Louise M. Judd

Impaired Renal NaCl Absorption in Mice Lacking the ROMK Potassium Channel, a Model for Type II Bartter's Syndrome

ROMK is an apical K+channel expressed in the thick ascending limb of Henle (TALH) and throughout the distal nephron of the kidney. Null mutations in theROMK gene cause type II Bartters syndrome, in which abnormalities of electrolyte, acid-base, and fluid-volume homeostasis occur because of defective NaCl reabsorption in the TALH. To understand better the pathogenesis of type II Bartters syndrome, we developed a mouse lacking ROMK and examined its phenotype. Young null mutants had hydronephrosis, were severely dehydrated, and ∼95% died before 3 weeks of age. ROMK-deficient mice that survived beyond weaning grew to adulthood; however, they had metabolic acidosis, elevated blood concentrations of Na+ and Cl−, reduced blood pressure, polydipsia, polyuria, and poor urinary concentrating ability. Whole kidney glomerular filtration rate was sharply reduced, apparently as a result of hydronephrosis, and fractional excretion of electrolytes was elevated. Micropuncture analysis revealed that the single nephron glomerular filtration rate was relatively normal, absorption of NaCl in the TALH was reduced but not eliminated, and tubuloglomerular feedback was severely impaired. These data show that the loss of ROMK in the mouse causes perturbations of electrolyte, acid-base, and fluid-volume homeostasis, reduced absorption of NaCl in the TALH, and impaired tubuloglomerular feedback.

Gastric H+, K+-adenosine triphosphatase β subunit is required for normal function, development, and membrane structure of mouse parietal cells

BACKGROUND & AIMS Parietal cells of the gastric mucosa contain a complex and extensive secretory membrane system that harbors gastric H(+),K(+)-adenosine triphosphatase (ATPase), the enzyme primarily responsible for acidification of the gastric lumen. We have produced mice deficient in the H(+),K(+)-ATPase beta subunit to determine the role of the protein in the biosynthesis of this membrane system and the biology of gastric mucosa. METHODS Mice deficient in the H(+), K(+)-ATPase beta subunit were produced by gene targeting. RESULTS The stomachs of H(+),K(+)-ATPase beta subunit-deficient mice were achlorhydric. Histological and immunocytochemical analyses with antibodies to the H(+),K(+)-ATPase alpha subunit revealed that parietal cell development during ontogeny was retarded in H(+), K(+)-ATPase beta subunit-deficient mice. In 15-day-old mice, cells with secretory canaliculi were observed in wild-type but not in H(+), K(+)-ATPase beta subunit-deficient mice. Parietal cells of H(+), K(+)-ATPase beta subunit-deficient mice 17 days and older contained an abnormal canaliculus that was dilated and contained fewer and shorter microvilli than normal. In older parietal cells, the abnormal canaliculus was massive (25 micrometer in diameter) and contained few microvilli. We did not observe typical tubulovesicular membranes in any parietal cell from H(+),K(+)-ATPase beta subunit-deficient mice. Histopathologic alterations were only observed in the stomach. CONCLUSIONS The H(+),K(+)-ATPase beta subunit is required for acid-secretory activity of parietal cells in vivo, normal development and cellular homeostasis of the gastric mucosa, and attainment of the normal structure of the secretory membranes.

Mice with a Targeted Disruption of the AE2 Exchanger Are Achlorhydric

The AE2 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2-/-) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2-/- mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2-/- mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2-/- gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads

The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate “hybrid” assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

Augmented gp130-mediated cytokine signalling accompanies human gastric cancer progression

H. pylori infection accounts for most cases of gastric cancer, but the initiating events remain unclear. The principal H. pylori pathogenicity‐associated CagA protein disrupts intracellular SHP‐2 signalling pathways including those used by the IL‐6 family cytokines, IL‐6 and IL‐11. Imbalanced IL‐6 family cytokine signalling in the gp130757FF mouse model of gastric cancer arising from hyperactivation of oncogenic STAT3 after altered SHP‐2 : ERK1/2 signalling produces dysplastic antral tumours preceded by gastritis and metaplasia. In a cohort of patient gastric biopsies with known H. pylori and CagA status, we investigated whether (i) STAT3 and ERK1/2 activation is altered in H. pylori‐dependent gastritis; (ii) these profiles are more pronounced in CagA+ H. pylori infection; and (iii) the expression of pro‐inflammatory cytokines that activate STAT3 and ERK 1/2 pathways is associated with progression to gastric cancer. IL‐6, IL‐11, and activated STAT3 and ERK1/2 were quantified in antral biopsies from gastritic stomach, metaplastic tissue, and resected gastric cancer tissues. We observed significantly increased STAT3 and ERK1/2 activation (p = 0.001) in H. pylori‐dependent gastritis, which was further enhanced in the presence of CagA+ H. pylori strains. Of known gastric ligands that drive STAT3 activation, IL‐6 expression was increased after H. pylori infection and both IL‐6 and IL‐11 were strongly up‐regulated in the gastric cancer biopsies. This suggests a mechanism by which IL‐11 drives STAT3 activation and proliferation during gastric cancer progression. We addressed this using an in vitro approach, demonstrating that recombinant human IL‐11 activates STAT3 and concomitantly increases proliferation of MKN28 gastric epithelial cells. In summary, we show increased STAT3 and ERK1/2 activation in H. pylori‐dependent gastritis that is likely driven in an IL‐6‐dependent fashion. IL‐11 expression is associated with adenocarcinoma development, but not gastritic lesions, and we identify a novel mechanism for IL‐11 as a potent inducer of proliferation in the human gastric cancer setting. Copyright

Inactivation of IL11 Signaling Causes Craniosynostosis, Delayed Tooth Eruption, and Supernumerary Teeth

Craniosynostosis and supernumerary teeth most often occur as isolated developmental anomalies, but they are also separately manifested in several malformation syndromes. Here, we describe a human syndrome featuring craniosynostosis, maxillary hypoplasia, delayed tooth eruption, and supernumerary teeth. We performed homozygosity mapping in three unrelated consanguineous Pakistani families and localized the syndrome to a region in chromosome 9. Mutational analysis of candidate genes in the region revealed that all affected children harbored homozygous missense mutations (c.662C>G [p.Pro221Arg], c.734C>G [p.Ser245Cys], or c.886C>T [p.Arg296Trp]) in IL11RA (encoding interleukin 11 receptor, alpha) on chromosome 9p13.3. In addition, a homozygous nonsense mutation, c.475C>T (p.Gln159X), and a homozygous duplication, c.916_924dup (p.Thr306_Ser308dup), were observed in two north European families. In cell-transfection experiments, the p.Arg296Trp mutation rendered the receptor unable to mediate the IL11 signal, indicating that the mutation causes loss of IL11RA function. We also observed disturbed cranial growth and suture activity in the Il11ra null mutant mice, in which reduced size and remodeling of limb bones has been previously described. We conclude that IL11 signaling is essential for the normal development of craniofacial bones and teeth and that its function is to restrict suture fusion and tooth number. The results open up the possibility of modulation of IL11 signaling for the treatment of craniosynostosis.

The Interleukin-6 Family Cytokine Interleukin-11 Regulates Homeostatic Epithelial Cell Turnover and Promotes Gastric Tumor Development

BACKGROUND & AIMS Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. METHODS We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. RESULTS IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor alpha significantly reduced HKbeta-/- mouse fundic hyperplasia and ablated gp130(757F/F) mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-beta, RegIII-gamma, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130(757F/F) and HKbeta-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. CONCLUSIONS Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.

Autoimmune gastritis results in disruption of gastric epithelial cell development

We have investigated the underlying basis of the lesion in murine autoimmune gastritis, a model of the human disease pernicious anemia. The disease is mediated by T lymphocytes and characterized by selective depletion of parietal and zymogenic cells from the gastric unit (gland) together with gastric epithelial cell hyperplasia. The gastric units of gastritic stomachs contained 2.3-fold more cells than normal and accumulated rapidly dividing, short-lived gastric epithelial stem cells and mucous neck cells. Most of these immature cells failed to differentiate into end-stage cells but rather appeared to die by apoptosis. We also found no correlation between anti-parietal cell autoantibody titers and the degree of gastric pathology, providing further evidence that autoantibodies do not play a direct role in the pathogenesis of gastritis. Taken together, the normal developmental pathways of the gastric mucosa are disrupted in autoimmune gastritis, resulting in an amplification of immature cell types. The differentiation of these immature cells appears to be blocked, contributing to depletion of end-stage cells. This scenario provides an explanation for depletion of not only parietal cells but also zymogenic cells even though they are not directly targeted by the immune system.We have investigated the underlying basis of the lesion in murine autoimmune gastritis, a model of the human disease pernicious anemia. The disease is mediated by T lymphocytes and characterized by selective depletion of parietal and zymogenic cells from the gastric unit (gland) together with gastric epithelial cell hyperplasia. The gastric units of gastritic stomachs contained 2.3-fold more cells than normal and accumulated rapidly dividing, short-lived gastric epithelial stem cells and mucous neck cells. Most of these immature cells failed to differentiate into end-stage cells but rather appeared to die by apoptosis. We also found no correlation between anti-parietal cell autoantibody titers and the degree of gastric pathology, providing further evidence that autoantibodies do not play a direct role in the pathogenesis of gastritis. Taken together, the normal developmental pathways of the gastric mucosa are disrupted in autoimmune gastritis, resulting in an amplification of immature cell types. The differentiation of these immature cells appears to be blocked, contributing to depletion of end-stage cells. This scenario provides an explanation for depletion of not only parietal cells but also zymogenic cells even though they are not directly targeted by the immune system.

Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(–/–)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors

Background:  Gastric neoplasia is common in humans, yet controversy remains over contributions of chronic achlorhydria, gastrinemia and hyperplasia, to cancer risk. To study this, mice lacking the gastric H/K‐ATPase (Atp4a(–/–) mice) were used to determine whether chronic loss of acid secretion, with attendant hypergastrinemia, predisposes to cancer phenotype.