Management of paraesophageal hiatus hernia: recommendations following a European expert Delphi consensus.

AIMS: There is considerable controversy regarding optimal management of patients with paraesophageal hiatus hernia (pHH). This survey aims at identifying recommended strategies for work-up, surgical therapy, and postoperative follow-up using Delphi methodology. METHODS: We conducted a 2-round, 33-question, web-based Delphi survey on perioperative management (preoperative work-up, surgical procedure and follow-up) of non-revisional, elective pHH among European surgeons with expertise in upper-GI. Responses were graded on a 5-point Likert scale and analyzed using descriptive statistics. Items from the questionnaire were defined as "recommended" or "discouraged" if positive or negative concordance among participants was > 75%. Items with lower concordance levels were labelled "acceptable" (neither recommended nor discouraged). RESULTS: Seventy-two surgeons with a median (IQR) experience of 23 (14-30) years from 17 European countries participated (response rate 60%). The annual median (IQR) individual and institutional caseload was 25 (15-36) and 40 (28-60) pHH-surgeries, respectively. After Delphi round 2, "recommended" strategies were defined for preoperative work-up (endoscopy), indication for surgery (typical symptoms and/or chronic anemia), surgical dissection (hernia sac dissection and resection, preservation of the vagal nerves, crural fascia and pleura, resection of retrocardial lipoma) and reconstruction (posterior crurorrhaphy with single stitches, lower esophageal sphincter augmentation (Nissen or Toupet), and postoperative follow-up (contrast radiography). In addition, we identified "discouraged" strategies for preoperative work-up (endosonography), and surgical reconstruction (crurorrhaphy with running sutures, tension-free hiatus repair with mesh only). In contrast, many items from the questionnaire including most details of mesh augmentation (indication, material, shape, placement, and fixation technique) were "acceptable". CONCLUSIONS: This multinational European Delphi survey represents the first expert-led process to identify recommended strategies for the management of pHH. Our work may be useful in clinical practice to guide the diagnostic process, increase procedural consistency and standardization, and to foster collaborative research.

Longer-term outcomes of gastroesophageal reflux disease treated with magnetic sphincter augmentation.

Surgical intervention for gastroesophageal reflux disease (GERD) has historically been limited to fundoplication. Magnetic sphincter augmentation (MSA) is a less invasive alternative that was introduced 15 years ago, and it may have a superior side-effect profile. To date, however, there has been just a single published study reporting outcomes in a UK population. This study reports quality-of-life (QOL) outcomes and antacid use in patients undergoing MSA, with a particular focus on postoperative symptoms and those with severe reflux. A single-center cohort study was carried out to assess the QOL outcomes and report long-term safety outcomes in patients undergoing MSA. GERD-health-related quality of life (GERD-HRQL) and Reflux Symptom Index (RSI) scores were collected preoperatively, and immediately postoperatively, at 1-, 2-, 3-, and 5-year follow-up time points. All patients underwent preoperative esophagogastroduodenoscopy, impedance, and manometry. Two hundred and two patients underwent laparoscopic MSA over 9 years. The median preoperative GERD-HRQL score was 31, and the median RSI score was 17. There was a reduction in all scores from preoperative values to each time point, which was sustained at 5-year follow-up; 13% of patients had a preoperative DeMeester score of >50, and their median preoperative GERD-HRQL and RSI scores were 32 and 15.5, respectively. These were reduced to 0 at the most recent follow-up. There was a significant reduction in antacid use at all postoperative time points. Postoperative dilatation was necessary in 7.4% of patients, and the device was removed in 1.4%. Erosion occurred in no patients. MSA is safe and effective at reducing symptom burden and improving QOL scores in patients with both esophageal and laryngopharyngeal symptoms, including those with severe reflux.

The prevalence of intestinal dysbiosis in patients referred for antireflux surgery.

BACKGROUND: Prior to antireflux surgery, most patients with symptoms of gastroesophageal reflux disease (GERD) have been taking long-term proton pump inhibitors (PPIs). PPIs have been shown to cause changes to the intestinal microbiota, such as small intestinal bacterial overgrowth (SIBO), which is characterised by symptoms of gas bloating. Patients undergoing antireflux surgery are not routinely screened for SIBO, yet many patients experience gas-related symptoms postoperatively. METHODS: Data from consecutive patients (n = 104) referred to a speciality reflux centre were retrospectively assessed. Patients underwent a routine diagnostic workup for GERD including history, endoscopy, oesophageal manometry and 24-h pH-impedance monitoring off PPIs. Intestinal dysbiosis was determined by hydrogen and methane breath testing with a hydrogen-positive result indicative of SIBO and a methane-positive result indicative of intestinal methanogen overgrowth (IMO). RESULTS: 60.6% of patients had intestinal dysbiosis (39.4% had SIBO and 35.6% had IMO). Patients with dysbiosis were more likely to report bloating (74.6% vs 48.8%; P = 0.01) and belching (60.3% vs 34.1%; P = 0.01). The oesophageal acid exposure time and number of reflux episodes were similar between dysbiosis and non-dysbiosis groups, but patients with dysbiosis were more likely to have a positive reflux-symptom association (76.2% vs 31.7%; P < 0.001), especially for regurgitation in those with SIBO (P = 0.01). Hydrogen gas production was significantly greater in patients with a positive reflux-symptom association for regurgitation (228.8 ppm vs 129.1 ppm, P = 0.004) and belching (mean AUC 214.8 ppm vs 135.9 ppm, P = 0.02). CONCLUSIONS: The prevalence of intestinal dysbiosis is high in patients with GERD, and these patients are more likely to report gas-related symptoms prior to antireflux surgery. Independently, SIBO may be a contributory factor to refractory reflux symptoms and gas bloating in antireflux surgery candidates.

Reduction of microglial progranulin does not exacerbate pathology or behavioral deficits in neuronal progranulin-insufficient mice.

Loss-of-function mutations in progranulin (GRN), most of which cause progranulin haploinsufficiency, are a major autosomal dominant cause of frontotemporal dementia (FTD). Individuals with loss-of-function mutations on both GRN alleles develop neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. Progranulin is a secreted glycoprotein expressed by a variety of cell types throughout the body, including neurons and microglia in the brain. Understanding the relative importance of neuronal and microglial progranulin insufficiency in FTD pathogenesis may guide development of therapies. In this study, we used mouse models to investigate the role of neuronal and microglial progranulin insufficiency in the development of FTD-like pathology and behavioral deficits. Grn-/- mice model aspects of FTD and NCL, developing lipofuscinosis and gliosis throughout the brain, as well as deficits in social behavior. We have previously shown that selective depletion of neuronal progranulin disrupts social behavior, but does not produce lipofuscinosis or gliosis. We hypothesized that reduction of microglial progranulin would induce lipofuscinosis and gliosis, and exacerbate behavioral deficits, in neuronal progranulin-deficient mice. To test this hypothesis, we crossed Grnfl/fl mice with mice expressing Cre transgenes targeting neurons (CaMKII-Cre) and myeloid cells/microglia (LysM-Cre). CaMKII-Cre, which is expressed in forebrain excitatory neurons, reduced cortical progranulin protein levels by around 50%. LysM-Cre strongly reduced progranulin immunolabeling in many microglia, but did not reduce total brain progranulin levels, suggesting that, at least under resting conditions, microglia contribute less than neurons to overall brain progranulin levels. Mice with depletion of both neuronal and microglial progranulin failed to develop lipofuscinosis or gliosis, suggesting that progranulin from extracellular sources prevented pathology in cells targeted by the Cre transgenes. Reduction of microglial progranulin also did not exacerbate the social deficits of neuronal progranulin-insufficient mice. These results do not support the hypothesis of synergistic effects between progranulin-deficient neurons and microglia. Nearly complete progranulin deficiency appears to be required to induce lipofuscinosis and gliosis in mice, while partial progranulin insufficiency is sufficient to produce behavioral deficits.

Development of small-molecule Tau-SH3 interaction inhibitors that prevent amyloid-ß toxicity and network hyperexcitability.

Alzheimer's disease (AD) is the leading cause of dementia and lacks highly effective treatments. Tau-based therapies hold promise. Tau reduction prevents amyloid-ß-induced dysfunction in preclinical models of AD and also prevents amyloid-ß-independent dysfunction in diverse disease models, especially those with network hyperexcitability, suggesting that strategies exploiting the mechanisms underlying Tau reduction may extend beyond AD. Tau binds several SH3 domain-containing proteins implicated in AD via its central proline-rich domain. We previously used a peptide inhibitor to demonstrate that blocking Tau interactions with SH3 domain-containing proteins ameliorates amyloid-ß-induced dysfunction. Here, we identify a top hit from high-throughput screening for small molecules that inhibit Tau-FynSH3 interactions and describe its optimization with medicinal chemistry. The resulting lead compound is a potent cell-permeable Tau-SH3 interaction inhibitor that binds Tau and prevents amyloid-ß-induced dysfunction, including network hyperexcitability. These data support the potential of using small molecule Tau-SH3 interaction inhibitors as a novel therapeutic approach to AD.

Preclinical Interventions in Mouse Models of Frontotemporal Dementia Due to Progranulin Mutations.

Heterozygous loss-of-function mutations in progranulin (GRN) cause frontotemporal dementia (FTD), a leading cause of early-onset dementia characterized clinically by behavioral, social, and language deficits. There are currently no FDA-approved therapeutics for FTD-GRN, but this has been an active area of investigation, and several approaches are now in clinical trials. Here, we review preclinical development of therapies for FTD-GRN with a focus on testing in mouse models. Since most FTD-GRN-associated mutations cause progranulin haploinsufficiency, these approaches focus on raising progranulin levels. We begin by considering the disorders associated with altered progranulin levels, and then review the basics of progranulin biology including its lysosomal, neurotrophic, and immunomodulatory functions. We discuss mouse models of progranulin insufficiency and how they have been used in preclinical studies on a variety of therapeutic approaches. These include approaches to raise progranulin expression from the normal allele or facilitate progranulin production by the mutant allele, as well as approaches to directly increase progranulin levels by delivery across the blood-brain barrier or by gene therapy. Several of these approaches have entered clinical trials, providing hope that new therapies for FTD-GRN may be the next frontier in the treatment of neurodegenerative disease.

Patients with sporadic FTLD exhibit similar increases in lysosomal proteins and storage material as patients with FTD due to GRN mutations.

Loss of function progranulin (GRN) mutations are a major autosomal dominant cause of frontotemporal dementia (FTD). Patients with FTD due to GRN mutations (FTD-GRN) develop frontotemporal lobar degeneration with TDP-43 pathology type A (FTLD-TDP type A) and exhibit elevated levels of lysosomal proteins and storage material in frontal cortex, perhaps indicating lysosomal dysfunction as a mechanism of disease. To investigate whether patients with sporadic FTLD exhibit similar signs of lysosomal dysfunction, we compared lysosomal protein levels, transcript levels, and storage material in patients with FTD-GRN or sporadic FTLD-TDP type A. We analyzed samples from frontal cortex, a degenerated brain region, and occipital cortex, a relatively spared brain region. In frontal cortex, patients with sporadic FTLD-TDP type A exhibited similar increases in lysosomal protein levels, transcript levels, and storage material as patients with FTD-GRN. In occipital cortex of both patient groups, most lysosomal measures did not differ from controls. Frontal cortex from a transgenic mouse model of TDP-opathy had similar increases in cathepsin D and lysosomal storage material, showing that TDP-opathy and neurodegeneration can drive these changes independently of progranulin. To investigate these changes in additional FTLD subtypes, we analyzed frontal cortical samples from patients with sporadic FTLD-TDP type C or Pick's disease, an FTLD-tau subtype. All sporadic FTLD groups had similar increases in cathepsin D activity, lysosomal membrane proteins, and storage material as FTD-GRN patients. However, patients with FTLD-TDP type C or Pick's disease did not have similar increases in lysosomal transcripts as patients with FTD-GRN or sporadic FTLD-TDP type A. Based on these data, accumulation of lysosomal proteins and storage material may be a common aspect of end-stage FTLD. However, the unique changes in gene expression in patients with FTD-GRN or sporadic FTLD-TDP type A may indicate distinct underlying lysosomal changes among FTLD subtypes.

Impaired ß-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations.

Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only ß-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature ß-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated ß-glucocerebrosidase. Immunostaining revealed loss of neuronal ß-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on ß-glucocerebrosidase outside of the context of neurodegeneration, we investigated ß-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn-/- mice had lower ß-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs ß-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired ß-glucocerebrosidase processing.

Carbonic anhydrase inhibitors. Interaction of 2-N,N-dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide with 12 mammalian isoforms: kinetic and X-ray crystallographic studies.

2-N,N-Dimethylamino-1,3,4-thiadiazole-5-methanesulfonamide was tested for its interaction with the 12 catalytically active mammalian carbonic anhydrase (CA, EC 4.2.1.1) isozymes, CA I-XIV. The compound is a potent inhibitor of CA IV, VII, IX, XII, and XIII (K(I)s of 0.61-39 nM), a medium potency inhibitor of CA II and VA (K(I)s of 121-438 nM), and a weak inhibitor against the other isoforms (CA III, VB, VI, and XIV), making it a very interesting candidate for situations in which a strong/selective inhibition of certain isozymes is needed. The crystal structure of the hCA II adduct of this sulfonamide revealed interesting interactions between the inhibitor and the enzyme which are quite different from those observed in the adducts of CA II with the structurally related aliphatic derivatives zonisamide, 2-amino-1,3,4-thiadiazolyl-5-difluoromethanesulfonamide, and 2-dimethylamino-5-[sulfonamido-(aminomethyl)]-1,3,4-thiadiazole reported earlier.

Difluoromethylenediphosphonate: a convenient, scalable, and high-yielding synthesis.

[reaction: see text] Since the first disclosure of difluoromethylenediphosphonate, 2, almost 40 years ago, interest in this compound has flourished in several research areas. In this paper, we present a convenient, high-yielding (99% overall) method for the preparation of milligram to multigram quantities of 2 (as the bis(tributylammonium salt, 2b) in a solid form that is easy to handle.

X-ray crystallographic studies reveal that the incorporation of spacer groups in carbonic anhydrase inhibitors causes alternate binding modes.

Human carbonic anhydrases (CAs) are well studied targets for the development of inhibitors for pharmaceutical applications. The crystal structure of human CA II has been determined in complex with two CA inhibitors (CAIs) containing conventional sulfonamide and thiadiazole moieties separated by a -CF2- or -CHNH2- spacer group. The structures presented here reveal that these spacer groups allow novel binding modes for the thiadiazole moiety compared with conventional CAIs.

Leveraging human genomic information to identify nonhuman primate sequences for expression array development.

BACKGROUND: Nonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. RESULTS: We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. CONCLUSION: This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.

Formats for combinatorial synthesis: solid-phase, liquid-phase and surface.

Methods for combinatorial and parallel synthesis continue to evolve in order to meet the demands of modern synthetic organic chemistry. The nature of the support, while typically overlooked, is a key consideration for successful combinatorial organic synthesis. Developments in combinatorial synthesis technologies such as the 'lab-on-a-chip' concept and 96-well-plate-compatible resin plugs have been reported, which should contribute to meeting the increasing challenges of this field.

Synthesis of 2',3'-dideoxynucleoside 5'-alpha-P-borano-beta,gamma-(difluoromethylene)triphosphates and their inhibition of HIV-1 reverse transcriptase.

The triphosphates of antiviral 2',3'-dideoxynucleosides (ddNs) are the active chemical species that inhibit viral DNA synthesis. The inhibition involves incorporation of ddNMP into DNA and subsequent chain termination. A conceivable strategy for antiviral drugs is to employ nucleoside 5'-triphosphate mimics that can entirely bypass cellular phosphorylation. AZT 5'-alpha-R(P)-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaB-betagammaCF(2)TP) has been identified as a potent inhibitor of HIV-1 reverse transcriptase (HIV-1 RT). This work was aimed at confirming that 5'-alphaB-betagammaCF(2)TP is a useful generic triphosphate moiety and can render antiviral ddNs with potent inhibitory effects on HIV-1 RT. Thus, 10 ddNs were converted to their 5'-alphaB-betagammaCF(2)TPs via a sequence (one-pot) of reactions: formation of an activated phosphite, formation of a cyclic triphosphate, boronation, and hydrolysis. Other synthetic routes were also explored. All ddN 5'-alphaB-betagammaCF(2)TPs tested exhibited essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs. A conclusion can be made that 5'-alphaB-betagammaCF(2)TP is a generic and promising triphosphate mimic (P3M) concerning HIV-1 RT inhibition and serum stability. It is anticipated that use of 5'-alphaB-betagammaCF(2)TP as P3M moiety will lead to the discovery of a new class of anti-HIV agents.

2',3'-dideoxynucleoside 5'-beta, gamma-(difluoromethylene) triphosphates with alpha-P-thio or alpha-P-seleno modifications: synthesis and their inhibition of HIV-1 reverse transcriptase.

Nucleoside reverse transcriptase inhibitors (NRTIs) are prodrugs which require three intracellular phosphorylation steps to yield their corresponding, biologically active, nucleoside triphosphate. In order to circumvent this often inefficient phosphorylation cascade, a plausible approach is to provide the active species directly in the form of a stabilized nucleoside triphosphate mimic. We have previously shown that such a mimic, namely 5'-alpha-Rp-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaBCF2TP) is a generic triphosphate mimic that is biologically stable and can render antiviral ddNs with potent inhibitory activity against HIV-1 RT. Herein we report the synthesis and activity against HIV-1 RT of several ddN 5'-alpha-modified-beta,gamma(difluoromethylene)triphosphate mimics with either a non-bridging calphaP-thio (5'-alphaSCF2TP) or alpha-P-seleno (5'-alpha SeCF2TP) modification. One compound, namely, AZT-5'-alpha-P-seleno-beta,gamma-(difluoromethylene)triphosphate (diastereomer I), was identified as a potent inhibitor of HIV-1 RT (Ki = 64 nM) and represents the first report of HIV-1 RT inhibition data for a nucleotide bearing an alpha-P-seleno modification. These triphosphate mimics may be useful in the investigation of enzyme mechanism and may have interesting properties with respect to drug resistance and polymerase selectivity.

Synthesis of AZT 5'-triphosphate mimics and their inhibitory effects on HIV-1 reverse transcriptase.

In search of active nucleoside 5'-triphosphate mimics, we have synthesized a series of AZT triphosphate mimics (AZT P3Ms) and evaluated their inhibitory effects on HIV-1 reverse transcriptase as well as their stability in fetal calf serum and in CEM cell extracts. Reaction of AZT with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by treatment of the phosphite intermediate 2 with pyrophosphate analogues, yielded the cyclic triphosphate intermediates 4b-4f, which were subjected to boronation and subsequent hydrolysis to give AZT 5'-alpha-borano-beta,gamma-bridge-modified triphosphates 6b-6f in moderate to good yields. Reaction of the cyclic intermediate 4d with iodine, followed by treatment with a series of nucleophiles, afforded the AZT 5'-beta,gamma-difluoromethylene-gamma-substituted triphosphates (7b-7i). Several different types of AZT P3Ms containing alpha-P-thio (or dithio) and beta,gamma-difluoromethylene (13,14), alpha,beta-difluoromethylene and gamma-P-methyl(or phenyl) (15,16), and alpha-borano-beta,gamma-difluoromethylene and gamma-O-methyl/phenyl (11,12) were also synthesized. The effectiveness of the compounds as inhibitors of HIV-1 reverse transcriptase was determined using a fluorometric assay and a poly(A) homopolymer as a template. A number of AZT P3Ms exhibited very potent inhibition of HIV-1 reverse transcriptase. Modifications at the beta,gamma-bridge of triphosphate rendered the AZT P3Ms 6b-6f with varied activities (K(i) from 9.5 to >>500 nM) while modification at the alpha,beta-bridge of triphosphate led to weak AZT P3M inhibitors. The results imply that the AZT P3Ms were substrate inhibitors, as is AZT triphosphate. The most active compound, AZT 5'-alpha-R(p)()-borano-beta,gamma-(difluoromethylene)triphosphate (AZT 5'-alphaB-betagammaCF(2)TP) (6d-I), is as potent as AZT triphosphate with a K(i)() value of 9.5 nM and at least 20-fold more stable than AZT triphosphate in the serum and cell extracts. Therefore, for the first time, a highly active and stable nucleoside triphosphate mimic has been identified, which is potentially useful as a new type of antiviral drug. The promising triphosphate mimic, 5'-alpha-borano-beta,gamma-(difluoromethylene)triphosphate, is expected to be valuable to the discovery of nucleotide mimic antiviral drugs.

Peptidomimetics and peptide backbone modifications.

The replacement of the amide bond in a peptide backbone is a widely used form of peptide mimicry. Several of the most common amide bond surrogates, including peptidomimetic work done in this laboratory, and their biological applications are presented in this review.