Schistosoma mansoni Coinfection Attenuates Murine Toxoplasma gondii-Induced Crohn's-Like Ileitis by Preserving the Epithelial Barrier and Downregulating the Inflammatory Response.

Background and aims: Mice orally infected with T. gondii develop Crohn's disease (CD)-like enteritis associated with severe mucosal damage and a systemic inflammatory response, resulting in high morbidity and mortality. Previously, helminthic infections have shown therapeutic potential in experimental colitis. However, the role of S. mansoni in T. gondii-induced CD-like enteritis has not been elucidated. Our study investigated the mechanisms underlying T. gondii-induced ileitis and the potential therapeutic effect of S. mansoni coinfection. Methods: C57BL/6 mice were infected by subcutaneous injection of cercariae of the BH strain of S. mansoni, and 7-9 weeks later, they were orally infected with cysts of the ME49 strain of T. gondii. After euthanasia, the ileum was removed for histopathological analysis; staining for goblet cells; immunohistochemistry characterizing mononuclear cells, lysozyme expression, apoptotic cells, and intracellular pathway activation; and measuring gene expression levels by real-time PCR. Cytokine concentrations were measured in the serial serum samples and culture supernatants of the ileal explants, in addition to myeloperoxidase (MPO) activity. Results:T. gondii-monoinfected mice presented dense inflammatory cell infiltrates and ulcerations in the terminal ileum, with abundant cell extrusion, apoptotic bodies, and necrosis; these effects were absent in S. mansoni-infected or coinfected animals. Coinfection preserved goblet cells and Paneth cells, remarkably depleted in T. gondii-infected mice. Densities of CD4- and CD11b-positive cells were increased in T. gondii- compared to S. mansoni-infected mice and controls. MPO was significantly increased among T. gondii-mice, while attenuated in coinfected animals. In T. gondii-infected mice, the culture supernatants of the explants showed increased concentrations of TNF-alpha, IFN-gamma, and IL-17, and the ileal tissue revealed increased expression of the mRNA transcripts for IL-1 beta, NOS2, HMOX1, MMP3, and MMP9 and activation of NF-kappa B and p38 MAPK signaling, all of which were counterregulated by S. mansoni coinfection. Conclusion:S. mansoni coinfection attenuates T. gondii-induced ileitis by preserving mucosal integrity and downregulating the local inflammatory response based on the activation of NF-kappa B and MAPK. The protective function of prior S. mansoni infection suggests the involvement of innate immune mechanisms and supports a conceptually new approach to the treatment of chronic inflammatory diseases, including CD.

Allantoin reduces cell death induced by cisplatin: possible implications for tumor lysis syndrome management.

Massive lysis of tumor mass in cancer patients under chemotherapy regimens generates high levels of uric acid, leading to what is known as tumor lysis syndrome (TLS). Rasburicase, a recombinant urate oxidase, converts urate to allantoin, which is readily excreted by the kidneys. Even though there is a high production of allantoin from urate in cancer patients following rasburicase treatment, there are no studies on how allantoin excess could interfere with chemotherapy. We have evaluated allantoin interference with cisplatin efficiency on the lung cancer cell line H460 in vitro. The cells were treated with cisplatin (33 µM), with or without allantoin, for 48 h, in the presence or absence of UV light, and N-acetyl-L-cysteine (NAC) for 24 h. Cell viability, cell cycle, ROS production, apoptosis and immunoblot assays were performed. We showed that allantoin reduced the apoptosis induced by cisplatin in the H460 cell line. However, the activity of carboplatin and oxaliplatin, betulinic acid, TIBA, UV and H2O2 was not affected by allantoin. NMR spectroscopy showed that allantoin reduces cisplatin activity through direct interaction with cisplatin.

Canonical WNT Signaling Pathway is Altered in Mesenchymal Stromal Cells From Acute Myeloid Leukemia Patients And Is Implicated in BMP4 Down-Regulation.

Mesenchymal stromal cells (hMSCs) are key components of the bone marrow microenvironment (BMM). A molecular signature in hMSCs from Acute myeloid leukemia patients (hMSC-AML) has been proposed where BMP4 is decreased and could be regulated by WNT signaling pathway. Therefore, the aim of this work was to verify whether the WNT signaling pathway can regulate the BMP4 gene in hMSCs. The results showed differentially expressed genes in the WNT canonical pathway between hMSC-AML and hMSCs from healthy donors and a real-time quantitative assay corroborated with these findings. Moreover, the main WNT canonical pathway regulators were decreased in hMSC-AML, such as LEF-1, ß-catenin and the ß-catenin/TCF-LEF regulatory complex in the nucleus. This result, together with functional assays, suggests that the induction of BMP4 expression by the WNT signaling pathway is decreased in hMSC-AML. Overall, the WNT canonical pathway is able to regulate the BMP4 gene in hMSC-AML and its reduced activation could also lead to the lower expression of BMP4 in hMSC-AML. Due to the important role of the BMM, changes in BMP4 expression through the WNT canonical pathway may be a potential mechanism of leukemogenesis.

Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-ß oligomers in mice.

It is increasingly recognized that sleep disturbances and Alzheimer's disease (AD) share a bidirectional relationship. AD patients exhibit sleep problems and alterations in the regulation of circadian rhythms; conversely, poor quality of sleep increases the risk of development of AD. The aim of the current study was to determine whether chronic sleep restriction potentiates the brain impact of amyloid-ß oligomers (AßOs), toxins that build up in AD brains and are thought to underlie synapse damage and memory impairment. We further investigated whether alterations in levels of pro-inflammatory mediators could play a role in memory impairment in sleep-restricted mice. We found that a single intracerebroventricular (i.c.v.) infusion of AßOs disturbed sleep pattern in mice. Conversely, chronically sleep-restricted mice exhibited higher brain expression of pro-inflammatory mediators, reductions in levels of pre- and post-synaptic marker proteins, and exhibited increased susceptibility to the impact of i.c.v. infusion of a sub-toxic dose of AßOs (1pmol) on performance in the novel object recognition memory task. Sleep-restricted mice further exhibited an increase in brain TNF-α levels in response to AßOs. Interestingly, memory impairment in sleep-restricted AßO-infused mice was prevented by treatment with the TNF-α neutralizing monoclonal antibody, infliximab. Results substantiate the notion of a dual relationship between sleep and AD, whereby AßOs disrupt sleep/wake patterns and chronic sleep restriction increases brain vulnerability to AßOs, and point to a key role of brain inflammation in increased susceptibility to AßOs in sleep-restricted mice.

Hedgehog pathway signaling regulates human colon carcinoma HT-29 epithelial cell line apoptosis and cytokine secretion.

The Hedgehog (Hh) pathway is involved in embryogenesis and physiologic processes including cell survival and proliferation. We used the HT-29 and other human colon carcinoma cell lines to investigate Hh signaling and biological functions in colonic epithelial cells. HT-29 cells were cultured under different conditions and exposed to various stimuli. The expression of Hh pathway components and related genes and proteins were assessed by real-time PCR and immunofluorescence. Viability, apoptosis and cell proliferation were measured by the MTT assay, Annexin-V/7-AAD staining and BrdU uptake, respectively. Chemokines production was measured by ELISA in culture supernatants. Indian and Sonic Hh mRNA levels and the downstream transcription factors Gli-1 and Gli-2 increased following treatment with Hh agonists and butyrate, but decreased upon exposure to cyclopamine or GANT61. BMP4 and BMP7 expression increased after stimulation with Hh agonists. Gli-1 protein expression increased after Hh agonists and decreased following cyclopamine. Exposure to Hh agonists promoted ß-catenin reduction and subcellular redistribution. Levels of IL-8 and MCP-1 decreased upon exposure to Hh agonists compared to Hh antagonists, LPS, IFN-γ or EGF. Monocyte chemotaxis decreased upon exposure to supernatants of HT-29 cells treated with Shh compared to Hh antagonists, LPS and IFN-γ. Cellular incorporation of BrdU and cell viability decreased following Hh blockade. Hh agonists abrogated the anti-CD95 induced apoptosis. Hh pathway is a key controller of colon cancer cells, as demonstrated by its effect in dampening inflammatory signals and antagonizing apoptosis. The differential expression of Hh components may underlie abnormalities in the local immune response and in epithelial barrier integrity, with potential homeostatic implications for the development of colonic inflammation and malignancies.

Use of butyrate or glutamine in enema solution reduces inflammation and fibrosis in experimental diversion colitis.

AIM: To investigate whether butyrate or glutamine enemas could diminish inflammation in experimental diversion colitis. METHODS: Wistar specific pathogen-free rats were submitted to a Hartmann's end colostomy and treated with enemas containing glutamine, butyrate, or saline. Enemas were administered twice a week in the excluded segment of the colon from 4 to 12 wk after the surgical procedure. Follow-up colonoscopy was performed every 4 wk for 12 wk. The effect of treatment was evaluated using video-endoscopic and histologic scores and measuring interleukin-1ß, tumor necrosis factor-alpha, and transforming growth factor beta production in organ cultures by enzyme linked immunosorbent assay. RESULTS: Colonoscopies of the diverted segment showed mucosa with hyperemia, increased number of vessels, bleeding and mucus discharge. Treatment with either glutamine or butyrate induced significant reductions in both colonoscopic (P < 0.02) and histological scores (P < 0.01) and restored the densities of collagen fibers in tissue (P = 0.015; P = 0.001), the number of goblet cells (P = 0.021; P = 0.029), and the rate of apoptosis within the epithelium (P = 0.043; P = 0.011) to normal values. The high levels of cytokines in colon explants from rats with diversion colitis significantly decreased to normal values after treatment with butyrate or glutamine. CONCLUSION: The improvement of experimental diversion colitis following glutamine or butyrate enemas highlights the importance of specific luminal nutrients in the homeostasis of the colonic mucosa and supports their utilization for the treatment of human diversion colitis.

Wnt/ß-catenin pathway regulates ABCB1 transcription in chronic myeloid leukemia.

BACKGROUND: The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of ABCB1, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by ABCB1) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to ABCB1 regulation through its canonical pathway, which is mediated by ß-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/ß-catenin pathway in the regulation of ABCB1 transcription in CML, as the basal promoter of ABCB1 has several ß-catenin binding sites. ß-catenin is the mediator of canonical Wnt signaling, which is important for CML progression. METHODS: In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used. RESULTS: ß-catenin was present in the protein complex on the basal promoter of ABCB1 in both cell lines in vitro, but its binding was more pronounced in the resistant cell line in vivo. Lucena cells also exhibited higher ß-catenin levels compared to its parental cell line. Wnt1 and ß-catenin depletion and overexpression of nuclear ß-catenin, together with TCF binding sites activation demonstrated that ABCB1 is positively regulated by the canonical pathway of Wnt signaling. CONCLUSIONS: These results suggest, for the first time, that the Wnt/ß-catenin pathway regulates ABCB1 in CML.

Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis.

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. RESULTS: Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1ß decreased, while VEGF and TGF-ß did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. CONCLUSIONS: Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Unfractionated heparin and new heparin analogues from ascidians (chordate-tunicate) ameliorate colitis in rats.

The anti-inflammatory effect of mammalian heparin analogues, named dermatan sulfate and heparin, isolated from the ascidian Styela plicata was accessed in a TNBS-induced colitis model in rats. Subcutaneous administration of the invertebrate compounds during a 7-day period drastically reduced inflammation as observed by the normalization of the macroscopic and histological characteristics of the colon. At the molecular level, a decrease in the production of TNF-alpha, TGF-beta, and VEGF was observed, as well as a reduction of NF-kappaB and MAPK kinase activation. At the cellular level, the heparin analogues attenuated lymphocyte and macrophage recruitment and epithelial cell apoptosis. A drastic reduction in collagen-mediated fibrosis was also observed. No hemorrhagic events were observed after glycan treatment. These results strongly indicate the potential therapeutic use of these compounds for the treatment of colonic inflammation with a lower risk of hemorrhage when compared with mammalian heparin.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae).

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to approximately 80% and 20% of the total GAGs, respectively. In the fish eye, KS represents approximately 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by DeltaUA-1-->3-GalNAc(4SO4) (59.4%), DeltaUA-1-->3-GalNAc(6SO4) (23.1%), and DeltaUA-1-->3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

Distinct patterns of mucosal apoptosis in H pylori-associated gastric ulcer are associated with altered FasL and perforin cytotoxic pathways.

AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer. METHODS: Antral specimens from patients with H pylori-related active gastric ulcer (GU), H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser. RESULTS: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradication therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylori-gastritis. Co-expression of Fas with T-cell and macrophage markers was reduced in GU. FasL- and perforin-expressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylori-gastritis. CONCLUSION: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforin-expression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection.

Apoptosis and expression of anti- and pro-apoptotic proteins in peripheral blood mononuclear cells of Fanconi anaemia patients: a study of 73 cases.

Fanconi anaemia (FA) is a rare genetic disease whose patients have a high predisposition to haematological abnormalities and cancer. Fas expression levels in peripheral blood lymphocytes samples of 73 FA patients were measured to verify if alterations in Fas expression could lead to predisposition/resistance to spontaneous or PHA induced apoptosis, as well as, to reflect some haematological features of this disease. The anti- and pro-apoptotic proteins Bcl-2 and Bax were also evaluated. FA patients samples could be divided into three different groups based on Fas expression: 20 samples had low, 32 normal and 21 increased Fas levels when compared to 41 control samples. No correlation was found between Fas and Bcl-2 expression but a good association was obtained with Bax, in the subgroup with increased Fas expression. The best correlation was seen between Bax expression and apoptosis. Out of the 15 samples with high Bax expression, 11 underwent apoptosis whereas only one out of seven samples with low levels of Bax displayed increased induced apoptosis. Most patients with normal haematological features expressed Fas within normal levels. It is difficult to establish, however, if Fas-expression is involved in the cause or is a consequence of the effects observed.

Biochemical and immunohistochemical analysis of glycosaminoglycans in inflamed and non-inflamed intestinal mucosa of patients with Crohn's disease.

OBJECTIVES: Changes in extracellular matrix glycosaminoglycans (GAGs) of the intestinal mucosa have been associated with inflammatory bowel disease. The aim of the present study was to follow the changes in GAGs metabolism during the progression from non-inflamed to inflamed intestinal colon of patients with Crohn's disease (CD), using direct biochemical analysis and specific immunohistochemistry against chondroitin/dermatan sulfate and heparan sulfate. DESIGN AND METHODS: The content of GAGs from inflamed and non-inflamed colon of eight patients with active CD was estimated by uronic acid per dry weight of tissue and analyzed by agarose gel electrophoresis and ion-exchange chromatography. Intestinal sections were stained using antibodies against dermatan sulfate/chondroitin 4-sulfate (DS/CS), heparan sulfate (HS), and ICAM-1 (CD54), and analyzed by confocal microscopy. RESULTS: There was a reduction in the amount of GAGs in the non-inflamed colon of patients with CD. In the inflamed colon, HS, CS and DS showed increased concentrations compared with the non-inflamed colon. GAGs showed a diffuse distribution in the lamina propria and in the basement membrane of both inflamed and non-inflamed mucosa of patients with CD. CONCLUSION: We observed a marked reduction in GAGs with altered patterns of distribution in the non-inflamed colon of patients with CD. The increase in the synthesis of GAGs observed in the inflamed colon may be a compensatory mechanism for the restoration of the integrity of the intestinal mucosa.

Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease: evidence of altered expression of FasL and perforin cytotoxic pathways.

BACKGROUND AND AIMS: Abnormal apoptosis may result in the persistence of activated intestinal T-cells in inflammatory bowel disease (IBD). We investigated apoptosis in distinct mucosal compartments, and the expression of Fas/Fas ligand and perforin in the inflamed and non-inflamed intestinal mucosa of patients with IBD. METHODS: Colon specimens from 15 patients with ulcerative colitis (UC) and inflamed and non-inflamed mucosa from 15 patients with Crohn's disease (CD) were analysed for densities and distribution of apoptotic cells determined by the terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick-end labelling (TUNEL) method. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68, by double immunofluorescence with confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser. RESULTS: Colonic lamina propria (LP) and epithelium from patients with UC showed higher rates of apoptosis than controls, but no difference was shown regarding patients with CD. In LP, co-expression of Fas was reduced with T-cells in inflamed CD mucosa, and with macrophages in all patients with IBD. No difference was found in the expression of Fas on B-cells. Rates of FasL-expressing cells in LP were higher in IBD than in controls, with no correlation with the rates of apoptosis. Rates of perforin-expressing cells in LP were greater in UC than in controls, and correlated to the rates of apoptosis. No difference was shown regarding the inflamed and non-inflamed CD mucosa. Rates of FasL and perforin-expressing intra-epithelial lymphocytes showed no difference among groups. CONCLUSIONS: Increased expression of FasL in IBD colonic LP not parallelled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells. Expression of perforin is correlated to the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in UC.