Macrophage secretion of miR-106b-5p causes renin-dependent hypertension.

Myeloid cells are known mediators of hypertension, but their role in initiating renin-induced hypertension has not been studied. Vitamin D deficiency causes pro-inflammatory macrophage infiltration in metabolic tissues and is linked to renin-mediated hypertension. We tested the hypothesis that impaired vitamin D signaling in macrophages causes hypertension using conditional knockout of the myeloid vitamin D receptor in mice (KODMAC). These mice develop renin-dependent hypertension due to macrophage infiltration of the vasculature and direct activation of renal juxtaglomerular (JG) cell renin production. Induction of endoplasmic reticulum stress in knockout macrophages increases miR-106b-5p secretion, which stimulates JG cell renin production via repression of transcription factors E2f1 and Pde3b. Moreover, in wild-type recipient mice of KODMAC/miR106b-/- bone marrow, knockout of miR-106b-5p prevents the hypertension and JG cell renin production induced by KODMAC macrophages, suggesting myeloid-specific, miR-106b-5p-dependent effects. These findings confirm macrophage miR-106b-5p secretion from impaired vitamin D receptor signaling causes inflammation-induced hypertension.

Production of ectopic gastric intrinsic factor in gastric mucosa of humans with chronic gastritis.

BACKGROUND: Ectopic expression of gastric intrinsic factor (IF) has been described in rodent models of chronic gastritis. AIMS: The current study undertook to determine if ectopic IF was also present in chronic gastritis in humans and might identify the process of ectopic protein expression as part of the response to chronic injury. METHODS: Archived biopsies from mid-body, angularis and prepylorus of 9 patients with and without chronic gastritis and food-cobalamin malabsorption were examined in a blinded fashion by immunocytochemistry as were biopsies from 5 normal subjects. Cells with ectopic IF were further examined with antibodies against pepsin or with Griffonia simplicifolia II (GSII) to identity cells in the mucous neck cell compartment. RESULTS: Ectopic IF production in non-parietal cells was identified in cells that were H(+),K(+)-ATPase-negative but IF-positive in 7 of the 9 patients (6/9 in the angularis and/or prepylorus biopsies and 1/9 only in the mid-body). These included 5 of the 6 H. pylori-infected patients and all 5 patients with severe food-cobalamin malabsorption. No normal control subjects demonstrated ectopic IF. The cells with ectopic IF were pepsinogen-positive peptic cells and were not GSII-positive. Expression was most extensive in patients and gastric regions with inflammation. In all but one sample, ectopic IF was observed near anatomical mucosal junctions, such as antral/body and prepylorus/duodenum junctions. CONCLUSIONS: These data in humans with and without gastritis are consistent with the hypothesis that local factors influence ectopic gastric IF expression, arising from either the anatomical location, the focal inflammation, or both.

Identification of genetic diversity of hepatitis E virus (HEV) and determination of the seroprevalence of HEV in eastern China.

Hepatitis E, caused by the hepatitis E virus (HEV), is endemic in China. However, the molecular characteristics of HEV circulating in eastern China and the seroprevalence of HEV infection in eastern China are relatively unknown. In this study, 25 HEV strains, isolated from sporadic hepatitis E cases in eastern China, were sequenced in the RNA-dependent RNA polymerase region and subjected to phylogenetic analysis. These HEV strains were 74.6-98.7% identical in nucleotides and were all clustered into HEV genotype 4. Most of them formed new sub-genotypes and revealed a high degree of genetic variance. In addition, 12,052 serum samples were collected from people of different ages, living in urban or rural areas in eastern China. Anti-HEV IgG activity was detected in 2073 (17.20%). The prevalence of anti-HEV IgG significantly increased with age (P<0.0001), ranging from 7.92% in children (<10 years old) to 21.48% among older persons (>or=60 years old). Moreover, statistical analysis showed that there was a significant difference between rural and urban areas, with higher prevalence for people living in rural neighborhoods (P<0.001).

Production of gastric intrinsic factor, transcobalamin, and haptocorrin in opossum kidney cells.

Opossum kidney epithelial cells were shown previously to synthesize and secrete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis that renal tubular cells also produce intrinsic factor (IF), and this production provides an explanation for the presence of IF in urine. By using antisera raised against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein was blocked by recombinant human IF. When proteins secreted into the media were separated electrophoretically under nondenaturing conditions after binding with [(57)Co]Cbl, a broad major band migrated at a relative front independently of recombinant IF or TCII, and probably represents Hc, as the Cbl binding is blocked by cobinamide. Small amounts of bound [(57)Co]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The presence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming the ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragment of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western blot confirmed the presence of IF protein. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other epithelia.

Effect of tissue non-specific alkaline phosphatase in maintenance of structure of murine colon and stomach.

The gastrointestinal tract of mammals secretes a phospholipid-rich membrane that is enriched in alkaline phosphatase (AP) and surfactant proteins (surfactant-like particle, SLP). The production of this particle is stimulated in the small intestine by fat feeding and in cultured cells in vitro by transfection with intestinal alkaline phosphatase (IAP). To test whether tissue non-specific alkaline phosphatase (TNAP) was a factor in stimulating surfactant-like particle production in stomach and colon (tissues expressing TNAP), mice lacking this enzyme were studied. Mice were harvested at 8 days of life, when body weight of homozygous animals (TNAP -/-) was about half that of congenic controls (TNAP +/+) or heterozygotes (TNAP +/-), but before seizures had begun. No difference in content of the major SLP protein (65 kDa) by Western blotting or immunocytochemistry was seen in stomach or colon of TNAP -/- vs. TNAP +/+ animals, but the content was only about half in the IAP-expressing small bowel. Transmission electron microscopy of the TNAP -/- small bowel showed large dilated lysosomes and residual bodies. Colonocytes and gastric surface epithelial cells from the same animals showed mitochondria containing homogeneous dense inclusions, consistent with neutral lipid. In the underweight homozygous animals, there was a decrease in the neuronal content of submucosal ganglia in the jejunum and ileum and of myenteric ganglia in the jejunum of TNAP -/- animals. These findings suggest that (1) TNAP is not important in maintaining surfactant-like particle content of tissues that express TNAP, (2) normal fat absorption is important in maintaining SLP content in the small intestine, and (3) TNAP is important in the maintenance of some intestinal structures, and perhaps their function.

Rat intestinal alpha1-antitrypsin secretion is regulated by triacylglycerol feeding.

alpha1-Antitrypsin (AAT) is secreted by the enterocyte, but its regulation of expression, intramucosal distribution, and functional status are unclear. After corn oil gavage (plus Pluronic L-81 to block chylomicron release), rat intestine was examined for mRNA encoding AAT, immunoreactivity by light and electron microscopy, and protein content by Western blot. Species-specific antisera used were raised against both AAT and surfactant-like particle (SLP), a membrane secreted by the enterocyte in response to fat feeding. Purified luminal SLP was fractionated by Bio-Gel P-200 chromatography to assess its interaction with AAT. Triacylglycerol feeding maximally increased mucosal mRNA-encoding AAT and AAT intracellular protein content by 3 and 5 h, respectively. Immunocytochemistry revealed predominance of AAT in basolateral spaces around enterocytes and Pluronic-blocked extracellular accumulation of AAT, patterns nearly identical to those of secreted SLP. About 10% of AAT was reversibly associated with SLP. Luminal AAT was smaller (51 kDa) than mature AAT (55 kDa) and did not form a complex with pancreatic elastase. When the common bile duct was tied, excluding pancreatic proteases from the lumen, mature AAT that was cleaved by pancreatic elastase was secreted. The luminal secretion of AAT and its reversible association with SLP suggest an intracellular association and a possible role for AAT during lipid digestion and absorption.

Sulphydryl blocker-induced rat colitis is ameliorated by intravenous injection of antibody to colonic surfactant-like particle.

A rat model of experimental colitis and jejunitis induced by iodoacetamide (IA), a sulphydryl blocker is accompanied by increased leukotriene, prostaglandin E2 (PGE2) generation, and nitric oxide synthase (NOS) activity. Rat small intestinal and colonic surfactant-like particles (SLP) that accumulate on the apical surface of mucosal cells have been identified and specific antibodies to them have been produced. The aim of this study was to evaluate a possible role of SLP in IA-induced colitis and jejunitis. Inflammation was induced in Sprague-Dawley rats either by intracolonic administration of 3% IA (0.1 ml) or by intrajejunal administration of 2% IA (0.1 ml). Antisera raised against either colonic SLP, pulmonary SP-A (a major protein associated with colonic SLP), or small intestinal SLP were injected into the tail vein of rats 48 h before, simultaneous with, or 24 h after IA administration. Rats were killed 2 or 10 days after IA was given, their colon or small intestine was isolated and rinsed, and a segment of colon (10 cm) or small bowel (30 cm) was weighed and processed for microscopy, NOS and myeloperoxidase (MPO) activities, and PGE2 generation. Intracolonic or jejunal IA resulted after 48 h in extensive macroscopic and microscopic damage, accompanied by increased segmental weight, MPO and NOS activity, and PGE2 generation. Colonic SLP antibody administration, either 48 h before or at the time of damage induction, significantly decreased macroscopic as well as microscopic damage, segmental weight, MPO activity, and PGE2 generation, but had no effect on NOS activity. Neither control sera nor antisera against SP-A had any protective effect, nor did injection of anti-colonic SLP antisera given 24 h after IA. Small bowel SLP antibody offered no protection against IA jejunitis. IA-induced colitis but not jejunitis is ameliorated by intravenous injection of SLP antibody by a mechanism yet to be determined. These data provide further evidence of a physiologic role for gastrointestinal SLP.

Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.

Morphological and functional heterogeneity of parietal cells has been thought to be due to different maturation positions within the gastric gland. Morphodynamic studies have shown that 2% of parietal cells in mice derive from a pre-neck (chief) cell precursor. Intrinsic factor (IF) and pepsinogen, markers of rat chief cells, were used to determine if these proteins identified a subset of parietal cells that might reflect origin from the pre-neck cell lineage. The zymogenic region of the rat stomach and gradient-isolated fractions enriched in parietal and chief cells were fixed in 10% buffered Formalin or in Bouin's solution. Immunostaining was performed using indirect immunoperoxidase histochemistry and double-labeled immunofluorescence with antibodies raised against human IF, pepsinogen II, and H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). In intact tissue, parietal (H(+)-K(+)-ATPase-positive) cells were found starting at the upper edge of the isthmus, but parietal cells positive for IF and pepsinogen were only found from just below the isthmus and neck region to the base of the gastric gland. Three to four percent of isolated parietal cells were positive for these ectopic markers. This subset of cells was also positive for H(+)-K(+)-ATPase. Thus products of rat chief cells are expressed in a subset of parietal cells. The percentage of positive cells is similar to that predicted to be derived from the pre-neck (chief) precursor lineage in the mouse. The distribution of these cells to the lower neck and base of the gland suggests that the expression of chief cell products is consistent with either predetermination by lineage or parietal cell maturation or with both processes.

Apical expression of functional asialoglycoprotein receptor in the human intestinal cell line HT-29.

BACKGROUND & AIMS: The asialoglycoprotein receptor localizes to the basolateral membrane of hepatocytes and to the apical membrane of enterocytes. The aim of this study was to examine HT-29 cells as a polarized cell model for studying apically localized endogenous asialoglycoprotein receptor. METHODS: Subunits H1 and H2 (human) were detected by Western blot and immunoprecipitated using subunit-specific antisera against hepatic receptor peptides. Receptor function was assessed by uptake of iodinated asialo-orosomucoid, immunoglobulin (Ig) A1, and haptocorrin. Immunocytochemistry was analyzed by standard light and confocal microscopy. RESULTS: Receptor content of the minor subunit, H2, was predominant. HT-29 cells mediated specific uptake and degradation of 125I-asialo-orosomucoid. A high-affinity (0.6 x 10(-9) mmol/L) and a low-affinity binding site were present. The specific ligand binding capacity of the apical surface was approximately twice that of the basolateral surface. Immunocytochemistry revealed a predominant apical membrane location of the minor receptor subunit, with some intracellular receptor. The apical H2 subunit was preferentially labeled with amino acid precursors compared with basolaterally located subunit. Human IgA1 bound specifically to HT-29 cells with a molar ratio of 0.26 compared with asialo-orosomucoid; porcine haptocorrin bound with a molar ratio of 1.35. CONCLUSIONS: HT-29 cells produce a functional apically located asialoglycoprotein receptor and provide a model for receptor trafficking in the enterocyte.

Isolation and characterization of surfactant-like particles in rat and human colon.

The source of a phospholipid-rich layer recovered from the surface of the mammalian colon has been obscure. This report describes the isolation of a low-density membrane from the surface of rat and human colons (d = 1.07-1.08 g/ml), with a low cholesterol-to-phospholipid ratio and phosphatidylcholine as its major phospholipid. Electron microscopy shows unilamellar and partially coiled membranes. Compared with microvillous membranes isolated from underlying mucosa, this extracellular membrane is enriched for tissue-unspecific alkaline phosphatase and surfactant protein A. It does not contain small intestinal marker proteins (intestinal alkaline phosphatase and sucrase-isomaltase). The human membrane contains only traces of the colonic microvillous membrane marker, carcinoembryonic antigen. Antiserum against the rat colonic membrane does not recognize colonic microvillous membrane or small intestinal surfactant-like particle proteins. Antiserum against human colonic membrane identifies one protein in the surfactant-like particle from the adjacent small intestine and two proteins in the colonic microvillous membrane. These data show that the colonocyte microvillous membrane is covered by another membrane with a different protein composition. Enrichment for surfactant protein A suggests that this colonic membrane is another example of a surfactant-like particle sharing proteins with pulmonary surfactant.

Human gastric intrinsic factor expression is not restricted to parietal cells.

Gastric parietal cells have been accepted as the only site of intrinsic factor production in the human stomach. In animals, however, intrinsic factor has been localised to various other cell types of foregut origin, including chief and enteroendocrine cells in gastric mucosa, and duct cells from salivary glands and pancreas. The availability of recombinant human intrinsic factor has led to production of high titre, monospecific antiserum which was used to reexamine the distribution and subcellular localisation of intrinsic factor in the human stomach. Immunolight microscopy revealed that most positively stained cells were gastric parietal cells, but at the margins of the anatomical regions (e.g. cardia/fundus, body/antrum) clusters of gastric chief cells and individual enteroendocrine cells were found to contain intrinsic factor. Immunoelectron microscopy demonstrated the highest antigen density on endocytic and apical membranes of parietal cells. Exocrine secretory granules of a subpopulation of chief cells, the secretory granules of some enteroendocrine cells, and the plasma membranes and smooth vesicles of endothelial cells of the lamina propria capillaries underlying enteroendocrine cells were also positive for the antigen. Labelling in all cells was specific, as it was abolished by preabsorption of the antisera with purified recombinant human intrinsic factor. These findings demonstrate a potential for cellular expression of human intrinsic factor in nonparietal cells. Because such expression occurs normally at the margins of anatomical gastric regions, it suggests that local factors may influence expression of intrinsic factor.

Determination of T-lymphocyte subsets on site in rural Tanzania: results in HIV-1 infected and non-infected individuals.

With the FACSCount flow cytometer, counts of CD4, CD8 and CD3 lymphocytes and CD4/CD8 ratios were performed in a rural hospital in Tanzania. A total of 168 subjects (21 HIV-1 seropositive and 147 HIV-1 seronegative) were tested as part of a population-based serosurvey and AIDS education programme; 134 other subjects were hospitalized patients who had signs and symptoms suggestive of AIDS (69 HIV-1 seropositive and 65 HIV-seronegative). Mean values for the 147 HIV-1 seronegative subjects from the local population were 980 CD4 cells (95% CI 930, 1031), 598 CD8 cells (560, 635) and CD4/CD8 ratio 1.78 (1.68, 1.89). Seropositive subjects from the local population had significantly lower CD4 cell counts, higher CD8 counts and a lower CD4/CD8 ratio. CD4 cells were significantly lower and CD8 cells significantly higher in HIV-1 seropositive hospital patients compared to HIV-1 seronegative patients. However, 23 (35%) seronegative hospital patients had CD4 counts lower than 600. These results establish baseline values for the lymphocyte subsets in this population and indicate that this technique can be used in remote areas to monitor progress of HIV-infected individuals.

Immunolocalization of alkaline phosphatase and surfactant-like particle proteins in rat duodenum during fat absorption.

BACKGROUND & AIMS: In response to triacylglycerol feeding, rat duodenum secretes into the lumen and lamina propria phospholipid-rich membranes (surfactant-like particles) that are enriched with intestinal alkaline phosphatase. The purpose of this study was to determine whether the enzyme and particle proteins were coordinate in tissue distribution and in time. METHODS: Immunocytochemistry using specific polyclonal antisera against alkaline phosphatase and against the complex of particle proteins and its 40-kilodalton component was performed. RESULTS: Triacylglycerol feeding produced a peak between 3 and 5 hours in stain intensity for both antigens, intracellularly as well as in the paracellular space and lamina propria. In fasting animals, the microvillous membrane stained strongly for alkaline phosphatase; surfactant-like particle proteins were mainly localized to the lamina propria. Feeding Pluronic L-81 (BASF Wyandotte, Wyandotte, MI), a detergent that decreases transcellular triacylglycerol movement and surfactant-like particle secretion, produced a decrease in reactivity of both antigens in the paracellular space, lamina propria, and lumen and redistributed intracellular alkaline phosphatase and surfactant-like particle proteins from Golgi or cytosol to intracellular membranes, corresponding to the circumference of lipid droplets. CONCLUSIONS: These data support the hypothesis that some intestinal alkaline phosphatase is secreted from the cell associated with the surfactant-like particle and are consistent with a role for this particle in transepithelial transport of triacylglycerols in the enterocyte.