Search

Pregnancy after solid organ transplantation is becoming more common, with the largest recorded numbers in renal and liver transplant recipients. Intestinal transplantation is relatively new compared to other solid organs, and reports of successful pregnancy are far less frequent. All pregnancies reported to date in intestinal transplant recipients have been in women with stable graft function. The case reported here involves the first reported successful term pregnancy in an intestine-pancreas transplant recipient with chronic graft dysfunction and dependence on both transplant immunosuppression and parenteral nutrition (PN) at the time of conception. Pregnancy was unplanned and unexpected in the setting of chronic illness and menstrual irregularities, discovered incidentally on abdominal ultrasound at approximately 18 weeks' gestation. Rapamune was held, tacrolimus continued, and PN adjusted to maintain consistent weight gain. A healthy female infant was delivered vaginally at term. Medical complications during pregnancy included anemia and need for tunneled catheter replacements. Ascites and edema were improved from baseline, with recurrence of large volume ascites shortly after delivery. Successful pregnancy is possible in the setting of transplant immunosuppression, chronic intestinal graft dysfunction, and long-term PN requirement, but close monitoring is required to ensure the health of mother and child.

Summary: To robustly assess the antibacterial mechanisms of nanotopographies, it is critical to analyze the bacteria-nanotopography adhesion interface. Here, we utilize focused ion beam milling combined with scanning electron microscopy to generate three-dimensional reconstructions of Staphylococcus aureus or Escherichia coli interacting with nanotopographies. For the first time, 3D morphometric analysis has been exploited to quantify the intrinsic contact area between each nanostructure and the bacterial envelope, providing an objective framework from which to derive the possible antibacterial mechanisms of synthetic nanotopographies. Surfaces with nanostructure densities between 36 and 58 per μm2 and tip diameters between 27 and 50 nm mediated envelope deformation and penetration, while surfaces with higher nanostructure densities (137 per μm2) induced envelope penetration and mechanical rupture, leading to marked reductions in cell volume due to cytosolic leakage. On nanotopographies with densities of 8 per μm2 and tip diameters greater than 100 nm, bacteria predominantly adhered between nanostructures, resulting in cell impedance.

From its’ conception, 4-H has encouraged volunteerism and utilized volunteers to accomplish its’ mission - to assist youth in acquiring knowledge, developing life skills, and forming attitudes that will enable them to become self-directing, productive and contributing members of society. The purpose of this study was to explore and describe the factors that motivated [state] 4-H camp volunteers to volunteer with 4-H youth. The Volunteer Functions Inventory was used as the theoretical base for this study. The Values construct (M=3.68) of the VFI was the highest motivating factor of adult 4-H volunteers. The Understanding construct (M=3.39) ranked the second highest motivational factor in volunteerism followed by Social construct (M=3.28), the Enhancement construct (M = 3.05) and the Protective construct (M=2.78). The Career construct (M=2.61) was the least motivating factor to adult volunteers. It was also found that participants that were not involved with 4-H as a youth volunteered more days per year than did participants who were former 4-H members.

The function of NK cells is controlled by several activating and inhibitory receptors, including the family of Killer-immunoglobulin-like receptors (KIRs). The KIR gene 3DS1 (KIR3DS1) represents a conserved allelic variant of the KIR3DL1/S1 gene locus. 3DS1 belongs to the KIR haplotype group B along with other KIR genes encoding for activating KIRs and is present in all human populations. The encoded gene product KIR3DS1 exhibits three extracellular domains, a short cytoplasmic tail and a trans-membrane domain containing a positively charged residue. The latter allows recruitment of the ITAM-bearing adaptor molecule, DAP12, and thus conferring the activating quality to KIR3DS1. Although interaction with its putative ligand HLA-Bw4-I80 or any other HLA class I subtype has not been demonstrated to date, a growing number of associations between the presence of 3DS1 and the outcome of viral infections have been described. Especially, the protective role of KIR3DS1 in combination with HLA-Bw4-I80 in the context of progressive HIV infection has been studied intensively. In addition, a number of recent studies associated the presence or absence of KIR3DS1 with the occurrence and outcome of several malignancies, as well as of autoimmune diseases and graft-versus-host disease (GVHD). In this review, the present knowledge regarding the role of KIRD3S1 in various diseases will be summarized.

The history of intestinal transplantation can be traced back to the turn of the twentieth century. Although advancements have been made, the intestine still presents a greater challenge to transplantation than does that of other solid organs, experiencing higher rates of graft rejection and lower long-term survival. Increasingly, intestinal re-transplantation (re-ITx) is seen as a viable option and is now the fourth most common indication for ITx. Changes to immunosuppression protocols, technical modifications, and infectious disease monitoring have contributed to improved outcomes. The authors review the literature on re-ITx in regard to the history, management considerations, and future directions., Competing Interests: Disclosure The authors declare that they have no relevant or material financial interests that relate to the research described in this paper., (Published by Elsevier Inc.)

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori -induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.

Background/objectives: Rejection and infectious enteritis in intestinal transplant (ITx) patients present with virtually identical symptoms. Currently, the gold standard for differentiating between these two conditions is endoscopy, which is invasive and costly. Our primary aim was to identify differences in peripheral blood cytokines during episodes of acute cellular rejection (ACR) and infectious enteritis in patients with intestinal transplants., Methods: This was a prospective, cross-sectional study involving ITx patients transplanted between 2000 and 2016. We studied 63 blood samples collected from 29 ITx patients during periods of normal (n = 24) and abnormal (n = 17) allograft function. PBMCs from whole blood samples were cultured under unstimulated or stimulated conditions with phytohemagglutinin (PHA). The supernatant from these cultures were collected to measure cytokine and chemokine levels using a 38-plex luminex panel., Results: Our study found that cytokines and chemokines are differentially expressed in normal, ACR, and infectious enteritis samples under unstimulated conditions based on heatmap analysis. Although each cohort displayed distinctive signatures, only MDC (p = 0.037) was found to be significantly different between ACR and infectious enteritis. Upon stimulation of PBMCs, patients with ACR demonstrated increased immune reactivity compared to infectious enteritis; though this did not reach statistical significance., Conclusions: To our knowledge, this is the first comprehensive study comparing cytokine expression during acute rejection and infectious enteritis in intestinal transplant recipients. Our results suggest that cytokines have the potential to be used as clinical markers for risk stratification and/or diagnosis of ACR and infectious enteritis., (Published by Elsevier B.V.)

Monitoring of intestinal allograft function remains a challenge. While frequent endoscopies and biopsies are the gold standard, no single biomarker exists to screen for intestinal transplant rejection. The novel REG3α, an antimicrobial peptide secreted by intestinal enterocytes and Paneth cells, has been associated with inflammatory bowel disease as well as intestinal graft versus host disease. Our aim was to identify and describe a role of REG3α in monitoring or predicting acute allograft rejection after intestinal transplantation (ITx). Since 2019, we have incorporated REG3α into the standard monitoring of patients after ITx. We conducted a retrospective analysis of a prospectively maintained IRB-approved database and present, herein, the results of 2 adults with irreversible intestinal failure who underwent isolated ITx under this monitoring protocol. Increases in REG3α corresponded with acute allograft rejection in both cases and preceded acute allograft rejection by 1 week in one of the cases. We describe REG3α as a non-invasive marker of acute allograft rejection after adult isolated ITx which not only corresponded with acute allograft rejection but also preceded histopathological changes by 1 week., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The role of angiotensin II type-1 receptor (AT1R) antibodies in intestinal transplantation (ITx) is unclear. The aims were 1) to identify the prevalence of AT1R antibodies in pediatric ITx, compared to pediatric intestinal failure (IF), and 2) to determine whether AT1R antibodies were associated with graft dysfunction. 46 serum samples from 25 ITx patients (3 isolated ITx, 22 liver-inclusive ITx) were collected during routine visits >6 months apart and during episodes of graft dysfunction as a result of infectious enteritis or rejection. For comparison, samples were collected from 7 IF control patients. AT1R antibodies were considered positive for levels >17 U/mL. The median (range) AT1R antibody level for ITx patients was 40.0 U/mL (7.2-40.0), compared to 7.0 U/mL (5.7-40.0) for IF patients (p = .02). There was a trend toward higher prevalence of AT1R antibodies in ITx compared with IF patients (68% versus 29%, p = .09). Among ITx patients, the prevalence of AT1R antibodies was not different between periods of active graft dysfunction and normal health (83% versus 67%, p = .31). For 16 patients with >2 samples, AT1R antibodies remained positive in 67% cases, developed in 14% cases, disappeared in 10% cases, and remained negative in 10% cases. The changes in AT1R antibodies did not correlate with de/sensitizing events. This is the first study of AT1R antibodies in pediatric ITx. AT1R antibodies are highly prevalent after ITx and may be triggered by immune activation associated with the transplant. However, their pathogenicity and clinical utility remain in question., (© 2021 Wiley Periodicals LLC.)

Response to neoadjuvant chemotherapy (NAC) in triple negative breast cancer (TNBC) is highly prognostic and determines whether adjuvant chemotherapy is needed if residual tumor is found at surgery. To evaluate the predictive and prognostic values of circulating tumor DNA (ctDNA) in this setting, we analyzed tumor and serial bloods from 26 TNBC patients collected prior, during, and after NAC. Individual digital droplet PCR assays were developed for 121 variants (average 5/patient) identified from tumor sequencing, enabling ctDNA detection in 96% of patients at baseline. Mutant allele frequency at baseline was associated with clinical characteristics. Levels drastically fell after one cycle of NAC, especially in patients whose tumors would go on to have a pathological complete response (pCR), but then rose significantly before surgery in patients with significant residual tumor at surgery (p = 0.0001). The detection of ctDNA early during treatment and also late at the end of NAC before surgery was strongly predictive of residual tumor at surgery, but its absence was less predictive of pCR, especially when only TP53 variants are considered. ctDNA detection at the end of neoadjuvant chemotherapy indicated significantly worse relapse-free survival (HR = 0.29 (95% CI 0.08-0.98), p = 0.046), and overall survival (HR = 0.27 95% CI 0.075-0.96), p = 0.043). Hence, individualized multi-variant ctDNA testing during and after NAC prior to surgery has prognostic and predictive value in early TNBC patients.

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori -induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and β-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and β-subunits with ER chaperone BiP and impaired assembly of α/β-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori -induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori -induced Na-K-ATPase degradation will provide insights for protection against advanced disease. NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori -induced gastric injury.

Background: Temporary ileostomy during intestinal transplantation (ITx) is the standard technique for allograft monitoring. A detailed analysis of the ITx ileostomy has never been reported., Methods: A retrospective review of a single-center ITx database was performed. The analysis was divided into ileostomy formation and takedown episodes., Results: One hundred thirty-five grafts underwent ileostomy formation, and 79 underwent ileostomy takedown. Median age at ITx was 7.7 years and weight was 23 kg. Allograft types were intestine (22%), liver/intestine (55%), multivisceral (16%), and modified multivisceral (7%). Sixty-four percent had 1-stage ITx, whereas 36% required 2-staged ITx. Final ileostomy types were end (20%), loop (10%), distal blowhole (59%), and proximal blowhole (11%). Ileostomy formation: Thirty-one grafts had complications (23%), including prolapse (26%), ischemia (16%), and parastomal hernia (19%). Twelve required surgical revision. There were no significant differences in graft type, ileostomy type, survival, and ileostomy takedown rate between grafts with and without complications. Colon inclusive grafts had higher complication rates (P = 0.002). Ileostomy takedown: Ileostomy takedown occurred at a median of 422 days post-ITx. Twenty-five complications occurred after 22 takedowns (28%), including small bowel obstruction (27%) and abscess (18%). Fifteen grafts required surgical correction. Recipients with complications had longer hospital stay (17 versus 9 d; P = 0.001) than those without complications. Graft type, ileostomy type, and survival were not different., Conclusions: The first of its kind analysis of the surgical ileostomy after ITx reveals that most recipients can undergo successful ileostomy formation/takedown, complication rates are significant but within an acceptable range, and complications do not affect survival. This study demonstrates that the routine use of transplant ostomies remains an acceptable practice after ITx. However, true analysis of risk and benefit will require a randomized control trial.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells., (©2019 American Association for Cancer Research.)

Purpose: Lumpectomy (L) and breast radiotherapy (RT) results in equivalent outcomes in comparison to mastectomy (M) for early-stage breast cancer (BC) based on randomized controlled trials (RCT). Since 2004, RCT support that L without RT yields equivalent survival and acceptable local-regional outcomes in women ≥70-years old with T1N0 hormone-sensitive (ER+) BC on endocrine therapy. Based on this, we hypothesized that M rates should decrease substantially after 2004 in this low-risk elderly population. Methods: We used the Surveillance Epidemiology and End Results registry data to conduct this study. We included women with T1N0 ER+ BC from 2000 to 2014. We compared M rates in women diagnosed from 2000 to 2004 vs. 2005-2012 using the Chi-Square test. Logistic regression analyses was performed to examine demographic/clinical factors associated with mastectomy. Results: 67,506 women met the study criteria. In elderly Stage I ER+ BC, the M rate decreased by 6.3%: 29.0% before 2004 to 22.7% after 2004 ( p < 0.0001). M rates remained higher in elderly non-Hispanic black (NHB, 27.1%, p < 0.0001), non-Hispanic Asian-Pacific-Islander (NHAPI, 30.1%, p < 0.0001), and Hispanics (24.4%, p = 0.0004) vs. non-Hispanic White (NHW, 21.5%). Treatment in the modern cohort was associated with decreased odds of mastectomy (OR = 0.71, 95% CI 0.68-0.74, p < 0.0001) while NH-API race was associated with the highest increased odds of mastectomy (OR = 1.65, 95% 1.53-1.78, p < 0.0001). In the modern cohort specifically (2005-2014), Hispanic women (OR = 1.12, p = 0.014), NHB women (OR = 1.21, p < 0.0001), and NHAPI women (OR = 1.73, p < 0.0001) all had higher odds of undergoing mastectomy relative to NHW women after adjusting for all other patient and tumor related factors. Conclusions: In elderly patients with stage I, ER+ BC, M rates have decreased modestly since 2004. These trends are driven mostly be decreases in the M rate in NHW women, but M rates remain ~25% in Hispanic, NHB, and NHAPI women. Further research is needed to identify why M, which is associated with higher cost and morbidity than L alone, has not changed substantially in elderly, low-risk BC.

Objective: Recent studies show increased serum and esophageal IgG4 in patients with eosinophilic esophagitis (EoE), suggesting a possible IgG4-involved process. The role of IgG4 in pediatric EoE has not been extensively investigated. Our aim was to analyze IgG4 in esophageal tissue in children in parallel to that in adults with EoE., Methods: In a retrospective institutional review board-approved study, we performed immunohistochemical staining of IgG4 in esophageal biopsy specimens from 39 subjects: children with EoE (n = 16), adults with EoE (n = 15), children with reflux esophagitis (n = 4), and pediatric controls (n = 4). We assessed the relationships between IgG4 staining and clinical, endoscopic, and histopathologic characteristics., Results: Patients with EoE were significantly more likely to stain positively for IgG4 than children with reflux esophagitis or controls (P = 0.015). Fifteen of 31 (48%) EoE cases stained positively for IgG4. None of the reflux esophagitis or control cases stained positively. IgG4 staining had 48% sensitivity and 100% specificity for EoE. There was a trend toward IgG4 staining being associated with foreign body/food impaction (P = 0.153). There was a strong association between distal IgG4 staining and basal zone hyperplasia (P = 0.003)., Conclusions: Our study suggests IgG4 is not a consistent finding of EoE at disease diagnosis. Although IgG4 staining was specific for EoE, it had a poor sensitivity with positive staining in only 48% of EoE patients. Further studies are warranted to fully elucidate the role of IgG4 in EoE.

Purpose: Damaged central venous catheters (CVCs) are commonly repaired to avoid line replacement and preserve vascular access. However, limited data suggest an increased risk for central line-associated bloodstream infections (CLABSIs) associated with the repair procedure. The purpose of this study was to describe outcomes of CVC repairs among parenteral nutrition (PN) dependent children with intestinal failure (IF)., Methods: A 2-year retrospective review was performed on children with IF on home PN > 6 months. Outcomes of interest were repair success and postrepair CLABSI incidence. Descriptive statistics included medians and frequencies., Results: A total of 36 pediatric IF patients underwent 96 CVC repairs during the study period. The median CVC repair count was 1.5 repairs/patient (range, 1 to 16 repairs/patient) with >1 repair in half the patients. Ninety-four broken catheters (98%) were successfully repaired with restoration of function. Of the unsuccessful repairs (2%), the two catheters eventually required surgical removal and replacement. One repair (1%) was followed by a CLABSI with Enterococcus faecalis in an immunocompromised patient., Conclusion: CVC repair is a highly successful procedure with a low risk for infection. Catheter repair should be considered whenever possible as it may extend the lifetime of the catheter and decrease the risk for vascular access loss., Level of Evidence: Treatment study; level IV., (Published by Elsevier Inc.)

Background: Though intestinal failure (IF) after bariatric surgery (BS) is uncommon, its prevalence is increasing. However, data on the outcomes for these patients are limited., Objectives: To analyze the outcomes of treatment for patients with IF after BS., Setting: University hospital., Methods: A single-center analysis (1991-2016) of outcomes according to treatment arms established by a multidisciplinary team., Results: Twenty-five IF patients were identified (median age 45 yr). BS was 92% Roux-en-Y gastric bypass. The major cause of IF was volvulus/internal hernia (72%). Median time from BS to IF was 48 months. Treatment arms were intestinal rehabilitation (IR, n = 15), transplantation (TXP, n = 5), and parenteral nutrition (PN, n = 5). For IR, median bowel length was 60 cm. Forty-six percent ultimately discontinued PN. Twenty-seven percent were partially weaned PN and 27% failed IR. Common surgical rehabilitation was Roux-en-Y gastric bypass reversal and restoration of gastrointestinal continuity. The 5-year overall survival was 74%. For TXP, 7 patients were listed for TXP (5 initially and 2 after failed IR). Three underwent TXP, 2 isolated intestine and 1 isolated liver. Three were delisted (1 improvement and 2 death). For PN, 6 patients required long-term PN (5 initially and 1 after failed IR). Four patients are alive currently., Conclusions: IF after BS is an increasing problem facing IR centers. Internal hernia is the major cause. Surgical IR is the first-line therapy and affords the best outcome. TXP is reserved for rescuing patients who failed IR or develop PN complications. Long-term PN is suitable for patients in whom IR or TXP is impractical., (Copyright © 2018 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.)

Immunophenotyping of peripheral blood mononuclear cells has been shown to be a useful, non-invasive method of predicting acute cellular rejection (ACR) following intestinal transplantation (ITx). Our objectives were to characterize differences in the T cell immunophenotype of ITx recipients in peripheral blood samples (1) collected late versus early after ITx and (1) associated with episodes of ACR and infectious enteritis. An IRB-approved, cross-sectional study of ITx recipients was performed. Peripheral blood samples were collected during normal visits and episodes of allograft dysfunction. A total of 38 patients were included in the analysis: 31 ITx recipients (87% liver-inclusive allografts) and 7 intestinal failure control patients. Of the ITx patients, 26 patients were pediatric patients (<21 years). A total of 70 samples were analyzed from ITx recipients, including 51 during normal visits and 19 during episodes of allograft dysfunction (median of 2 samples per patient; range of 1-6 samples per patient). In the late (n = 32) versus early post-ITx (n = 19) normal samples, there was a significantly higher percentage of central memory CD4 T cells (p = .001). In the ACR (n = 5) versus infectious enteritis (n = 14) samples, there was a higher percentage of CD8 T cells expressing HLA-DR (p = .002), CD57 (p < .001), and KLRG1 (p < .001) and a higher percentage of CD4 T cells expressing CD57 (p = .03). Additional studies are needed with larger cohorts to validate these changes in the T cell immunophenotype. Further elucidating T cell immunophenotypes in ITx will lead to a better understanding of immune mechanisms of allograft dysfunction, identification of potential biomarkers in ITx, and optimized selection of immunosuppressive therapies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Post-transplant lymphoproliferative disease (PTLD) has the highest incidence following intestinal transplantation (ITx). Our center has seen a recent increase in PTLD. Our aim was to review a single-center PTLD experience with a focus on clinical characteristics and outcomes. We completed a retrospective review of biopsy-proven PTLD cases using a prospectively maintained database of 115 ITx recipients transplanted between 1991 and 2014. Nineteen (17%) ITx recipients developed 25 PTLD cases during a median follow-up time of 6.4 (1.6-14.6) years. The incidence of early PTLD was 6% (n = 7). There was a trend toward increased risk of PTLD in children compared with adults (P = .11) and a significantly increased risk of PTLD in re-ITx compared with primary ITx recipients (P = .03). Most PTLD cases were diagnosed between 2010 and 2014 (n = 14). All early PTLD cases were EBV+ on in situ hybridization. Overall graft and patient survival are 68% and 74%, respectively. Second episodes of PTLD were diagnosed in 43% of surviving pediatric patients. Our program has a low incidence of early PTLD with overall excellent graft and patient survival following diagnosis. However, we have also seen a rising incidence of late PTLD. The cause of the increase is unknown as no major changes in immunosuppression protocols have occurred since 1999., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Few studies have examined the epidemiology and risk factors for the development of outpatient-acquired catheter-related bloodstream infections (CRBSIs) in children receiving home parenteral nutrition. This study aimed to (1) characterize the incidence, clinical presentation, and epidemiology of CRBSIs and (2) identify risk factors for CRBSIs in children receiving home parenteral nutrition., Methods: A longitudinal database approved by our Institutional Review Board was created to prospectively track CRBSIs in the UCLA pediatric population from January to December 2012. Eligible patients included those < 18 years old receiving home parenteral nutrition., Results: Thirty of 60 patients (50%) were diagnosed with 66 CRBSIs, for an overall CRBSI rate of 3.6 per 1000 catheter days. Of the CRBSIs, 73% were due to single microorganisms and 27% were polymicrobial. There was a significant difference in median (range) time for blood cultures to turn positive depending on type of CRBSIs (p = 0.03), with polymicrobial infections detected at 13.4 (8.7-24.3) hours, gram-negative infections at 16.5 (9-30.8) hours, and gram-positive infections at 18.9 (8.4-37.1) hours. The most common presenting symptom was fever (82%), followed by gastrointestinal symptoms (42%) and chills (29%). The only significant multivariate risk factor for CRBSIs was presence of a feeding tube (2.3-fold increase in CRBSI risk, p = 0.04)., Discussion: Outpatient-acquired CRBSIs are common in children receiving home parenteral nutrition. CRBSIs typically present with fever, but are also associated with gastrointestinal and/or respiratory symptoms. The presence of feeding tubes may predispose children on home parenteral nutrition to developing CRBSIs., (© 2018 American Society for Parenteral and Enteral Nutrition.)

Helicobacter pylori is an organism known to colonize the normal human stomach. Previous studies have shown that the bacterium does this by elevating its periplasmic pH via the hydrolysis of urea. However, the value of the periplasmic pH was calculated indirectly from the proton motive force equation. To measure the periplasmic pH directly in H. pylori , we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA from H. pylori and TorA from Escherichia coli The fusion proteins were expressed in the H. pylori genome under the control of the cagA promoter. Confocal microscopic and cell fractionation/immunoblotting analyses detected TorA-EGFP in the periplasm and KapA-EGFP in both the periplasm and cytoplasm, while the mature form of HydA-EGFP was seen at low levels in the periplasm, with major cytoplasmic retention of the precursor form. With H. pylori expressing TorA-EGFP, we established a system to directly measure periplasmic pH based on the pH-sensitive fluorimetry of EGFP. These measurements demonstrated that the addition of 5 mM urea has little effect on the periplasmic pH at a medium pH higher than pH 6.5 but rapidly increases the periplasmic pH to pH 6.1 at an acidic medium pH (pH 5.0), corresponding to the opening of the proton-gated channel, UreI, and confirming the basis of gastric colonization. Measurements of the periplasmic pH in an HP0244 (FlgS)-deficient mutant of H. pylori expressing TorA-EGFP revealed a significant loss of the urea-dependent increase in the periplasmic pH at an acidic medium pH, providing additional evidence that FlgS is responsible for recruitment of urease to the inner membrane in association with UreI. IMPORTANCE Helicobacter pylori has been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection with H. pylori , which, if untreated, lasts for the lifetime of an infected individual, predisposes one to gastric malignancies, such as adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A unique feature of the neutralophilic bacterium H. pylori is its ability to survive in the extremely acidic environment of the stomach through its acid acclimation mechanism. The presented results on measurements of periplasmic pH in H. pylori based on fluorimetry of fully active green fluorescent protein fusion proteins exported with the twin-arginine translocase system provide a reliable and rapid tool for the investigation of acid acclimation in H. pylori ., (Copyright © 2018 American Society for Microbiology.)

Background: The pathogen Helicobacter pylori encounters many stressors as it transits to and infects the gastric epithelium. Gastric acidity is the predominate stressor encountered by the bacterium during initial infection and establishment of persistent infection. H. pylori initiates a rapid response to acid to maintain intracellular pH and proton motive force appropriate for a neutralophile. However, acid sensing by H. pylori may also serve as a transcriptional trigger to increase the levels of other pathogenic factors needed to subvert host defenses such as acid acclimation, antioxidants, flagellar synthesis and assembly, and CagA secretion., Materials and Methods: Helicobacter pylori were acid challenged at pH 3.0, 4.5, 6.0 vs nonacidic pH for 4 hours in the presence of urea, followed by RNA-seq analysis and qPCR. Cytoplasmic pH was monitored under the same conditions., Results: About 250 genes were induced, and an equal number were repressed at acidic pHs. Genes encoding for antioxidant proteins, flagellar structural proteins, particularly class 2 genes, T4SS/Cag-PAI, F o F 1 -ATPase, and proteins involved in acid acclimation were highly expressed at acidic pH. Cytoplasmic pH decreased from 7.8 at pH out of 8.0 to 6.0 at pH out of 3.0., Conclusions: These results suggest that increasing extracellular or intracellular acidity or both are detected by the bacterium and serve as a signal to initiate increased production of protective and pathogenic factors needed to counter host defenses for persistent infection. These changes are dependent on degree of acidity and time of acid exposure, triggering a coordinated response to the environment required for colonization., (© 2018 John Wiley & Sons Ltd.)

Background: Home parenteral nutrition (PN) is a lifesaving therapy for children with intestinal failure (IF). Our aims were to describe the prevalence of micronutrient deficiencies (vitamin D, zinc, copper, iron, selenium) in a diverse population of children with IF receiving PN and to identify and characterize risk factors associated with micronutrient deficiencies, including hematologic abnormalities., Methods: Data were collected on 60 eligible patients through retrospective chart review between May 2012 and February 2015. Descriptive statistics included frequencies, medians, interquartile ranges (IQRs), and odds ratios (ORs). Statistical analyses included χ 2 , Fisher's exact, t tests, and logistic, univariate, and multivariate regressions., Results: Patients were primarily young (median age, 3.3 years; IQR, 0.7-8.4), Latino (62%), and male (56%), with short bowel syndrome (70%). Of 60 study patients, 88% had ≥1 deficiency and 90% were anemic for age. Of 51 patients who had all 5 markers checked, 59% had multiple deficiencies (defined as ≥3). Multivariate analysis shows multiple deficiencies were associated with nonwhite race (OR, 9.4; P = .012) and higher body mass index z score (OR, 2.2; P = .016). Children with severe anemia (hemoglobin <8.5 g/dL) made up 50% of the cohort. Nonwhite race (OR, 6.6; P = .037) and zinc deficiency (OR, 11; P = .003) were multivariate predictors of severe anemia., Conclusions: Micronutrient deficiency and anemia are overwhelmingly prevalent in children with IF using chronic PN. This emphasizes the importance of universal surveillance and supplementation to potentially improve quality of life and developmental outcomes. Future research should investigate how racial disparities might contribute to nutrition outcomes for children using chronic PN., (© 2017 American Society for Parenteral and Enteral Nutrition.)

One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.

Background: Rejection remains the leading cause of allograft loss, and a major barrier to improving long-term outcomes after intestinal transplantation. Our aim is to define the prevalence and investigate the role of donor-specific antibody (DSA) on intestinal graft outcomes., Methods: The study includes 109 transplants performed in 95 recipients at a single center. Patients were screened for DSA pretransplant, monitored regularly posttransplant and when clinically indicated using the single-antigen bead Luminex assay. Standard induction immunosuppression was with interleukin-2 receptor antagonists, and antithymocyte globulin in high-risk recipients. Maintenance regimens were tacrolimus-based., Results: Pretransplant DSA was detected in 12 (11%) recipients with 50% continuing to have circulating antibodies posttransplant. An additional 24 (25%) patients developed de novo DSA, and of these, 71% had persistent antibodies. Recipients with preformed DSA demonstrated elevated risks of early graft failure, whereas those with de novo DSA experienced accelerated graft loss once DSA was detected, reaching a 28% failure rate within 2 years. HLA-DQ mismatch is a significant risk factor for de novo DSA emergence, whereas the persistence of antibodies is predicted by DSA strength and specificity. Although inclusion of the liver in the intestinal allograft imparts an immunological advantage against rejection-related graft loss, this protective effect was lost among recipients with persistent DSA., Conclusions: The presence of DSA is associated with inferior graft outcomes among intestinal transplant recipients. An enhanced understanding of the mechanisms by which DSA causes allograft injury, and effective strategies targeting humoral immune reactivity are needed to improve long-term intestinal graft outcomes.

Infection of the stomach by the gastric pathogen Helicobacter pylori results in chronic active gastritis and leads to the development of gastric and duodenal ulcer disease and gastric adenocarcinoma. Eradication of H. pylori infection improves or resolves the associated pathology. Current treatments of H. pylori infection rely on acid suppression in combination with at least two antibiotics. The role of acid suppression in eradication therapy has been variously attributed to antibacterial activity of proton pump inhibitors directly or through inhibition of urease activity or increased stability and activity of antibiotics. Here we discuss the effect of acid suppression on enhanced replicative capacity of H. pylori to permit the bactericidal activity of growth-dependent antibiotics. The future of eradication therapy will rely on improvement of acid inhibition along with current antibiotics or the development of novel compounds targeting the organism's ability to survive in acid., Competing Interests: Competing interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Helicobacter pylori infects about 50 % of the world's population, causing at a minimum chronic gastritis. A subset of infected patients will ultimately develop gastric or duodenal ulcer disease, gastric adenocarcinoma, or MALT (mucosa-associated lymphoid tissue) lymphoma. Eradication of H. pylori requires complex regimens that include acid suppression and multiple antibiotics. The efficacy of treatment using what were once considered standard regimens have declined in recent years, mainly due to widespread development of antibiotic resistance. Addition of bismuth to standard triple therapy regimens, use of alternate antibiotics, or development of alternative regimens using known therapies in novel combinations have improved treatment efficacy in specific populations, but overall success of eradication remains less than ideal. Novel regimens under investigation either in vivo or in vitro, involving increased acid suppression ideally with fewer antibiotics or development of non-antibiotic treatment targets, show promise for future therapy.

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1-β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1-β1 interactions, suggesting that the ratio between FXYD5 and α1-β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion., (© 2016. Published by The Company of Biologists Ltd.)

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Background: The pH-sensitive Helicobacter pylori ArsRS two-component system (TCS) aids survival of this neutralophile in the gastric environment by directly sensing and responding to environmental acidity. ArsS is required for acid-induced trafficking of urease and its accessory proteins to the inner membrane, allowing rapid, urea-dependent cytoplasmic and periplasmic buffering. Expression of ArsR, but not its phosphorylation, is essential for bacterial viability. The aim of this study was to characterize the roles of ArsS and ArsR in the response of H. pylori to acid., Materials and Methods: Wild-type H. pylori and an arsR(D52N) phosphorylation-deficient strain were incubated at acidic or neutral pH. Gene and protein expression, survival, membrane trafficking of urease proteins, urease activity, and internal pH were studied., Results: Phosphorylation of ArsR is not required for acid survival. ArsS-driven trafficking of urease proteins to the membrane in acid, required for recovery of internal pH, is independent of ArsR phosphorylation. ArsR phosphorylation increases expression of the urease gene cluster, and the loss of negative feedback in a phosphorylation-deficient mutant leads to an increase in total urease activity., Conclusions: ArsRS has a dual function in acid acclimation: regulation of urease trafficking to UreI at the cytoplasmic membrane, driven by ArsS, and regulation of urease gene cluster expression, driven by phosphorylation of ArsR. ArsS and ArsR work through phosphorylation-dependent and phosphorylation-independent regulatory mechanisms to impact acid acclimation and allow gastric colonization. Furthering understanding of the intricacies of acid acclimation will impact the future development of targeted, nonantibiotic treatment regimens., (© 2015 John Wiley & Sons Ltd.)

Objectives: Biopsies remain the criterion standard in the diagnosis of intestinal transplant (ITx) rejection, and gastrointestinal endoscopy plays a pivotal role in patient management. Herein, we describe a single-center 23-year endoscopic experience in pediatric ITx recipients., Methods: A retrospective review of endoscopy and pathology reports of all ITx recipients <18 years old transplanted between 1991 and 2013 was performed with the aim of describing the procedural indications, findings, and complications., Results: A total of 1770 endoscopic procedures within 1014 sessions were performed. A combination of esophagogastroduodenoscopy and ileoscopy was the most common procedure (36%). Increased stool output (35%) and surveillance endoscopy (32%) were the most common indications. A total of 162 episodes of biopsy-proven rejection were diagnosed. The first episode of rejection occurred at a median of 1 month after ITx. Of histology-proven rejections, 45% had normal-appearing endoscopies. The rate of procedural complications, including but not limited to bleeding and perforation, was 1.8%., Conclusions: Endoscopy with biopsy plays a significant role in the care of ITx recipients. Multiple procedures are required for graft surveillance, diagnosis of rejection, subsequent treatment, and follow-up of therapy. The gross endoscopic appearance, particularly in mild to moderate acute cellular rejection, does not correlate well with histology. Complex anatomy, complication rates that are higher than patients with non-ITx pediatric endoscopy, and timely histologic interpretation by experienced pathologists are reasons that these procedures should be performed at centers accustomed to caring for ITx recipients. The field would benefit from the development of a noninvasive biomarker to reliably and efficiently detect rejection.

Background: The gastric H(+) ,K(+) -ATPase is the preferred target for acid suppression. Until recently, the only drugs that effectively inhibited this ATPase were the proton pump inhibitors (PPIs). PPIs are acid-activated prodrugs that require acid protection. Once acid-activated, PPIs bind to cysteines of the ATPase, resulting in covalent, long-lasting inhibition. The short plasma half-life of PPIs and continual de novo synthesis of the H(+) ,K(+) -ATPase result in difficulty controlling night-time acid secretion. A new alternative to PPIs is the pyrrolo-pyridine, vonoprazan (TAK-438), a potassium-competitive acid blocker (PCAB) that does not require acid protection. In contrast to other PCABs, vonoprazan has a long duration of action, resulting in 24-h control of acid secretion, a high pKa of 9.37 and high affinity (Ki = 3.0 ηmol/L)., Aim: To determine binding selectivity of vonoprazan for the gastric H(+) ,K(+) -ATPase and to explain its slow dissociation., Methods: Gastric gland and parietal cell binding of vonoprazan was determined radiometrically. Molecular modelling explained the slow dissociation of vonoprazan from the H(+) ,K(+) -ATPase., Results: Vonoprazan binds selectively to the parietal cell, independent of acid secretion. Vonoprazan binds in a luminal vestibule between the surfaces of membrane helices 4, 5 and 6. Exit of the drug to the lumen is hindered by asp137 and asn138 in the loop between TM1 and TM2, which presents an electrostatic barrier to movement of the sulfonyl group of vonoprazan. This may explain its slow dissociation from the H(+) ,K(+) -ATPase and long-lasting inhibition., Conclusion: The binding model provides a template for design of novel potassium-competitive acid blockers., (© 2015 John Wiley & Sons Ltd.)

Background: Successful eradication of Helicobacter pylori is becoming more difficult, mainly due to emerging antibiotic resistance. Treatment regimens containing bismuth have increased efficacy, but the mechanism is unknown. Helicobacter pylori is a neutralophile adapted to survive the acidic gastric environment via acid acclimation, but demonstrates more robust growth at neutral pH. Many antibiotics used to treat H. pylori rely on bacterial growth., Aim: To investigate the mechanism of increased efficacy of bismuth-containing H. pylori treatment regimens., Methods: RNAseq and qPCR, urease activity in permeabilised and intact bacteria, internal pH and membrane potential were measured with and without colloidal bismuth subcitrate (CBS). Bacterial survival was assessed with CBS and/or ampicillin., Results: Genes involved with metabolism and growth were upregulated in the presence of CBS at acidic pH. Urease activity of permeabilised H. pylori at pH 7.4 and 4.5 decreased in the presence of CBS, but intact urease activity decreased only at acidic pH. The fall in cytoplasmic pH with external acidification was diminished by CBS. The increase in membrane potential in response to urea addition at acidic medium pH was unaffected by CBS. The impact of CBS and ampicillin on H. pylori survival was greater than either agent alone., Conclusions: Bismuth is not acting directly on urease or the urea channel. Colloidal bismuth subcitrate impedes proton entry into the bacteria, leading to a decrease in the expected fall in cytoplasmic pH. With cytoplasmic pH remaining within range for increased metabolic activity of a neutralophile, the efficacy of growth-dependent antibiotics is augmented., (© 2015 John Wiley & Sons Ltd.)

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Gastric infection by Helicobacter pylori is the most common cause of ulcer disease and gastric cancer. The mechanism of progression from gastritis and inflammation to ulcers and cancer in a fraction of those infected is not definitively known. Significant acidity is unique to the gastric environment and is required for ulcer development. The interplay between gastric acidity and H. pylori pathogenesis is important in progression to advanced disease. The aim of this study was to characterize the impact of acid on gastric epithelial integrity and cytokine release and how H. pylori infection alters these responses. Human gastric epithelial (HGE-20) cells were grown on porous inserts, and survival, barrier function, and cytokine release were studied at various apical pH levels in the presence and absence of H. pylori. With apical acidity, gastric epithelial cells demonstrate increased barrier function, as evidenced by increased transepithelial electrical resistance (TEER) and decreased paracellular permeability. This effect is reduced in the presence of wild-type, but not urease knockout, H. pylori. The epithelial inflammatory response is also modulated by acidity and H. pylori infection. Without H. pylori, epithelial IL-8 release decreases in acid, while IL-6 release increases. In the presence of H. pylori, acidic pH diminishes the magnitude of the previously reported increase in IL-8 and IL-6 release. H. pylori interferes with the gastric epithelial response to acid, contributing to altered barrier function and inflammatory response. H. pylori diminishes acid-induced tightening of cell junctions in a urease-dependent manner, suggesting that local pH elevation promotes barrier compromise and progression to mucosal damage.

Background: Helicobacter pylori, a neutralophile, colonizes the acidic environment of the human stomach by employing acid acclimation mechanisms that regulate periplasmic and cytoplasmic pH. The regulation of urease activity is central to acid acclimation. Inactive urease apoenzyme, UreA/B, requires nickel for activation. Accessory proteins UreE, F, G, and H are required for nickel insertion into apoenzyme. The ExbB/ExbD/TonB complex transfers energy from the inner to outer membrane, providing the driving force for nickel uptake. Therefore, the aim of this study was to determine the contribution of ExbD to pH homeostasis., Materials and Methods: A nonpolar exbD knockout was constructed and survival, growth, urease activity, and membrane potential were determined in comparison with wildtype., Results: Survival of the ΔexbD strain was significantly reduced at pH 3.0. Urease activity as a function of pH and UreI activation was similar to the wildtype strain, showing normal function of the proton-gated urea channel, UreI. The increase in total urease activity over time in acid seen in the wildtype strain was abolished in the ΔexbD strain, but recovered in the presence of supraphysiologic nickel concentrations, demonstrating that the effect of the ΔexbD mutant is due to loss of a necessary constant supply of nickel. In acid, ΔexbD also decreased its ability to maintain membrane potential and periplasmic buffering in the presence of urea., Conclusions: ExbD is essential for maintenance of periplasmic buffering and membrane potential by transferring energy required for nickel uptake, making it a potential nonantibiotic target for H. pylori eradication., (© 2013 John Wiley & Sons Ltd.)

Background: Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens., Aim: To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin., Methods: H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined., Results: At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0., Conclusions: Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication., (© 2012 Blackwell Publishing Ltd.)

Helicobacter pylori survives and grows at low pHs via acid acclimation mechanisms that enable periplasmic pH homeostasis. Important components include a cytoplasmic urease; a pH-gated urea channel, UreI; and periplasmic α-carbonic anhydrase. To allow the rapid adjustment of periplasmic pH, acid acclimation components are recruited to the inner membrane in acid. The ArsRS two-component system, in an acid-responsive manner, controls the transcription of the urease gene cluster and α-carbonic anhydrase. The aim of this study is to determine the role of ArsS in protein trafficking as a component of acid acclimation. H. pylori wild-type and ΔarsS bacteria were incubated at acidic and neutral pHs. Intact bacteria, purified membranes, and total protein were analyzed by Western blotting and urease activity measurements. The total urease activity level was decreased in the ΔarsS strain, but the acid activation of UreI was unaffected. A 30-min acid exposure increased the level and activity of urease proteins at the membrane in the wild type but not in the ΔarsS strain. The urease levels and activity of the ΔarsS strain after a 90-min acid exposure were similar to those of the wild type. ArsS, in addition to its role in urease gene transcription, is also involved in the recruitment of urease proteins to the inner membrane to augment acid acclimation during acute acid exposure. Urease membrane recruitment following prolonged acid exposure in the absence of ArsS was similar to that of the wild type, suggesting a compensatory mechanism, possibly regulated by FlgS, underscoring the importance of urease membrane recruitment and activation in periplasmic pH homeostasis.