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.

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.

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.

Quitting smoking raises whole blood glutathione.

Cigarette smoke contains numerous oxygen free radicals that may be important in smoking-related disease pathogenesis. These free radicals may overwhelm antioxidant defenses and produce a condition of oxidative stress that can result in damage to DNA and other cellular components. This study investigated whether or not indications of harmful oxidative stress decline following smoking cessation. Changes in whole blood glutathione (GSH), an index of oxidative stress level, were determined for 30 cigarette smokers who participated in an experimental smoking-cessation program. Measurements were taken during ad lib smoking and 3 weeks after smoking cessation. In 22 individuals who were continuously abstinent for 3 weeks, GSH levels rose significantly following smoking cessation, from 5.0 to 6.1 mumol/g Hb (p < 0.001). Individuals with the lowest GSH levels during ad lib smoking showed the greatest increases following cessation. Results suggest that oxidative stress and free-radical damage diminish soon after smoking cessation. Thus, some significant health benefits may appear rapidly when people quit smoking.

Small bowel biopsy through an enteroclysis catheter to augment findings at enteroclysis and hypotonic duodenography.

Proximal jejunal mucosal biopsy was performed by a radiologist through the nasojejunal catheter at the time of enteroclysis. Seventeen patients (10 men and seven women, aged 23-73 years [mean, 46 years]) were studied with enteroclysis because of clinical signs of malabsorption with suspected small bowel disease. In seven (41%) patients, results at biopsy were positive, and results in another seven (41%) were positive at enteroclysis. In 10 (59%) patients, results were positive at one or both tests. Performance of both small bowel biopsy and enteroclysis at the same session is feasible and offers additional clinically pertinent information than can be obtained at enteroclysis alone.

Menstrual cycle effects on caffeine elimination in the human female.

Increases in the levels of sex steroids due to pregnancy or oral contraceptive steroid use are known to decrease significantly the rate at which caffeine is eliminated from the body. An investigation has now been made into whether the changes in sex steroid levels that occur during normal menstrual cycling also affect the rate of caffeine elimination, especially whether hormonal shifts in the luteal phase are associated with slower elimination of caffeine. Repeated 24-hour caffeine elimination studies were conducted during the follicular and luteal phases of the menstrual cycle in 10 healthy women. Comparisons of the follicular and luteal phases revealed that systemic clearance of caffeine was slower in the luteal phase, although the t1/2 did not differ. The slowing effect was related to the proximity to onset of menstruation and to levels of progesterone. The evidence suggests that caffeine elimination may be slowed in the late luteal phase, prior to the onset of menstruation. Such a reduction would lead to increased accumulation of caffeine with repeated self-administration during the day, but the effect may be too small to be of clinical significance in the majority of women.

Physiological responses to catecholamine infusions in type A and type B men.

To determine whether there are basic biological differences between Type A and Type B men, we compared hemodynamic, electrophysiologic and neuroendocrine responses to equipotent doses of isoproterenol (ISO) and norepinephrine (NE) in 10 Type A and 10 Type B men ages 18 to 29. Results showed equal hemodynamic and neuroendocrine responses to graded ISO doses in Type A and Type B individuals. In contrast, Type A men showed a more prolonged decrease in electrocardiographic T-wave amplitude (TWA) than did Type B men. Post hoc analyses of the correlates of TWA recovery during high-dose ISO infusion provide preliminary evidence for a more robust parasympathetic antagonism of sympathetic nervous system effects in Type B men, especially those with low scores on the Cook-Medley Ho scale. These findings suggest that, in addition to cognitively mediated increases in sympathetic nervous system reactivity, Type As may also be placed at increased risk of developing coronary heart disease by reduced levels of parasympathetic antagonism of sympathetic effects.

Effects of cigarettes on saliva cortisol levels.

We determined the effects of cigarette deprivation and smoking on saliva cortisol levels in the presence and absence of an operant, monetarily reinforced work task. Subjects were randomly exposed to the following four experimental conditions over successive sessions: no smoking, smoking, no smoking + work, and smoking + work. Measurements of cortisol levels in saliva were determined before and after each daily session. Saliva cortisol levels declined from the beginning to the end of sessions and the end-of-session saliva cortisol levels were not affected by any of the four experimental conditions. Increased cigarette smoking in the presence of the work task also did not affect saliva cortisol levels. Our data do not support: reports of increased cortisol levels as a consequence of smoking or theories relating cortisol and endorphin release to nicotine habituation.