Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23-dependent murine model.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity: a randomized controlled trial.

BACKGROUND: Aerobic exercise is recommended for weight management but energy balance is often less negative than predicted from exercise energy expenditure (ExEE). OBJECTIVE: To examine effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity. METHODS: We randomized 130 younger, physically inactive women and men with overweight and obesity (body mass index: 25-35 kg m-2) to 6 months of habitual lifestyle (control; CON, n=18), active commuting (BIKE, n=35) or leisure-time exercise of moderate (MOD, 50% VO2peak reserve, n=39) or vigorous intensity (VIG, 70% VO2peak reserve, n=38). The primary outcome was change in fat mass measured by dual-energy X-ray absorptiometry, which was analyzed intention-to-treat. Accumulated energy balance was calculated based on changes in body composition, and ExEE was calculated based on heart rate monitoring during exercise. RESULTS: Testing at 3 and 6 months was completed by 95 and 90 participants, respectively. Fat mass was reduced after 3 and 6 months in BIKE (3 months: -3.6 (-5.5; -1.7) kg (mean (95% CI)); 6 months: -4.2 (-6.6; -1.9) kg; both: P<0.001), MOD (3 months: -2.2 (-3.9; -0.4) kg; 6 months: -2.6 (-4.8; -0.5) kg, both: P<0.02) and VIG (3 months: -3.4 (-5.2; -1.7) kg; 6 months: -4.5 (-6.6; -2.3) kg; both: P<0.001) compared with CON. Furthermore, fat loss was greater in VIG compared with MOD (6 months: -1.8 (-3.6; -0.1) kg, P=0.043). Based on the ExEE and the accumulated energy balance MOD compensated for the ExEE (77 (48; 106) %) but not BIKE (38 (-18; 95) %) and VIG (21 (-14; 55) %). CONCLUSIONS: A meaningful fat loss was obtained by 6 months of active commuting and leisure-time exercise, but fat loss was greater with vigorous compared with moderate intensity exercise. Active commuting is an alternative to leisure-time exercise in the management of overweight and obesity. The trial was registered at clinicaltrials.gov as NCT01962259 (main trial) and NCT01973686 (energy metabolism sub-study).

Double-blind randomized controlled trial of collagen mesh for the prevention of abdominal incisional hernia in patients having a vertical rectus abdominis myocutaneus flap during surgery for advanced pelvic malignancy.

AIM: The study investigated the effect of collagen mesh-assisted closure at the donor site in preventing the formation of incisional hernia following construction of a vertical rectus abdominis myocutaneus (VRAM) flap as part of pelvic surgery for recurrent colorectal cancer. METHOD: The study was a double-blinded randomized controlled superiority trial that was designed and performed according to the Consolidated Standards of Reporting Trials (CONSORT) Statement. Eligible patients undergoing surgery that included a VRAM flap for advanced colorectal pelvic malignancy were prospectively randomized to conventional abdominal wound closure or collagen mesh-assisted closure. The primary end-point was incisional herniation at 1 year confirmed by CT. Secondary end-points were CT-verified incisional herniation at 3 and 36 months, clinically recognizable incisional herniation, donor-site and reconstructive-site complications, surgical mortality, postoperative morbidity, postoperative recovery and survival. RESULTS: In total, 58 (29 conventional closure; 29 mesh-assisted closure) patients were included. At 1 year, incisional herniation on the CT scan was found in 12 (50%) of 24 patients in the conventional closure group, and in 8 (33%) of 24 in the mesh-assisted closure group (P = 0.38). No significant difference between the groups was found in surgical mortality, early or late complications or survival. Donor-site morbidity was comparable between the two groups. CONCLUSION: No preventative effect of collagen mesh-assisted closure was observed following VRAM flap reconstruction.

Radiological and pathological evaluation of the level of arterial division after colon cancer surgery.

AIM: In aiming to cure patients with colorectal cancer surgery, the surgeon must carefully dissect the mesocolon and mesorectum and divide the vascular pedicle near to its origin so as to include all local lymph nodes. This has been termed complete mesocolic excision. The distance from the distal vascular tie to the bowel wall in the fixed specimen is an indication as to the quality of surgery but this does not assess the length of the residual vascular pedicle and, by implication, residual lymph nodes. The aim of this study was to establish if our surgeons were carrying out complete mesocolic excision by assessing the length of the proximal arterial pedicle and relating this to arterial length in the fixed specimen. METHOD: This was a single centre prospective study of patients undergoing elective surgery for locally advanced colorectal cancer. An abdominal and pelvic CT scan was performed 2 days postoperatively and a radiologist blinded to the operative procedure measured the length of the residual arterial stump. Similarly, the length of the vessel in the fixed resected specimen and lymph node yield were also recorded. RESULTS: Fifty-two patients were recruited. The mean length of the residual arterial stump was 38 mm (95% CI: 33-43), which was significantly longer than the < 10 mm recommended in guidelines (P < 0.0001). The mean length was 31 mm (95% CI: 25-37) and 49 mm (95% CI: 40-57) for left and right sided resections respectively. There was no correlation between the residual arterial stump and the pathology. CONCLUSIONS: The residual arterial length was greater than suggested by guidelines and may indicate that our surgery is less radical than we planned. Caution should be taken when using pathological measurements of vascular ligation as it may not reflect the height of the pedicle division.

Transfer of liraglutide from blood to cerebrospinal fluid is minimal in patients with type 2 diabetes.

Treatment with liraglutide leads to weight loss. We investigated whether blood-to-cerebrospinal fluid (CSF) transfer of liraglutide occurs, and if so, whether it associates with clinical weight loss following liraglutide treatment in humans. We performed lumbar puncture and blood sampling in eight patients with type 2 diabetes (mean (range)): age 63 (54-79) years; actual body weight: 90 (75-118) kg treated with 1.8 mg liraglutide for 14 (5-22) months and with a treatment-induced weight loss of 8.4 (7-11) kg. We measured liraglutide in plasma and CSF with a radioimmunoassay specific for the N-terminus of the GLP-1 moiety of liraglutide. Mean plasma liraglutide was 31 (range: 21-63) nmol l(-1). The mean CSF-liraglutide concentration was 6.5 (range: 0.9-13.9) pmol l(-1). Ratio of CSF: plasma-liraglutide concentrations was 0.02 (range: 0.07-0.002)% and plasma liraglutide did not correlate with CSF-liraglutide levels (P=0.67). Body weight loss tended to correlate with plasma-liraglutide levels (P=0.06), but not with CSF-liraglutide levels (P=0.69). In conclusion, we measured very low concentrations of liraglutide in CSF, and the levels of CSF liraglutide did not correlate with the actual clinical weight loss in these patients. The amount of liraglutide in plasma tended to correlate with the clinical weight loss.

Changes in peak fat oxidation in response to different doses of endurance training.

The effect of different doses of endurance training on the capacity to oxidize fat during exercise in sedentary, overweight men and assessment of variables associated with changes in peak fat oxidation (PFO) were evaluated. Young, sedentary, overweight men were randomized to either the high-dose (HIGH, 600 kcal/day, n = 17) or moderate-dose (MOD, 300 kcal/day, n = 18) endurance training groups or controls (CON, n = 15). PFO and peak oxygen uptake (VO2 peak) were measured using indirect calorimetry, body composition using dual-energy x-ray absorptiometry, and protein levels of mitochondrial enzymes determined by Western blotting. PFO increased in both MOD [1.2 mg/kg fat-free mass (FFM)/min, 95% confidence interval (CI): 0.08:2.3, P = 0.03] and HIGH (1.8 mg/kg FFM/min, CI: 0.6:2.9, P < 0.001) compared with CON. Skeletal muscle expression of citrate synthase, ß-hydroxyacyl-CoA dehydrogenase, and mitochondrial oxphos complexes II-V increased similarly in MOD and HIGH. Stepwise multiple linear regression analysis with backward elimination of individual variables correlated with changes in PFO revealed increases in cycling efficiency, FFM, and VO2 peak as the remaining associated variables. In conclusion, PFO during exercise increased with both moderate- and high-dose endurance training. Increases in PFO were mainly predicted by changes in VO2 peak, FFM, and cycling efficiency, and less with skeletal muscle mitochondrial enzymes.

Exercise training favors increased insulin-stimulated glucose uptake in skeletal muscle in contrast to adipose tissue: a randomized study using FDG PET imaging.

Physical exercise increases peripheral insulin sensitivity, but regional differences are poorly elucidated in humans. We investigated the effect of aerobic exercise training on insulin-stimulated glucose uptake in five individual femoral muscle groups and four different adipose tissue regions, using dynamic (femoral region) and static (abdominal region) 2-deoxy-2-[¹8F]fluoro-d-glucose (FDG) PET/CT methodology during steady-state insulin infusion (40 mU·m⁻²·min⁻¹). Body composition was measured by dual X-ray absorptiometry and MRI. Sixty-one healthy, sedentary [V(O2max) 36(5) ml·kg⁻¹·min⁻¹; mean(SD)], moderately overweight [BMI 28.1(1.8) kg/m²], young [age: 30(6) yr] men were randomized to sedentary living (CON; n = 17 completers) or moderate (MOD; 300 kcal/day, n = 18) or high (HIGH; 600 kcal/day, n = 18) dose physical exercise for 11 wk. At baseline, insulin-stimulated glucose uptake was highest in femoral skeletal muscle followed by intraperitoneal visceral adipose tissue (VAT), retroperitoneal VAT, abdominal (anterior + posterior) subcutaneous adipose tissue (SAT), and femoral SAT (P < 0.0001 between tissues). Metabolic rate of glucose increased similarly (~30%) in the two exercise groups in femoral skeletal muscle (MOD 24[9, 39] µmol·kg⁻¹·min⁻¹, P = 0.004; HIGH 22[9, 35] µmol·kg⁻¹·min⁻¹, P = 0.003) (mean[95% CI]) and in five individual femoral muscle groups but not in femoral SAT. Standardized uptake value of FDG decreased ~24% in anterior abdominal SAT and ~20% in posterior abdominal SAT compared with CON but not in either intra- or retroperitoneal VAT. Total adipose tissue mass decreased in both exercise groups, and the decrease was distributed equally among subcutaneous and intra-abdominal depots. In conclusion, aerobic exercise training increases insulin-stimulated glucose uptake in skeletal muscle but not in adipose tissue, which demonstrates some interregional differences.

Maintenance of cardiorespiratory fitness, body composition, and a physically active lifestyle after structured exercise interventions in individuals with overweight and obesity: A mixed-method follow-up study.

Objectives: The aim of this mixed-method study was to explore maintenance of physical activity and health effects one year after completion of exercise interventions in transport and leisure-time domains of everyday life. We hypothesised that routinisation of active commuting would lead to better maintenance of physical activity and health effects compared with leisure-time exercise. Study design: Mixed-methods follow-up study. Methods: Individuals with overweight/obesity, who completed a 6-month exercise intervention (active commuting by bike (BIKE), moderate (MOD) or vigorous intensity leisure-time exercise (VIG)), were after one year invited to participate in a follow-up visit which included measurements of cardiorespiratory fitness during an incremental bicycle test and body composition using dual-energy X-ray absorptiometry. Variability in maintenance practices was assessed in a sub-sample of participants who experienced the greatest improvements ('VO2peak improvers') and reductions ('VO2peak reducers'), respectively, in cardiorespiratory fitness. Semi-structured interviews were conducted (15-30 min) and analysed using systematic text condensation to identify barriers and facilitators associated with maintenance of physical activity. Results: Out of the 74 participants completing an exercise intervention, 46 (62%) completed follow-up (BIKE: n = 14; MOD: n = 14; VIG: n = 18). Improvements in VO2peak and reductions in fat mass were maintained in BIKE and VIG. Body weight decreased in BIKE and fat free mass increased in VIG. Changes in VO2peak and anthropometry at follow-up did not differ between BIKE and MOD + VIG. Fat mass decreased and recreational physical activity increased in 'VO2peak improvers'. Findings from the interviews suggested that self-monitoring, collective exercising, and new personal exercise challenges facilitate maintenance of a physically active lifestyle. Conclusion: Completion of a structured exercise intervention consisting of 6 months of active commuting or vigorous intensity leisure-time exercise was associated with long-term maintenance of improvements in VO2peak and body composition, whereas moderate intensity leisure-time exercise was not. In contrast to our hypothesis, active commuting was not associated with better maintenance of physical activity and health effects after the intervention compared with leisure-time exercise.

Homozygous carriers of the G allele of rs4664447 of the glucagon gene (GCG) are characterised by decreased fasting and stimulated levels of insulin, glucagon and glucagon-like peptide (GLP)-1.

AIMS/HYPOTHESIS: The glucagon gene (GCG) encodes several hormones important for energy metabolism: glucagon, oxyntomodulin and glucagon-like peptide (GLP)-1 and -2. Variants in GCG may associate with type 2 diabetes, obesity and/or related metabolic traits. METHODS: GCG was re-sequenced as a candidate gene in 865 European individuals. Twenty-nine variants were identified. Four variants that were considered to have a likelihood for altered functionality: rs4664447, rs7581952, Ile158Val and Trp169Ter, were genotyped in 17,584 Danes. RESULTS: When examined in 5,760 treatment-naive individuals, homozygous carriers of the low frequency (minor allele frequency 2.3%) G allele of rs4664447, predicted to disrupt an essential splice enhancer binding site, had lower levels of fasting plasma glucose (mean ± SD, 4.8 ± 1.2 vs 5.5 ± 0.8 mmol/l, p = 0.004); fasting serum insulin (22 ± 14 vs 42 ± 27 pmol/l, p = 0.04); glucose-stimulated serum insulin (159 ± 83 vs 290 ± 183 pmol/l, p = 0.01) and adult height (165 ± 10 vs 172 ± 9 cm, p = 0.0009) compared with A allele carriers. During oral glucose tolerance and hyperglycaemic arginine stimulation tests, the plasma AUC for GLP-1 (730 ± 69 vs 1,334 ± 288 pmol/l × min, p = 0.0002) and basal and stimulated levels of serum insulin and plasma glucagon were ∼50% decreased (p < 0.001) among three homozygous carriers compared with nine matched wild-type carriers. rs7581952, Ile158Val and Trp169Ter (where 'Ter' indicates 'termination') variants of GCG did not significantly associate or co-segregate with the metabolic traits examined. CONCLUSIONS/INTERPRETATION: Re-sequencing of GCG revealed a low frequency intronic variant, rs4664447, and follow-up physiological studies suggest that this variant in homozygous form may cause decreased fasting and stimulated levels of insulin, glucagon and GLP-1. Overall, our findings suggest that variation in GCG has no major impact on carbohydrate metabolism in the study populations examined.

Do gastrointestinal transit times and colonic dimensions change with time since spinal cord injury?

STUDY DESIGN: Long-term follow-up study. OBJECTIVES: To determine whether gastrointestinal transit times (GITTs) and colonic dimensions change during the first or subsequent decades after spinal cord injury (SCI). SETTING: Aarhus University Hospital, Denmark. METHODS: GITT and colonic dimensions were evaluated by means of radio-opaque markers. Group A (n=12) was investigated 1 year after SCI and again 13 (range 11-14) years later. Group B (n=10) was studied 19 (range 9-36) years after injury and again 12 (range 11-12) years later. All had been treated with conservative bowel management. RESULTS: In group A, the median GITT 1 year after injury was 4.3 (range 1.1-6.5) days and 13 years later, it was 3.2 (range 1.3-6.5) days, P=0.96. In group B, the median GITT 19 year after injury was 3.4 (range 0.6-5.9) days and 12 years later, it was 3.2 (range 1.9-5.5) days, P=0.77. None of the two groups experienced a significant change in the diameter of the caecum/ascending colon, transverse colon, descending colon or the sigmoid during long-term follow-up. Megacolon was present in four patients at baseline and in five at follow-up. CONCLUSION: GITTs and colonic dimensions did not change, neither during the first decade nor long after SCI.

Fat oxidation at rest predicts peak fat oxidation during exercise and metabolic phenotype in overweight men.

OBJECTIVE: To elucidate if fat oxidation at rest predicts peak fat oxidation during exercise and/or metabolic phenotype in moderately overweight, sedentary men. DESIGN: Cross-sectional study. SUBJECTS: We measured respiratory exchange ratio (RER) at rest in 44 moderately overweight, normotensive and normoglycemic men and selected 8 subjects with a low RER (L-RER, body mass index (BMI): 27.9+/-0.9 kg m(-2), RER: 0.76+/-0.02) and 8 with a high RER (H-RER; BMI 28.1+/-1.1 kg m(-2), RER: 0.89+/-0.02). After an overnight fast, a venous blood sample was obtained and a graded exercise test was performed. Fat oxidation during exercise was quantified using indirect calorimetry. RESULTS: Peak fat oxidation during exercise was higher in L-RER than in H-RER (0.333+/-0.096 vs 0.169+/-0.028 g min(-1); P<0.01) and occurred at a higher relative intensity (36.2+/-6.6 vs 28.2+/-3.1% VO(2max), P<0.05). Using the International Diabetes Federation criteria, we found that there was a lower accumulation of metabolic risk factors in L-RER than in H-RER (1.6 vs 3.5, P=0.028), and no subjects in L-RER and four of eight subjects in H-RER had the metabolic syndrome. Resting RER was positively correlated with plasma triglycerides (P<0.01) and negatively with plasma free fatty acids (P<0.05), and peak fat oxidation during exercise was positively correlated with plasma free fatty acid concentration at rest (P<0.05). CONCLUSION: A low RER at rest predicts a high peak fat oxidation during exercise and a healthy metabolic phenotype in moderately overweight, sedentary men.

Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time approximately 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1-mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.

Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist.

The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.

A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors.

A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we highlight the emerging impact of these receptor on virus-mediated diseases.

Sacral nerve stimulation for faecal incontinence alters colorectal transport.

BACKGROUND: Sacral nerve stimulation reduces the frequency of defaecation in patients with faecal incontinence. The aim of this study was to examine the mechanism behind the reduced number of bowel movements in incontinent patients treated with sacral nerve stimulation. METHODS: The study included 20 patients with faecal incontinence and a positive percutaneous nerve evaluation test: 19 women and one man, with a median age of 63 (range 28-78) years. Colorectal scintigraphy was performed to assess colorectal emptying at defaecation before and after implantation. Segmental colorectal transit times were determined using radio-opaque markers. RESULTS: The median frequency of defaecation per 3 weeks decreased from 56 (range 19-136) to 26 (range 12-78) (P < 0.002). At defaecation, antegrade transport from the ascending colon decreased from a median score of 8 (range 0-23) to 0 (range 0-11) per cent (P = 0.001), while retrograde transport from the descending colon increased from a median score of 0 (range 0-14) to 2 (range 0-30) per cent (P = 0.039). The median defaecation score was unchanged. There was a non-significant increase in median total gastrointestinal transit time from 2.5 (range 0.9-6.2) to 3.3 (range 0.8-6.2) days (P = 0.079). CONCLUSION: Sacral nerve stimulation reduces antegrade transport from the ascending colon and increases retrograde transport from the descending colon at defaecation. This may prolong colonic transit time and increase the storage capacity of the colon.

EBI2 in splenic and local immune responses and in autoimmunity.

The seven transmembrane G protein-coupled receptor EBV-induced gene 2 (EBI2), also known as GPR183, is expressed in particular in immune cells. Activated by its endogenous ligands, which are a group of oxysterols, it functions as a chemo-attractant receptor, mediating cell migration. In coordination with other receptors, EBI2 plays important roles in controlling the migration of immune cells during the course of a T-dependent Ab response in the spleen. In recent years, it has become clear that EBI2 also has other roles to play in the immune system. Thus, EBI2 seems to be involved in innate immune responses, such as those mediated by TLR signaling, and it has been implicated in regional immune responses, including immune responses in the CNS. In this review, we describe the functions of EBI2 in B cells, T cells, and dendritic cells during the course of a T-dependent Ab response in the spleen. Furthermore, we review the existing evidence supporting a role for EBI2 in local immune responses and in autoimmune diseases, with a special focus on immune responses in the CNS. Finally, we discuss which type of role EBI2 may play in autoimmune diseases, and we give our opinion about the paths of future research in EBI2.

Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity.

ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

GIP(3-30)NH2 is a potent competitive antagonist of the GIP receptor and effectively inhibits GIP-mediated insulin, glucagon, and somatostatin release.

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH2, that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH2. We showed previously that GIP(3-30)NH2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH2 bound with high affinity (Ki of 17nM), in contrast to human GIP(3-30)NH2 (Ki of 250nM). In cAMP studies, rat GIP(3-30)NH2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH2 displayed weak, low-potent partial agonistic properties (EC50>1µM) with an efficacy of 9.4% at 0.32µM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH2, despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH2; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems.

Targeting the latent cytomegalovirus reservoir with an antiviral fusion toxin protein.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation.