Bringing Molecular Biology to Bear on Adhesion Prevention: Postsurgical Adhesion Reduction Using Intraperitoneal Inoculation of Hyaluronic Acid–Inducing Adenoviral Vector in a Murine Model

Objective: Seprafilm (Genzyme, Cambridge, MA) an absorbable adhesion barrier incorporating hyaluronic acid (HA), a high molecular mass glycosaminoglycan and important component of the extracellular matrix, has been shown to prevent adhesions in both experimental models and human subjects. Yet, the application of HA as a sheet at the time of surgery has several important logistic limitations. Recently, our laboratory has identified and cloned the genes encoding murine hyaluronic acid synthase 2 (mHAS2) and 3 (mHAS3) and engineered adenoviruses incorporating these genes, which, on intraperitoneal injection, significantly increases HA in peritoneal fluid. We hypothesized that intraperitoneal gene therapy with mHAS2 or mHAS3 via an adenoviral vector prior to a standardized cecal abrasion surgery would lead to a reduction in postoperative adhesion severity. Methods: Mice were assigned to one of four groups: (1) intraperitoneal inoculation with adenovirus encoding mHAS2; (2) mHAS3; (3) a control reporter adenovirus (RV) encoding GFP; or (4) intraoperative placement of a commercially available and murine-validated hyaluronic acid adhesion barrier (Seprafilm, SF). An a priori sample size calculation was performed. Mice in groups 1, 2, and 3 underwent injection of 2 x 107 viral particles in 1 ml of fluid on day -1. Sham injection was performed on group 4 SF mice. On day 0, laparotomy was performed in random sequence by surgeon blinded to the experimental group. On day 7, adhesion scores (0-3) were assigned independently by two blinded investigators. Results: Mean adhesion scores (n = 247) were 0.68 (mHAS2), 0.91 (mHAS3), 1.28 (RV), and 0.47 (SF). Pairwise comparisons using Wilcoxon rank-sum test revealed significant reduction in severity of adhesions between mHAS2, mHAS3, and SF compared to RV (p = 0.0004, 0.039, and 0.0001, respectively). Significance persisted despite correction for multiple comparisons (p = 0.0002, Kruskal-Wallis). There was a direct relationship between intraperitoneal HA concentration and adhesion reduction. Only one death (RV) was secondary to adhesive disease; differential risk of death between groups was statistically significant (p = 0.008) (highest in mHAS2 group). Conclusions: In a dose-response relationship, an intraperitoneal gene therapy approach to adhesion prevention in a murine model was successful, with adenoviruses most productive of HA resulting in the most significant reduction in adhesion scores compared to empty virus (RV). Although SF best reduced postoperative adhesions, the adenoviral gene delivery approach may prove to be more effective in clinical use when peritoneal injury is less localized or at laparoscopy where the application of SF is not possible. Further studies to elucidate the reason for the differential death rates (time bias may have played a role) and to validate results are in progress

Further evidence for the involvement of EFL1 in a Shwachman-Diamond-like syndrome and expansion of the phenotypic features.

Recent evidence has implicated EFL1 in a phenotype overlapping Shwachman-Diamond syndrome (SDS), with the functional interplay between EFL1 and the previously known causative gene SBDS accounting for the similarity in clinical features. Relatively little is known about the phenotypes associated with pathogenic variants in the EFL1 gene, but the initial indication was that phenotypes may be more severe, when compared with SDS. We report a pediatric patient who presented with a metaphyseal dysplasia and was found to have biallelic variants in EFL1 on reanalysis of trio whole-exome sequencing data. The variant had not been initially reported because of the research laboratory's focus on de novo variants. Subsequent phenotyping revealed variability in her manifestations. Although her metaphyseal abnormalities were more severe than in the original reported cohort with EFL1 variants, the bone marrow abnormalities were generally mild, and there was equivocal evidence for pancreatic insufficiency. Despite the limited number of reported patients, variants in EFL1 appear to cause a broader spectrum of symptoms that overlap with those seen in SDS. Our report adds to the evidence of EFL1 being associated with an SDS-like phenotype and provides information adding to our understanding of the phenotypic variability of this disorder. Our report also highlights the value of exome data reanalysis when a diagnosis is not initially apparent

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter

Amine Dynamics in Diamine-Appended Mg2(dobpdc) Metal-Organic Frameworks.

Variable-temperature 15N solid-state NMR spectroscopy is used to uncover the dynamics of three diamines appended to the metal-organic framework Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate), an important family of CO2 capture materials. The results imply both bound and free amine nitrogen environments exist when diamines are coordinated to the framework open Mg2+ sites. There are rapid exchanges between two nitrogen environments for all three diamines, the rates and energetics of which are quantified by 15N solid-state NMR data and corroborated by density functional theory calculations and molecular dynamics simulations. The activation energy for the exchange provides a measure of the metal-amine bond strength. The unexpected negative correlation between the metal-amine bond strength and CO2 adsorption step pressure reveals that metal-amine bond strength is not the only important factor in determining the CO2 adsorption properties of diamine-appended Mg2(dobpdc) metal-organic frameworks

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporte

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective: Barrett's oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods: Cell proliferation and migration within Barrett's glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results: Proliferation predominantly occurs in the middle of Barrett's glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barrett's mirrors pyloric glands and is preserved in Barrett's dysplasia. MUC2-positive goblet cells are localised above the neck in Barrett's glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barrett's glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barrett's glands are clonal, indicating derivation from a single stem cell. Conclusions: Barrett's shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barrett's oesophagus

Implementing new care models: learning from the Greater Manchester demonstrator pilot experience

Background: Current health policy focuses on improving accessibility, increasing integration and shifting resources from hospitals to community and primary care. Initiatives aimed at achieving these policy aims have supported the implementation of various ‘new models of care’, including general practice offering ‘additional availability’ appointments during evenings and at weekends. In Greater Manchester, six ‘demonstrator sites’ were funded: four sites delivered additional availability appointments, other services included case management and rapid response. The aim of this paper is to explore the factors influencing the implementation of services within a programme designed to improve access to primary care. The paper consists of a qualitative process evaluation undertaken within provider organisations, including general practices, hospitals and care homes. Methods: Semi-structured interviews, with the data subjected to thematic analysis. Results: Ninety-one people participated in interviews. Six key factors were identified as important for the establishment and running of the demonstrators: information technology; information governance; workforce and organisational development; communications and engagement; supporting infrastructure; federations and alliances. These factors brought to light challenges in the attempt to provide new or modify existing services. Underpinning all factors was the issue of trust; there was consensus amongst our participants that trusting relationships, particularly between general practices, were vital for collaboration. It was also crucial that general practices trusted in the integrity of anyone external who was to work with the practice, particularly if they were to access data on the practice computer system. A dialogical approach was required, which enabled staff to see themselves as active rather than passive participants. Conclusions: The research highlights various challenges presented by the context within which extended access is implemented. Trust was the fundamental underlying issue; there was consensus amongst participants that trusting relationships were vital for effective collaboration in primary care

Multiple mechanistically distinct modes of endocannabinoid mobilization at central amygdala glutamatergic synapses.

The central amygdala (CeA) is a key structure at the limbic-motor interface regulating stress responses and emotional learning. Endocannabinoid (eCB) signaling is heavily implicated in the regulation of stress-response physiology and emotional learning processes; however, the role of eCBs in the modulation of synaptic efficacy in the CeA is not well understood. Here we describe the subcellular localization of CB1 cannabinoid receptors and eCB synthetic machinery at glutamatergic synapses in the CeA and find that CeA neurons exhibit multiple mechanistically and temporally distinct modes of postsynaptic eCB mobilization. These data identify a prominent role for eCBs in the modulation of excitatory drive to CeA neurons and provide insight into the mechanisms by which eCB signaling and exogenous cannabinoids could regulate stress responses and emotional learning

Mutations in succinate dehydrogenase B (SDHB) enhance neutrophil survival independent of HIF-1α expression.

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Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice. METHODS: Using flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals. RESULTS: In vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed. CONCLUSION: The therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS