Jayleen Grams

Editing Machines: The Complexities of Trypanosome RNA Editing

The assembly and disassembly of ribonucleoprotein complexes containing substrate precursor mRNAs and guide RNAs is crucial to the initiation and propagation of RNA editing. We discuss here the composition of these complexes and how their assembly may regulate RNA editing.

Processing of polycistronic guide RNAs is associated with RNA editing complexes in Trypanosoma brucei

In kinetoplastid mitochondrial mRNA editing, post‐transcriptional insertion or deletion of uridines is templated by guide RNAs (gRNAs). Pre‐mRNAs are encoded by maxicircles, while gRNAs are encoded by both maxicircles and minicircles. We have investigated minicircle transcription and the processing of gRNAs in Trypanosoma brucei. We find that minicircles are transcribed polycistronically and that transcripts are accurately processed by an ∼19S complex. This gRNA processing activity co‐purifies with RNA editing complexes, and both remain associated in 19S complexes. Furthermore, we show that RNA editing complexes associate preferentially with a polycistronic gRNA over non‐processed RNAs. We propose that the ∼19S complexes initially described as RNA editing complex I are gRNA processing complexes that cleave polycistronic gRNA transcripts into monocistrons.

Association between Surgical Care Improvement Program venous thromboembolism measures and postoperative events

BACKGROUND In 2006, the Surgical Care Improvement Program (SCIP) implemented measures to reduce venous thromboembolism (VTE). There are little data on whether these measures reduce VTE rates. This study proposed to examine associations between SCIP-VTE adherence and VTE rates. METHODS SCIP-VTE adherence for 30,531 surgeries from 2006 to 2009 was linked with VA Surgical Quality Improvement Program data. Patient demographics, comorbidities, and surgical characteristics associated with VTE were summarized. VTE rates were compared by SCIP-VTE adherence. Multivariable logistic regression was used to model VTE by adherence, adjusting for multiple associated factors. RESULTS Of 30,531 surgeries, 89.9% adhered to SCIP-VTE; 1.4% experienced VTE. Logistic regression identified obesity, smoking, functional status, weight loss, emergent status, age older than 64 years, and surgical time as associated with VTE. SCIP-VTE was not associated with VTE (1.4% vs 1.33%; P = .3), even after adjustment. CONCLUSIONS This study identified several important risk factors for VTE but found no association with SCIP-VTE adherence.

Outcome of adrenocortical tumors in children

PURPOSE This study reviews adrenocortical tumors in children to determine factors that significantly affect outcome. METHODS An institutional review board-approved retrospective review from 1976 to 2005 identified 23 patients younger than 19 years old with histologic confirmation of adrenocortical carcinoma (ACC) and adenomas. RESULTS The mean age of the 23 children was 9.0 +/- 1.6 years; girls predominated (female-to-male ratio = 1.9:1) as did cancers (ACC 16, adenoma 7); tumor hormone production (74%); and advanced stage for disease (66%). All malignancies were more than 2.5 cm. Adrenalectomy, including en bloc resection of adjacent structures (35%) achieved grossly negative margins in 70% of patients. Three patients received chemotherapy or chemoradiation as primary treatment without surgery. There was no perioperative mortality; morbidity was 10% (pneumothorax, acute renal failure, chylous ascites, and thrombocytosis). Surgical cure without adjuvant therapy was achieved for all adenomas and ACC stages I and II. For ACC stage III and IV, median survival was 21 months, 5-year survival was 0%. All advanced-staged ACC received adjuvant therapy. Surgically negative margins conferred a survival advantage. CONCLUSIONS Children, especially females with ACC present with large advanced-staged tumors. Surgically negative margins with or without en bloc resection improves survival. The high percentage of children with functioning tumors suggests earlier detection is possible.

Carboxylated and uncarboxylated forms of osteocalcin directly modulate the glucose transport system and inflammation in adipocytes.

Osteocalcin is secreted by osteoblasts and improves insulin sensitivity in vivo, although mechanisms remain unclear. We tested the hypothesis that osteocalcin directly modulates cell biology in insulin-targeted peripheral tissues. In L-6 myocytes, osteocalcin stimulated glucose transport both in the absence (basal) and presence of insulin. Similarly, in primary cultured adipocytes, both carboxylated and uncarboxylated osteocalcin increased basal and insulin-stimulated glucose transport as well as insulin sensitivity. Osteocalcin also increased basal and insulin-stimulated glucose oxidation, though there was no effect on fatty acid synthesis or lipolysis. In primary-cultured adipocytes, both forms of osteocalcin suppressed secretion of tumor necrosis factor alpha into the media; however, only carboxylated osteocalcin suppressed interleukin 6 release, and neither form of osteocalcin modulated monocyte chemoattractant protein-1 secretion. Both carboxylated and uncarboxylated osteocalcin increased secretion of adiponectin and the anti-inflammatory cytokine interleukin 10. In conclusion, both carboxylated and uncarboxylated osteocalcin directly increase glucose transport in adipocytes and muscle cells, while suppressing proinflammatory cytokine secretion and stimulating interleukin 10 and adiponectin release. Thus, these results provide a mechanism for the insulin-sensitizing effects of osteocalcin and help elucidate the role that bone plays in regulating systemic metabolism.

Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages.

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.

Minimally invasive Heller's myotomy in children: safe and effective

PURPOSE The aim of the study was to review a single institution experience of minimally invasive Hellers myotomy in pediatric patients with achalasia. METHODS An institutional review board-approved retrospective review from 1999 to 2005 identified patients 18 years old and younger who underwent a minimally invasive Hellers myotomy for achalasia. RESULTS Twenty-six patients were identified with a mean age of 15 (range, 4-18 years). There were 11 female and 15 male patients. There were 3 intraoperative complications (2 esophageal mucosal injuries and 1 aspiration). There was no mortality. All 26 surgeries were completed laparoscopically. Two patients had Dor fundoplication, whereas 23 patients had Toupet fundoplication. Average length of hospital stay was 2.7 days (range, 1-4 days) excluding the 3 patients with intraoperative complications and 3.5 days for all patients (range, 1-17 days). Postoperative follow-up ranged from 0 to 75 months (mean, 20 months). Postoperatively, one patient developed reflux symptoms (incidence 4%). Seven patients (27%) had recurrence of symptoms at a mean of 13 months (range, 1-66 months) after their operation. CONCLUSIONS Laparoscopic Hellers myotomy with fundoplication is a safe and effective treatment of symptomatic achalasia in the pediatric population. Complications were low in this group of patients and comparable to other published reports in the literature.

Stromal Regulation of Human Gastric Dendritic Cells Restricts the Th1 Response to Helicobacter pylori

BACKGROUND & AIMS Mucosal dendritic cells (DCs) play a key role in initiating the T-helper (Th)1 response to Helicobacter pylori. To further elucidate the mucosal response to H pylori, we examined whether gastric stromal factors condition DCs to support tolerance to H pylori, analogous to intestinal stromal factor-driven macrophage tolerance to commensal bacteria. METHODS To model mucosal DC development, we isolated and cultured cell-depleted human stroma/extracellular matrix from fresh gastric and intestinal mucosa to generate stroma-conditioned media. We then analyzed the capacity of stroma-conditioned media-treated monocyte-derived DCs and primary human gastric and intestinal DCs pulsed in vitro with H pylori to induce T-cell proliferation and interferon gamma secretion. RESULTS Stromal factors in gastric mucosa suppressed H pylori-stimulated DC activation and the ability of DCs to drive a Th1 proliferative and cytokine response to H pylori. The ability of gastric stromal factors to down-regulate DC function was similar to that of intestinal stromal factors and was independent of transforming growth factor β, prostaglandin E₂, interleukin (IL)-10, and thymic stromal lymphopoietin. Stroma-conditioned media-induced reduction in DC-stimulated Th1 responses was associated with reduced DC release of IL-12. CONCLUSIONS Gastric stromal factors down-regulate DC responsiveness to H pylori, resulting in a dampened gastric Th1 response. We speculate that stroma-induced down-regulation of DC function contributes to the permissiveness of both gastric and intestinal mucosa to colonization by persistent residential microbes.

Human gastric epithelial cells contribute to gastric immune regulation by providing retinoic acid to dendritic cells

Despite the high prevalence of chronic gastritis caused by Helicobacter pylori, the gastric mucosa has received little investigative attention as a unique immune environment. Here, we analyzed whether retinoic acid (RA), an important homeostatic factor in the small intestinal mucosa, also contributes to gastric immune regulation. We report that human gastric tissue contains high levels of the RA precursor molecule retinol (ROL), and that gastric epithelial cells express both RA biosynthesis genes and RA response genes, indicative of active RA biosynthesis. Moreover, primary gastric epithelial cells cultured in the presence of ROL synthesized RA in vitro and induced RA biosynthesis in co-cultured monocytes through an RA-dependent mechanism, suggesting that gastric epithelial cells may also confer the ability to generate RA on gastric dendritic cells (DCs). Indeed, DCs purified from gastric mucosa had similar levels of aldehyde dehydrogenase activity and RA biosynthesis gene expression as small intestinal DCs, although gastric DCs lacked CD103. In H. pylori-infected gastric mucosa, gastric RA biosynthesis gene expression was severely disrupted, which may lead to reduced RA signaling and thus contribute to disease progression. Collectively, our results support a critical role for RA in human gastric immune regulation.

Helicobacter pylori Infection Inhibits Phagocyte Clearance of Apoptotic Gastric Epithelial Cells

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR+ mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori–induced macrophage TNF-α, which was expressed at higher levels in H. pylori–infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori–infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.