Individually monitoring ligand-induced changes in the structure of the GABAA receptor at benzodiazepine binding site and non-binding-site interfaces.

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (alpha/gamma interface) and at aligned positions in the alpha/beta interface. alpha(1)His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of alpha(1)H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of alpha(1)H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of alpha(1)H101C at the alpha/beta interface was significantly slower than modification of alpha(1)H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the alpha/gamma and alpha/beta interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near alpha(1)H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.

Adult Intestinal and Multivisceral Transplantation: Experience From a Single Center in the United Kingdom.

Cambridge is one of two designated adult intestinal transplant centers in the United Kingdom and has performed 60 transplants on 54 patients since 2007; 52% of these were undertaken in the last 3 years. This increasing trend is in contrast with that reported worldwide; 27% were small bowel grafts (SBT), 15% modified multivisceral (MMVT), and 58% multivisceral (MVT). Median recipient age was 47 years; the female-to-male ratio was 27/33. Primary diseases included visceral arterial thromboses (17%), Crohn's disease (17%), motility disorders (12%), visceral venous thromboses (12%), familial adenomatous polyposis (FAP)/desmoids (8%), alcoholic cirrhosis (3%), nonalcoholic fatty liver disease (3%), ulcerative colitis (2%), and other (15%). Indications for transplant included intestinal failure-associated liver disease (IFALD) (27%), loss of central venous access (17%), FAP/desmoid disease (5%), extensive portomesenteric venous thrombosis (PMVT) (20%), widespread mesenteric arterial ischemia (WMAI) (13%), re-transplant (8%), and other (10%). Overall 1-year/5-year patient survival rates were 77%/62%. One-year/5-year patient survival rates were 92%/83%, 85%/65%, and 71%/33% for SBT, MMVT, and MVT. One-year/5-year survival rates for patients with IFALD, PMVT, and other indications who underwent MVT were 80%/20%, 65%/55%, and 55%/35%. The greatest proportion of patient deaths occurred during the first year after transplant (50% in year 1, 23% in year 2, 9% in year 3, 5% in year 4, and 14% in year 5), particularly in the MVT group. Referrals to our United Kingdom center are increasing, and the indications for transplant are becoming more diverse. Our patient survival rates remain comparable with figures reported worldwide.

Cytomegalovirus Infection and Rates of Antiviral Resistance Following Intestinal and Multivisceral Transplantation.

BACKGROUND: Cytomegalovirus (CMV) disease is a common and clinically significant complication following intestinal or multivisceral transplantation. CMV disease is more common in cases of serologic mismatch between donor and recipient. Though in some cases it may be asymptomatic, in the immunosuppressed population it often manifests with evidence of systemic infection or end-organ disease. METHODS: We conducted a retrospective review of all patients undergoing intestinal or multivisceral transplantation over 8 years at our institution. RESULTS: Forty-eight transplantations were performed, with 40% of the patients (19/48) having ≥1 episode of CMV viremia, which rose to 90% in the "donor-positive, recipient-negative" (DPRN) serologic mismatch group. The median time to 1st episode following transplantation was 22.3 weeks (range, 1-78) and median duration of each episode was 4.9 weeks (range, 1.6-37.4). Six of the 19 viremic patients (31.6%) developed virologic resistance with 4 of these occurring in the DPRN group. Four of the 6 patients with drug-resistant CMV died with CMV viremia. All patients with drug resistance acquired ganciclovir resistance; these patients were more challenging to manage with second-line toxicity-limited treatments, including foscarnet, cidofovir, and leflunomide. CMV immunoglobulin has been used and we briefly discuss the use of CMV-specific adoptive T-lymphocyte transfer in the management of 1 case. CONCLUSIONS: Post-transplantation CMV disease continues to be challenging to manage, and there is little consensus on optimal management strategies in this patient group, with a significant requirement for novel therapies; these may be pharmacologic or cell based. Extensive multidisciplinary discussion is important for most cases, but particularly for those patients who acquire virologic resistance.

The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes.

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.

Occult invasive aspergillosis infection following multivisceral transplantation.

Patients undergoing multivisceral transplantation are particularly susceptible to post-operative infections due to immunosuppression and the inclusion of bowel in the transplanted graft. These patients typically receive broad-spectrum antimicrobial and antifungal agents as prophylaxis and treatment. However, evidence for this is limited due to the small number of patients undergoing the procedure. We present a case of occult disseminated invasive aspergillosis infection in a patient who underwent multivisceral transplantation.

Optimising delivery of care in coeliac disease - comparison of the benefits of repeat biopsy and serological follow-up.

BACKGROUND: The majority of deleterious health consequences of coeliac disease (CD) are most likely to be secondary to intestinal inflammation; hence, mucosal recovery is a desirable goal of therapy. Follow-up in CD is controversial and serological response is often used as a surrogate for histological recovery. AIMS: To inform the clinical management of CD using comparative serological and histological data from a biopsy-driven pathway of care. METHODS: A retrospective analysis of the Cambridge Coeliac Clinic database of 595 patients routinely followed up by biopsy and serology. RESULTS: Paired biopsy results were available for 391 patients (15% seronegative). Persisting villous atrophy (VA) occurred in 182 patients (47%). The sensitivity of anti-tissue transglutaminase (TTG) antibody for ongoing VA was only 43.6%. Information on dietetic management and further biopsy to assess response was available for 94 initially unresponsive patients, in whom targeted dietetic intervention by removal of identified gluten sources or avoidance of trace amounts of gluten led to resolution of persistent VA in 50%. The effects of institution of a formal care pathway are analysed in 298 patients. Discharge to primary care and clinical management was facilitated by the information derived from repeat biopsy. CONCLUSIONS: Serology appears to be a poor surrogate marker for mucosal recovery on a gluten-free diet; dietary assessment fails to identify a potential gluten source in many patients with ongoing villous atrophy. The benefits of re-biopsy on diet include stratification of patients with coeliac disease suitable for early discharge from secondary care or those requiring more intensive clinical management.