP65.

The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2.

BACKGROUND AND AIMS: Cyclooxygenase 2 (COX-2) is upregulated in most colorectal cancers and is responsible for metabolism of the endogenous cannabinoid, anandamide, into prostaglandin-ethanolamides (PG-EAs). The aims of this study were to determine whether anandamide and PG-EAs induce cell death in colorectal carcinoma (CRC) cells, and whether high levels of COX-2 in CRC cells could be utilised for their specific targeting for cell death by anandamide. METHODS: We determined the effect of anandamide on human CRC cell growth by measuring cell growth and cell death, whether this was dependent on COX-2 protein expression or enzyme activity, and the potential involvement of PG-EAs in induction of cell death. RESULTS: Anandamide inhibited the growth of CRC cell lines HT29 and HCA7/C29 (moderate and high COX-2 expressors, respectively) but had little effect on the very low COX-2 expressing CRC cell line, SW480. Induction of cell death in HT29 and HCA7/C29 cell lines was partially rescued by the COX-2 selective inhibitor NS398. Cell death induced by anandamide was neither apoptosis nor necrosis. Furthermore, inhibition of fatty acid amide hydrolase potentiated the non-apoptotic cell death, indicating that anandamide induced cell death was mediated via metabolism of anandamide by COX-2, rather than its degradation into arachidonic acid and ethanolamine. Interestingly, both PGE2-EA and PGD2-EA induced classical apoptosis. CONCLUSIONS: These findings suggest anandamide may be a useful chemopreventive/therapeutic agent for colorectal cancer as it targets cells that are high expressors of COX-2, and may also be used in the eradication of tumour cells that have become resistant to apoptosis.

Integrative noetic therapies as adjuncts to percutaneous intervention during unstable coronary syndromes: Monitoring and Actualization of Noetic Training (MANTRA) feasibility pilot.

BACKGROUND: Patients undergoing percutaneous coronary intervention (PCI) for unstable coronary syndromes have substantial emotional and spiritual distress that may promote procedural complications. Noetic (nonpharmacologic) therapies may reduce anxiety, pain and distress, enhance the efficacy of pharmacologic agents, or affect short- and long-term procedural outcomes. METHODS: The Monitoring and Actualization of Noetic Training (MANTRA) pilot study examined the feasibility of applying 4 noetic therapies-stress relaxation, imagery, touch therapy, and prayer-to patients in the setting of acute coronary interventions. Eligible patients had acute coronary syndromes and invasive angiography or PCI. Patients were randomized across 5 treatment groups: the 4 noetic and standard therapies. Questionnaires completed before PCI reflected patients' religious beliefs and anxiety. Index hospitalization end points included post-PCI ischemia, death, myocardial infarction, heart failure, and urgent revascularization. Mortality was followed up for 6 months after hospitalization. RESULTS: Of eligible patients, 88% gave informed consent. Of 150 patients enrolled, 120 were assigned to noetic therapy; 118 (98%) completed their therapeutic assignments. All clinical end points were available for 100% of patients. Results were not statistically significant for any outcomes comparisons. There was a 25% to 30% absolute reduction in adverse periprocedural outcomes in patients treated with any noetic therapy compared with standard therapy. The lowest absolute complication rates were observed in patients assigned to off-site prayer. All mortality by 6-month follow-up was in the noetic therapies group. In patients with questionnaire scores indicating a high level of spiritual belief, a high level of personal spiritual activity, a low level of community-based religious involvement, or a high level of anxiety, noetic therapies appeared to show greater reduction in absolute in-hospital complication rates compared with standard therapy. CONCLUSIONS: Acceptance of noetic adjuncts to invasive therapy for acute coronary syndromes was excellent, and logistics were feasible. No outcomes differences were significant; however, index hospitalization data consistently suggested a therapeutic benefit with noetic therapy. Of all noetic therapies, off-site intercessory prayer had the lowest short- and long-term absolute complication rates. Definitive demonstration of treatment effects of this magnitude would be feasible in a patient population about 4 times that of this pilot study. Absolute mortality differences make safety considerations a mandatory feature of future clinical trials in this area.

Depressed mood is a factor in glycemic control in type 1 diabetes.

OBJECTIVE: The diabetes literature contains conflicting evidence on the relationship between depression and glycemic control. This may be due, in part, to the fact that past studies failed to distinguish between patients with type 1 and type 2 diabetes. Because these are actually completely different diseases that are often treated differently and consequently make different demands on patients, the relationship between glycemic control and depressed mood in type 1 and type 2 diabetes was examined separately. METHODS: The relationship between Beck Depression Inventory (BDI) scores and HbA1c, as an index of long-term glycemic control, was measured in samples of 30 patients with type 1 and 34 patients with type 2 diabetes. RESULTS: Groups of patients with type 1 and type 2 diabetes did not differ in mean BDI score or HbA1c level. Correlation analysis revealed a significant positive relationship between BDI scores and HbA1c in the type 1 group (r = .44, p < .02) but not in the type 2 group (r = -0.06, p > .05). This relationship was evident throughout the entire range of BDI scores and was not restricted to scores indicative of clinical depression. Patients with type 1 diabetes who had higher HbA1c and BDI scores reported a lower frequency of home blood glucose monitoring. CONCLUSIONS: Variations in depressive mood, below the level of clinical depression, are associated with meaningful differences in glycemic control in type 1 but not type 2 diabetes. Preliminary data analysis suggests that this effect may be mediated, at least in part, by decreased self-care behaviors in patients with more depressed mood.

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

Individual differences in smoking reward from de-nicotinized cigarettes.

Most studies of cigarette smoking and smoking cessation have focused on the psychopharmacological effects of nicotine; relatively few have explored the role of sensory aspects of cigarette smoke. Sensory aspects of cigarette smoke play a role in the maintenance of smoking behavior, and may be particularly important for certain smokers. This paper presents the results of a pooled analysis of nine studies conducted in our laboratory, in order to explore the influence of demographic and smoking-related variables on ratings of de-nicotinized as compared to nicotine-containing cigarettes. A major finding of this analysis is that ratings of smoking derived from de-nicotinized, but not nicotine-containing, cigarettes appear to vary with level of tobacco dependence, suggesting that sensory factors may be more important to highly dependent, as compared to less-dependent, smokers. The implications of these findings for smoking cessation treatment and for future research are discussed.

Real-time identification of Pseudomonas aeruginosa direct from clinical samples using a rapid extraction method and polymerase chain reaction (PCR).

Pseudomonas aeruginosa has emerged as one of the most problematic Gram-negative nosocomial pathogens. Bacteremia caused by P. aeruginosa is clinically indistinguishable from other Gram-negative infections although the mortality rate is higher. This microorganism is also inherently resistant to common antibiotics. Standard bacterial identification and susceptibility testing is normally a 48-hour process and difficulty sometimes exists in rapidly and accurately identifying antimicrobial resistance. The Polymerase Chain Reaction (PCR) is a rapid and simple process for the amplification of target DNA sequences. However, many sample preparation methods are unsuitable for the clinical laboratory because they are not cost effective, take too long to perform, or do not provide a good template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis. In this report, we have utilized our rapid DNA extraction method to generate bacterial DNA direct from clinical samples for PCR. The lower detection level for P. aeruginosa was estimated to be 10 CFU/ml. In addition, we wanted to compare the results of a new rapid-cycle DNA thermocycler that uses continuous fluorescence monitoring with the results of standard thermocycling. We tested 40 clinical isolates of P. aeruginosa and 18 non-P. aeruginosa isolates received in a blinded fashion. Coded data revealed that there was 100% correlation in both the rapid-cycle DNA thermocycling and standard thermocycling when compared to standard clinical laboratory results. In addition, total results turn-around time was less than 1 hour. Specific identification of P. aeruginosa was determined using intragenic primer sets for bacterial 16S rRNA and Pseudomonas outer-membrane lipoprotein gene sequences. The total cost of our extraction method and PCR was $2.22 per sample. The accuracy and rapidness of this DNA-extraction method, with its PCR-based identification system, make it an ideal candidate for use in the clinical laboratory.

Personality correlates of glycemic control in type 2 diabetes.

OBJECTIVE: To determine whether traits of normal personality are associated with variations in glycemic control in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: A longitudinal cohort study was conducted using data from 105 type 2 diabetic patients in a clinical trial of a stress management intervention. Before treatment assignment, patients completed the NEO Personality Inventory, Revised, which is a questionnaire inventory measuring 5 major domains of normal personality and 30 important traits that define these domains. Glycemic control was assessed by measures of HbA1c and average blood glucose levels based on 7 days of self-monitoring at baseline and at 6 and 12 months. Relationships between personality traits and measures of glycemic control were examined by correlation and linear regression models that were adjusted for age, sex, race, duration of diabetes, medication status, and experimental treatment. RESULTS: Lower average blood glucose values at baseline were associated with higher scores for the personality domain of neuroticism and several specific traits including anxiety, angry hostility depression, self-consciousness, and vulnerability but were associated with lower scores for the trait of altruism. Results were similar for HbA1c but were not as strong. Follow-up results were similar but were less consistent. CONCLUSIONS: Personality traits may offer new insights into variations in glycemic control in patients with type 2 diabetes undergoing standard management. The relative tendency to experience fewer negative emotions and to focus on the needs of others instead of oneself could prove to be a risk factor for poor glycemic control.

Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR.

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134), and coagulase-negative Staphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.

Renal biopsy in high-risk patients with medical diseases of the kidney.

The number of high-risk patients undergoing renal biopsy is likely to increase in the near future because of the increased use of anticoagulants for such conditions as atrial fibrillation, combined liver and kidney disease caused by hepatitis C, and the aging of the population. Nephrologists need to become increasingly familiar with evaluating such patients through both specialized management of percutaneous kidney biopsy and alternate methods of renal biopsy, which primarily consist of open (surgical) biopsy, transjugular (transvenous) biopsy, and laparoscopic biopsy. The indications, complications, and general approach to such patients are discussed. This is a US government work. There are no restrictions on its use.

Microtubule-based endoplasmic reticulum motility in Xenopus laevis: activation of membrane-associated kinesin during development.

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute approximately 19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower ( approximately 0.4 microm/s) than minus end-directed motility ( approximately 1.3 microm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.

Phosphorylation of microtubule-associated proteins from the ovaries of hemipteran insects by MPF and MAP kinase: possible roles in the regulation of microtubules during oogenesis.

Nutritive tubes that link the developing oocytes to the nurse cells in ovarioles of hemipteran insects contain extensive arrays of microtubules. These are established, then later depolymerised, by developmentally regulated processes. Breakdown of the microtubules corresponds with the activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase), later in oogenesis, as the oocytes proceed to arrest at the first meiotic metaphase [Lane and Stebbings, Roux's Arch Dev Biol 205:150-159 (1995)]. The mechanisms that lead to the breakdown of nutritive tube microtubules are unknown. Here, we have investigated the possibility that the insect ovarian microtubules are regulated by MPF- or MAP kinase-dependent phosphorylation, focusing upon the prominent high molecular weight microtubule-associated protein (HMW MAP) enriched in this system, which is a potential target for protein kinase activity in vivo. We have purified the prominent HMW MAPs from the ovaries of two species of hemipterans, and have shown them to be substrates in vitro for the activities of MPF and MAP kinase. However, although the catalytic component of MPF (p34cdc2) is present within microtubule-rich portions of hemipteran ovarioles, we have found that neither this protein nor its regulatory partner (cyclin B) co-purify with microtubules during taxol-mediated microtubule isolation.

Caffeine deprivation affects vigilance performance and mood.

The effects of brief caffeine deprivation on vigilance performance, mood, and symptoms of caffeine withdrawal were studied in habitual coffee drinkers. Thirty male and female coffee drinkers were tested twice at midday (1130 to 1330 hours) after mornings in which they either consumed caffeinated beverages ad lib or abstained. Vigilance performance was tested with a 30-min computerized visual monitoring task. Mood and withdrawal symptom reports were collected by questionnaires. Caffeine deprivation was associated with impaired vigilance performance characterized by a reduction in the percentage of targets detected and an increase in response time, and by subjective reports of decreased vigor and increased fatigue and symptoms characterized by sleepiness, headache, and reduced ability to work. Even short periods of caffeine deprivation, equivalent in length to skipping regular morning coffee, can produce deficits in sustained attention and noticeable unpleasant caffeine-withdrawal symptoms in habitual coffee drinkers. Such symptoms may be a common side-effect of habitual caffeine consumption that contributes to the maintenance of this behavior.

Caffeine raises blood pressure at work.

OBJECTIVE: The study investigated the effects of moderate doses of caffeine on ambulatory blood pressure and heart rate during workday activities. METHODS: Healthy, nonsmoking, habitual coffee drinkers (N = 21) received daily doses of 100 mg and 500 mg of caffeine on 2 days in a crossover design. Treatment order was random and counterbalanced, and administration was double-blind. Ambulatory monitoring was conducted for 6 to 9 hours during normal workday activities and diary entries were completed at each measurement. Ambulatory data were analyzed for the effects of caffeine dose, controlling for variations in posture, physical activity, and perceived stress. RESULTS: The average workday blood pressure and heart rate were significantly higher when the higher dose of caffeine was consumed. Controlling for other factors, dose-related differences were 4 mm Hg for systolic and 3 mm Hg for diastolic blood pressure, and were 3 bpm for heart rate. CONCLUSIONS: Results support earlier evidence that caffeine raises blood pressure at work, and demonstrate that these pressor effects are independent of changes in posture, physical activity, or stress. Daily blood pressure increases associated with caffeine consumption could increase the risk of developing cardiovascular diseases. In addition, caffeine consumption effects might confound ambulatory investigations of the cardiovascular effects of other psychosocial, personality, or health-behavior factors.

Binaural auditory beats affect vigilance performance and mood.

When two tones of slightly different frequency are presented separately to the left and right ears the listener perceives a single tone that varies in amplitude at a frequency equal to the frequency difference between the two tones, a perceptual phenomenon known as the binaural auditory beat. Anecdotal reports suggest that binaural auditory beats within the electroencephalograph frequency range can entrain EEG activity and may affect states of consciousness, although few scientific studies have been published. This study compared the effects of binaural auditory beats in the EEG beta and EEG theta/delta frequency ranges on mood and on performance of a vigilance task to investigate their effects on subjective and objective measures of arousal. Participants (n = 29) performed a 30-min visual vigilance task on three different days while listening to pink noise containing simple tones or binaural beats either in the beta range (16 and 24 Hz) or the theta/delta range (1.5 and 4 Hz). However, participants were kept blind to the presence of binaural beats to control expectation effects. Presentation of beta-frequency binaural beats yielded more correct target detections and fewer false alarms than presentation of theta/delta frequency binaural beats. In addition, the beta-frequency beats were associated with less negative mood. Results suggest that the presentation of binaural auditory beats can affect psychomotor performance and mood. This technology may have applications for the control of attention and arousal and the enhancement of human performance.

Caffeine withdrawal symptoms following brief caffeine deprivation.

The effects of short-term caffeine deprivation on mood, withdrawal symptoms and psychomotor performance were studied in habitual coffee drinkers. Thirty-one male and female coffee drinkers were tested twice at midday (1130 to 1330 h) 4 h after double-blind administration of 250 mg of caffeine or placebo. Mood and withdrawal symptoms reports were collected by questionnaires. Psychomotor performance was tested with a brief computerized test battery, and causal blood pressure was measured. Caffeine deprivation was associated with decreased vigor and increased fatigue and with symptoms including sleepiness and yawning. Blood pressure was lower by 5-6 mm Hg. No changes in psychomotor performance were observed. Even short periods of caffeine deprivation, equivalent in length to missing regular morning coffee, can produce noticeable unpleasant caffeine withdrawal symptoms in habitual coffee drinkers. Such symptoms may be common side effects of habitual caffeine consumption that contribute to the maintenance of this behavior.