Prognostic significance of red blood cell distribution width in gastrointestinal disorders.

The red blood cell distribution width (RDW) is a routinely measured and automatically reported blood parameter, which reflects the degree of anisocytosis. Recently, the baseline RDW was found to have clinical significance for assessing clinical outcome and severity of various pathological conditions including cardiovascular diseases, sepsis, cancers, leukemia, renal dysfunction and respiratory diseases. A myriad of factors, most of which ill-defined, have an impact on the red cell population dynamics (i.e., production, maturation and turnover). A delay in the red blood cell clearance in pathological conditions represents one of the leading determinants of increased anisocytosis. Further study of RDW may reveal new insight into inflammation mechanisms. In this review, we specifically discuss the current literature about the association of RDW in various disease conditions involving the gastrointestinal and hepatobiliary systems. We also present some of the related measurements for their value in predicting clinical outcomes in such conditions. According to our data, RDW was found to be a valuable prognostic index in gastrointestinal disorders along with additional inflammatory biomarkers (i.e., C reactive protein, erythrocyte sedimentation rate, and platelet count) and current disease severity indices used in clinical practice.

Suppression of islet homeostasis protein thwarts diabetes mellitus progression.

During progression to type 1 diabetes, insulin-producing ß-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered ß-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

miR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology.

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system in vitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. In vivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical for the proliferation and migration of HCC cells and OC response.

A disintegrin and metalloprotease with thrombospondin type I motif 7: a new protease for connective tissue growth factor in hepatic progenitor/oval cell niche.

Hepatic progenitor/oval cell (OC) activation occurs when hepatocyte proliferation is inhibited and is tightly associated with the fibrogenic response during severe liver damage. Connective tissue growth factor (CTGF) is important for OC activation and contributes to the pathogenesis of liver fibrosis. By using the Yeast Two-Hybrid approach, we identified a disintegrin and metalloproteinase with thrombospondin repeat 7 (ADAMTS7) as a CTGF binding protein. In vitro characterization demonstrated CTGF binding and processing by ADAMTS7. Moreover, Adamts7 mRNA was induced during OC activation, after the implantation of 2-acetylaminofluorene with partial hepatectomy in rats or on feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet in mice. X-Gal staining showed Adamts7 expression in hepatocyte nuclear factor 4α(+) hepatocytes and desmin(+) myofibroblasts surrounding reactive ducts in DDC-treated Adamts7(-/-) mice carrying a knocked-in LacZ gene. Adamts7 deficiency was associated with higher transcriptional levels of Ctgf and OC markers and enhanced OC proliferation compared to Adamts7(+/+) controls during DDC-induced liver injury. We also observed increased α-smooth muscle actin and procollagen type I mRNAs, large fibrotic areas in α-smooth muscle actin and Sirius red staining, and increased production of hepatic collagen by hydroxyproline measurement. These results suggest that ADAMTS7 is a new protease for CTGF protein and a novel regulator in the OC compartment, where its absence causes CTGF accumulation, leading to increased OC activation and biliary fibrosis.

Febrile pleuropericarditis, a potentially life-threatening adverse event of balsalazide--case report and literature review of the side effects of 5-aminosalicylates.

Ulcerative colitis (UC) is an idiopathic chronic inflammatory disorder that affects the colonic mucosa. One class among the drugs used for its treatment is the 5-aminosalicylates (5-ASAs). While highly efficacious in treating mild-to-moderate UC, 5-ASAs are associated with rare but potentially life-threatening side effects such as pericarditis, myocarditis and pneumonitis. These adverse events appear to be caused by a hypersensitivity reaction and resolve after cessation of 5-ASA drugs. This article presents a case report of febrile pleuropericarditis in a UC patient treated with balsalazide, and provides a thorough literature review of the rare side effects of 5-ASAs, their incidence, clinical presentation, differential diagnosis and treatment. In conclusion, the clinicians should be aware that this type of adverse events to 5-ASA compounds can be easily overlooked but it has significant morbidity if not promptly diagnosed.

Influence of host immunoregulatory genes, ER stress and gut microbiota on the shared pathogenesis of inflammatory bowel disease and Type 1 diabetes.

Inflammatory bowel disease (IBD) with its two distinct entities, Crohn's disease and ulcerative colitis, and Type 1 diabetes mellitus (T1D) are autoimmune diseases. The prevalence of these diseases continues to rapidly rise in the industrialized world. Despite the identification of several genetic loci that are associated with both IBD and T1D, thus far, there is a paucity of epidemiological data to support a clinical overlap. In an effort to better understand the underlying pathogenic mechanisms of both IBD and T1D, this review summarizes the literature about these related autoimmune diseases, describes the most recent advances in their etiopathogenesis and emphasizes the genetic and nongenetic factors that exercise a differential influence. Genome-wide association studies have identified genetic loci with a role in immune response regulation that are linked to both IBD (particularly Crohn's disease) and T1D. Some of these genetic loci (e.g., IL-18RAP) have a divergent role, conferring risk for one disease and protection for the other. Recent evidence highlights an important role of gut microbiota and cellular responses (e.g., endoplasmic reticulum stress) in the pathogenesis of both IBD and T1D.

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models.

RATIONALE: Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "off-target" effect of enhancing the enzymatic activity of ACE2 in vitro. OBJECTIVES: To evaluate the pharmacological actions of DIZE in experimental models of PH. METHODS: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. MEASUREMENTS AND MAIN RESULTS: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1α, which was corrected by in vitro DIZE treatment. CONCLUSIONS: Our results identify a therapeutic potential of DIZE in PH therapy.

Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter.

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor-4-independent pathway.

The FOXA (forkhead box A) proteins (FOXA1, FOXA2, and FOXA3) play a critical role in the development of the liver, and they also regulate metabolism in adult hepatic tissue. The liver responds to changes in nutrient availability by initiating a number of stress signaling pathways. The present studies demonstrated that in mouse dams fed a low-protein diet hepatic expression of FOXA2 and FOXA3 messenger RNA, but not FOXA1, was induced. Conversely, fetal liver did not exhibit this regulation. Amino acid deprivation of HepG2 hepatoma cells also enhanced transcription from the FOXA2 and FOXA3 genes. In contrast, endoplasmic reticulum stress inhibited the expression of FOXA1, only slightly induced FOXA2, and had no effect on FOXA3. The FOXA2 and FOXA3 messenger RNA induction by amino acid deprivation did not require activating transcription factor 4, a critical component of the conventional amino acid response (AAR) pathway, but their induction was partially dependent on CCAAT/enhancer-binding protein beta. Simultaneous knockdown of both FOXA2 and FOXA3 by small interfering RNA did not affect the activation of other amino acid responsive genes, suggesting that the FOXA proteins are not required for the known AAR pathway. Collectively, the results document that the hepatic FOXA family of genes are differentially regulated by amino acid availability.

Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway.

The UPR (unfolded protein response) pathway comprises three signalling cascades mediated by the ER (endoplasmic reticulum) stress-sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPbeta (CCAAT/enhancer-binding protein beta) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared with triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box-binding protein 1), ATF6alpha or ATF6beta expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, ASNS NSRE-1 and NSRE-2, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian ER-stress-responsive sequences.

Activated transcription via mammalian amino acid response elements does not require enhanced recruitment of the Mediator complex.

It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.

Despite increased ATF4 binding at the C/EBP-ATF composite site following activation of the unfolded protein response, system A transporter 2 (SNAT2) transcription activity is repressed in HepG2 cells.

The activated amino acid response (AAR) and unfolded protein response (UPR) stress signaling pathways converge at the phosphorylation of translation initiation factor eIF2alpha. This eIF2alpha modification suppresses global protein synthesis but enhances translation of selected mRNAs such as that for activating transcription factor 4 (ATF4). An ATF4 target gene, SNAT2 (system A sodium-dependent neutral amino acid transporter 2), contains a C/EBP-ATF site that binds ATF4 and triggers increased transcription during the AAR. However, the present studies show that despite increased ATF4 binding to the SNAT2 gene during UPR activation in HepG2 human hepatoma cells, transcription activity was not enhanced. Hyperacetylation of histone H3 and recruitment of the general transcription factors at the HepG2 SNAT2 promoter occurred in response to the AAR but not the UPR. In contrast, the UPR did enhance transcription from a plasmid-based reporter gene driven by a SNAT2 genomic fragment containing the C/EBP-ATF site. Simultaneous activation of the AAR and the UPR pathways revealed that the UPR actually suppressed the increased SNAT2 transcription by the AAR pathway, demonstrating that the UPR pathway generates a repressive signal that acts downstream of ATF4 binding.

MEK signaling is required for phosphorylation of eIF2alpha following amino acid limitation of HepG2 human hepatoma cells.

The mammalian amino acid response (AAR) pathway is up-regulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of the translation initiation factor eIF2alpha (eukaryotic initiation factor 2alpha), and, through subsequent translational control, enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation in HepG2 human hepatoma cells by PD98059 or U0126 blocked the increased phosphorylation of eIF2alpha and ATF4 synthesis triggered by amino acid limitation, showing that the AAR requires activation of the MEK-ERK pathway. Inhibitors of the JNK or p38 MAPK pathways were ineffective. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by overexpression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity and eIF2alpha phosphorylation. Inhibition of protein phosphatase 1 action on phospho-eIF2alpha by knockdown of GADD34 did not block the sensitivity to PD98059, suggesting that MEK functions to enhance GCN2-dependent eIF2alpha phosphorylation rather than suppressing dephosphorylation. Collectively, these results document a critical interdependence between the MEK-ERK MAPK signaling pathway and the amino acid stress-activated pathway.

Amino-acid limitation induces transcription from the human C/EBPbeta gene via an enhancer activity located downstream of the protein coding sequence.

For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPbeta (CCAAT/enhancer-binding protein beta) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPbeta genomic fragments containing the 8451 bp 5' upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3' downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPbeta enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPbeta promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPbeta itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.