Iron deficiency-induced thrombocytosis increases thrombotic tendency in rats.

Iron deficiency (ID) is globally prevalent, and apart from anemia is associated with thrombocytosis. While considered benign, studies linking thrombotic events with prior ID anemia suggest otherwise. Herein we used animal models to assess the influence of ID on thrombotic tendency. Sprague-Dawley rats were fed control or iron deficient diets and ferric carboxymaltose was used to reverse ID. Thrombosis was induced via stenosis of the inferior vena cava or damage to the right carotid artery using ferric chloride. Thrombi were evaluated histologically and via high frequency ultrasound in the venous model. ID consistently induced thrombocytosis alongside anemia. Venous thrombus growth and final dimensions in both arterial and venous thrombi were largest in ID. In both models, platelet numbers correlated with the final thrombus size, with ID thrombi having the largest platelet areas. Platelet function was also evaluated in surgically naive rats. Coagulability on thromboelastography and hemostasis on tail transection were augmented in ID. Platelet and plasma P-selectin expression were both higher in ID. Platelet adhesion and aggregation in ID was impaired under shear flow but was intact on static assays. Iron replacement therapy reversed all ID-related changes in hematological parameters, thrombus dimensions, and platelet assays. In summary, ID alone increases thrombotic tendency. Iron replacement therapy reverses these changes, making it a viable strategy for prevention of ID-related thrombotic disease. This may be of importance in patients with chronic illnesses which may already be at increased risk for thrombosis such as inflammatory bowel disease, chronic kidney disease, or cancer.

Iron deficiency, depression, and fatigue in inflammatory bowel diseases.

BACKGROUND: Iron deficiency and anemia are common findings in IBD. Treatment of anemia improves quality of life. Neurological symptoms like depression or anxiety are also common in IBD; however, their relationship with ID has not been studied in detail. METHODS: Prospective, single center, non-interventional trial in an IBD cohort (n = 98), which is generally at risk for ID. Quality of sleep (using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale, and Insomnia Severity Index) and the presence of fatigue (Piper fatigue scale), depression (Self-rating Depression Scale [SDS]) or anxiety (Self-rating Anxiety Scale [SAS]) were related to ID (ferritin, transferrin saturation), anemia (hemoglobin), and inflammatory disease activity (CRP). RESULTS: ID was present in 35 %, anemia in 16 %, and inflammation in 30 %. The overall quality of sleep in this cohort was similar to that reported for the general population. ID, anemia, or inflammation had no influence on the PSQI (median 4.0 [CI 3.0-5.0]), the ESS 5.5 (5.0-7.0), and the ISI 4.00 (2.5-5.5). Fatigue (PFS; present in 30 %), anxiety (SAS; present in 24 %), and depression (SDS; present in 33 %) were more common than in the general population. Iron deficient and anemic patients were more likely to be depressed (p = 0.02 and p < 0.01) and showed a trend towards presence of fatigue (p = 0.06 and 0.07). Systemic inflammation as measured by CRP had no effect on any of these conditions. CONCLUSION: In this IBD cohort, ID and anemia affect depression and possibly fatigue independent of the presence of inflammation.

Management of Iron Deficiency Anaemia in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a group of chronic relapsing inflammatory disorders affecting the large and small intestine, with a rising worldwide incidence and prevalence. Anaemia is the most common extraintestinal manifestation of IBD, correlating with disease activity, and tending to relapse even after successful therapy. Iron deficiency is the most common cause; however, it often manifests in combination with anaemia of inflammation. As such, multiple parameters are used for the diagnosis of iron deficiency anaemia in IBD. Timely recognition and selection of appropriate therapy leads to an improvement in the quality of life and prevention of potential sequelae. Oral iron can be effective under specific circumstances; however, as luminal iron changes microbiota and bacterial metabolism, oral administration should be avoided. Intravenous iron is preferred as it bypasses the sites of inflammation. Nevertheless, the optimization of IBD treatment should occur simultaneously, as this improves both patient condition and response to iron therapy. Herein, we discuss the screening, diagnosis, selection of therapy, and follow-up for iron deficiency anaemia in IBD.

Identification of PMN-released mutagenic factors in a co-culture model for colitis-associated cancer.

Microsatellite instability (MSI) is present in ulcerative colitis (UC) and colitis-associated colorectal cancers (CAC). Certain factors released by polymorphonuclear cells (PMNs) may drive mucosal frameshift mutations resulting in MSI and cancer. Here, we applied a co-culture system with PMNs and colon epithelial cells to identify such culprit factors. Subjecting HCT116 + chr3 and human colonic epithelial cells (HCEC)-1CT MSI-reporter cell lines harboring mono-, di- or tetranucleotide DNA repeats linked to enhanced green fluorescent protein (EGFP) to activated PMNs induced frameshift mutations within all repeats, as quantified by flow cytometry. Activated PMNs released superoxide and hydrogen peroxide (H2O2), as measured by lucigenin-amplified chemiluminescence and fluorometry, respectively. Catalase, which scavenges H2O2, reduced such PMN-induced MSI. The NADPH-oxidase inhibitor apocynin, which blocks the oxidative burst in PMNs, similarly inhibited PMN-induced MSI. A bead-based multiplex assay revealed that PMNs release a wide range of cytokines such as interleukin (IL)-8, IL-6 and tumor necrosis factor-α (TNF-α). In vitro, these cytokines increased MSI in colon epithelial cells, and the Janus kinase (JAK) inhibitor tofacitinib abolished IL-6-induced or PMN-induced MSI. Intracellular reactive oxygen species (ROS) formation, as measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, was induced upon cytokine treatment. DNA oxidation upon IL-6 was present, as detected by formamidopyrimidine glycosylase (FPG)-modified comet assay. In conclusion, activated PMNs induce frameshift mutations in colon epithelial cells resulting in MSI. Both oxidative burst with release of ROS and PMN-secreted cytokines, such as IL-8, IL-6 or TNF-α, contribute to MSI. ROS scavengers and/or specific inhibitors of cytokine signaling may delay or prevent cancer development in the setting of colitis.

[Diagnostic approach to iron deficiency anemia]. / Algorithmus zur Diagnose von Eisenmangelanämie.

Anemia affects a fourth of the global population, with iron deficiency remaining the primary cause. It is associated with diminished work capacity, fatigue, impaired cognitive function, and can negatively impact the course of diseases like chronic heart failure or chronic kidney disease. Treatment options include oral and intravenous iron; however, conditions such as inflammatory bowel disease, celiac disease, or autoimmune gastritis can diminish the efficacy of oral iron. Timely recognition of iron deficiency anemia and administration of appropriate therapy not only improves quality of life, but also reduces the need for blood transfusions. Proper selection of iron-deficient patients for whom further diagnostic testing is necessary facilitates identification of underlying diseases that require specific treatment, and avoids unnecessary invasive testing.

Iron deficiency alters megakaryopoiesis and platelet phenotype independent of thrombopoietin.

Iron deficiency is a common cause of reactive thrombocytosis, however, the exact pathways have not been revealed. Here we aimed to study the mechanisms behind iron deficiency-induced thrombocytosis. Within few weeks, iron-depleted diet caused iron deficiency in young Sprague-Dawley rats, as reflected by a drop in hemoglobin, mean corpuscular volume, hepatic iron content and hepcidin mRNA in the liver. Thrombocytosis established in parallel. Moreover, platelets produced in iron deficient animals displayed a higher mean platelet volume and increased aggregation. Bone marrow studies revealed subtle alterations that are suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte ploidy and accelerated megakaryocyte differentiation. Iron deficiency did not alter the production of hematopoietic growth factors such as thrombopoietin, interleukin 6 or interleukin 11. Megakaryocytic cell lines grown in iron-depleted conditions exhibited reduced proliferation but increased ploidy and cell size. Our data suggest that iron deficiency increases megakaryopoietic differentiation and alters platelet phenotype without changes in megakaryocyte growth factors, specifically TPO. Iron deficiency-induced thrombocytosis may have evolved to maintain or increase the coagulation capacity in conditions with chronic bleeding.

Nfe2l2/NRF2 Deletion Attenuates Tumorigenesis and Increases Bacterial Diversity in a Mouse Model of Lynch Syndrome.

Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer. Its early onset and high lifetime risk for colorectal cancer emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds induce NRF2. However, although NRF2 counteracts oxidative stress, it is also overexpressed in colorectal cancer and may promote tumorigenesis. In this study, we evaluated the role of NRF2 in the prevention of LS-associated neoplasia. We found increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelium-specific Msh2 deletion (MSH2ΔIEC) compared with C57BL/6 (wild-type) mice, as well as an increase in downstream NRF2 targets NAD(P)H dehydrogenase (quinone 1) and glutamate-cysteine ligase catalytic subunit. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared with healthy human controls. In silico analysis of a publicly accessible RNA sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null (MSH2ΔIECNrf2null) mice, we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null mice compared with MSH2ΔIEC mice after 40 weeks, which occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. The loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/ß-catenin signaling, but apoptosis was unaffected. Microbial α-diversity increased over time with the loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS. Prevention Relevance: Patients with LS have an early onset and high lifetime risk for colorectal cancer. In this study, we show that NRF2 protein levels are increased in MSH2 deficiency and associated neoplasia, but the loss of NRF2 attenuates tumorigenesis. This suggests that NRF2 may not be a tumor suppressor in this specific context.

Thymoquinone attenuates tumor growth in ApcMin mice by interference with Wnt-signaling.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) are at increased risk for the development of colorectal cancer. Surgery and chemoprevention are the most effective means to prevent cancer development. Thymoquinone (TQ) is considered the main compound of the volatile Nigella sativa seed oil and has been reported to possess anticarcinogenic properties. In this study we evaluated the chemopreventive properties of TQ in a mouse model of FAP. METHODS: APCMin mice were fed with chow containing 37.5 mg/kg or 375 mg/kg TQ for 12 weeks. H&E stained intestine tissue sections were assessed for tumor number, localization, size, and grade. Immunohistochemistry for ß-catenin, c-myc, Ki-67 and TUNEL-staining was performed to investigate TQ's effect on major colorectal cancer pathways. TQ's impact on GSK-3ß and ß-catenin were studied in RKO cells. RESULTS: 375 mg/kg but not 37.5 mg/kg TQ decreased the number of large polyps in the small intestine of APCMin mice. TQ induced apoptosis in the neoplastic tissue but not in the normal mucosa. Furthermore, upon TQ treatment, ß-catenin was retained at the membrane and c-myc decreased in the nucleus, which was associated with a reduced cell proliferation in the villi. In vitro, TQ activated GSK-3ß, which induced membranous localization of ß-catenin and reduced nuclear c-myc expression. CONCLUSIONS: In summary, TQ interferes with polyp progression in ApcMin mice through induction of tumor-cell specific apoptosis and by modulating Wnt signaling through activation of GSK-3ß. Nigella sativa oil (or TQ) might be useful as nutritional supplement to complement surgery and chemoprevention in FAP.

Rates of substance and polysubstance use through universal maternal testing at the time of delivery.

OBJECTIVE: To report substance and polysubstance use at the time of delivery. STUDY DESIGN: A cross-sectional study was performed on mothers consented for universal drug testing (99%) during hospital admission at six delivery hospitals in Cincinnati, Ohio. Mass spectrometry urinalysis detected positivity rates of 46 substances. Rates of positive drug tests for individual and common co-occurring substances measured were reported. RESULTS: 2531 maternal samples were tested (88%) and 33% contained cotinine, 11.3% THC, 7.2% opioids, 3.8% cocaine, and 1.9% methamphetamines. Polysubstance use prevalence was as high as 15%. Among mothers testing positive for methadone or buprenorphine, 93% also tested positive for cotinine and 39% tested positive for a third substance in addition to cotinine. CONCLUSIONS: Substance use at delivery is more prevalent than previously reported. Many mothers testing positive for opioids also test positive for other substances, which may increase overdose risk and exacerbate neonatal opioid withdrawal syndrome (NOWS).

A Novel PAK1-Notch1 Axis Regulates Crypt Homeostasis in Intestinal Inflammation.

BACKGROUND & AIMS: p21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/ß-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice. METHODS: IL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT. RESULTS: When compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased ß-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation. CONCLUSIONS: PAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

Regional comparison of self-reported late pregnancy cigarette smoking to mass spectrometry analysis.

OBJECTIVE: To report a more accurate prevalence estimate of late pregnancy nicotine exposures. STUDY DESIGN: A cross-sectional study during a 2-month period in 2019. Participants were women delivering in any of the six county maternity hospitals who consented to universal drug testing at the time of delivery as part of routine hospital admission. RESULTS: Of 2531 tested samples, 18.7% tested positive for high levels of cotinine indicating primary smoking or other primary use of tobacco products. Together, 33.0% of the study population tested positive for nicotine exposure during late pregnancy compared to vital records which reported 8.2% cigarette smoking during the third trimester of pregnancy and 10.5% cigarette smoking at any time during pregnancy through maternal self-report. CONCLUSION: Captured vital birth smoking measures vastly underreport actual primary exposures to nicotine products. Vital birth data also fail to capture secondhand exposures which constitute a significant proportion of the population.

Carbon and nitrogen pools and mobile fractions in surface soils across a mangrove saltmarsh ecotone.

In the subtropics, climate change is pushing woody mangrove forests into herbaceous saltmarshes, altering soil carbon (C) and nitrogen (N) pools, with implications for coastal wetland productivity and C and N exports. We quantified total C and N pools, and mobile fractions including extractable mineral N, extractable organic C and N, and active (aerobically mineralizable) C and N, in surface soils (top 7.6 cm) of adjacent mangrove (primarily Avicennia germinans) and saltmarsh (Juncus roemerianus) vegetation zones in tidal wetlands of west-central Florida (USA). We tested whether surface-soil accumulations of C, N, and their potentially mobile fractions are greater in mangrove than in saltmarsh owing to greater accumulations in the mangrove zone of soil organic matter (SOM) and fine mineral particles (C- and N-retaining soil constituents). Extractable organic fractions were 39-45% more concentrated in mangrove than in saltmarsh surface soil, and they scaled steeply and positively with SOM and fine mineral particle (silt + clay) concentrations, which themselves were likewise greater in mangrove soil. Elevation may drive this linkage. Mangrove locations were generally at lower elevations, which tended to have greater fine particle content in the surface soil. Active C and extractable mineral N were marginally (p < 0.1) greater in mangrove soil, while active N, total N, and total C showed no statistical differences between zones. Extractable organic C and N fractions composed greater shares of total C and N pools in mangrove than in saltmarsh surface soils, which is meaningful for ecosystem function, as persistent leaching of this fraction can perpetuate nutrient limitation. The active (mineralizable) C and N fractions we observed constituted a relatively small component of total C and N pools, suggesting that mangrove surface soils may export less C and N than would be expected from their large total C and N pools.

Mesalamine and azathioprine modulate junctional complexes and restore epithelial barrier function in intestinal inflammation.

Disruption of mucosal structure and barrier function contribute to the pathogenesis of inflammatory bowel disease (IBD). Efficacy of therapy in IBD is based on endoscopic mucosal healing, which occurs by a dynamic interplay of epithelial cell regeneration, migration and differentiation. Both mesalamine (5-ASA) and azathioprine (AZTP) promote this process through mechanisms not clearly understood. We examined molecular pathways implicated in epithelial barrier function that were altered by 5-ASA and AZTP. Paracellular permeability induced by inflammatory mediators was mitigated by both compounds through restoration of cellular anchoring complexes. 5-ASA and AZTP induced rearrangement and membranous localization of junctional proteins and modulated genes involved in tight junctions. Intestinal organoids from wildtype-mice treated with TNF-α and IL-10- deficient-mice displayed impaired epithelial barrier with loss of membranous E-cadherin and reduced Desmoglein-2 expression. These effects were counteracted by 5-ASA and AZTP. Unlike AZTP that exhibited antiproliferative effects, 5-ASA promoted wound healing in colon epithelial cells. Both affected cellular senescence, cell cycle distribution and restricted cells in G1 or S phase without inducing apoptosis. This study provides mechanistic evidence that molecular actions of 5-ASA and AZTP on intestinal epithelia are fundamental in the resolution of barrier dysfunction.

Management of Iron Deficiency Anemia.

Anemia affects one-fourth of the world's population, and iron deficiency is the predominant cause. Anemia is associated with chronic fatigue, impaired cognitive function, and diminished well-being. Patients with iron deficiency anemia of unknown etiology are frequently referred to a gastroenterologist because in the majority of cases the condition has a gastrointestinal origin. Proper management improves quality of life, alleviates the symptoms of iron deficiency, and reduces the need for blood transfusions. Treatment options include oral and intravenous iron therapy; however, the efficacy of oral iron is limited in certain gastrointestinal conditions, such as inflammatory bowel disease, celiac disease, and autoimmune gastritis. This article provides a critical summary of the diagnosis and treatment of iron deficiency anemia. In addition, it includes a management algorithm that can help the clinician determine which patients are in need of further gastrointestinal evaluation. This facilitates the identification and treatment of the underlying condition and avoids the unnecessary use of invasive methods and their associated risks.