Maria Russo

Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity

BackgroundCeliac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal symptoms similar to those in CD, but the overall clinical picture is generally less severe and is not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune comorbidities. By studying and comparing mucosal expression of genes associated with intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we sought to better understand the similarities and differences between these two gluten-associated disorders.MethodsCD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens were collected to study the expression of genes involved in barrier function and immunity.ResultsUnlike CD, GS is not associated with increased intestinal permeability. In fact, this was significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2 was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients (P = 0.0293).ConclusionsThis study shows that the two gluten-associated disorders, CD and GS, are different clinical entities, and it contributes to the characterization of GS as a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

Dietary polyphenols in cancer prevention: the example of the flavonoid quercetin in leukemia.

Increased consumption of fruit and vegetables can represent an easy strategy to significantly reduce the incidence of cancer. We recently demonstrated that the flavonoid quercetin, naturally present in the diet and belonging to the class of phytochemicals, is able to sensitize several leukemia cell lines and B cells isolated from patients affected by chronic lymphocytic leukemia (B‐CLL), in addition to apoptotic inducers (anti‐CD95 and rTRAIL). Further, it potentiates the effect of fludarabine, a first‐line chemotherapeutic drug used against CLL. The proapoptotic activity of quercetin in cell lines and B‐CLL is related to the expression and activity of Mcl‐1–antiapoptotic proteins belonging to the Bcl‐2 family. Quercetin downregulates Mcl‐1 mRNA and protein levels acting on mRNA stability and protein degradation. Considering the low toxicity of the flavonoids toward normal peripheral blood cells, our experimental results are in favor of a potential use of quercetin in adjuvant chemotherapy in CLL or other types of cancer.

Quercetin: A Pleiotropic Kinase Inhibitor Against Cancer

Increased consumption of fruits and vegetables can represent an easy strategy to significantly reduce the incidence of cancer. From this observation, derived mostly from epidemiological data, the new field of chemoprevention has emerged in the primary and secondary prevention of cancer. Chemoprevention is defined as the use of natural or synthetic compounds able to stop, reverse, or delay the process of tumorigenesis in its early stages. A large number of phytochemicals are potentially capable of simultaneously inhibiting and modulating several key factors regulating cell proliferation in cancer cells. Quercetin is a flavonoid possessing potential chemopreventive properties. It is a functionally pleiotropic molecule, possessing multiple intracellular targets, affecting different cell signaling processes usually altered in cancer cells, with limited toxicity on normal cells. Simultaneously targeting multiple pathways may help to kill malignant cells and slow down the onset of drug resistance. Among the different substrates triggered by quercetin, we have reviewed the ability of the molecule to inhibit protein kinases involved in deregulated cell growth in cancer cells.

Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates

Objectives: Helicobacter pylori infection is a major health problem worldwide, and effective eradication of the infection is mandatory. The efficacy of recommended eradication regimens is approximately 70%. To avoid treatment failure and the consequent development of secondary resistance(s), it is important to choose the most appropriate first-line treatment regimen. This choice should also be made based on the knowledge of the antimicrobial resistance peculiar to a given geographical area. We evaluated the prevalence of antimicrobial-resistant H pylori strains isolated from naive patients and from patients with previous unsuccessful treatments. Methods: This study examined 109 H pylori-infected subjects (Group 1) who had never received an eradication treatment and 104 H pylori-infected subjects (Group 2) who had failed one or more eradication treatments. Resistance to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET) and levofloxacin (LEV) was determined using the epsilometer test. The significance of differences was evaluated by the χ2 test. Results: The prevalence of antimicrobial resistance was 0% versus 3.1% to AMO, 0% versus 2% to TET, 27% versus 41.3% to MET (p<0.05), 18% versus 45.8% to CLA (p<0.05) and 3% versus 14.6% to LEV (p<0.05) in Group 1 vs Group 2, respectively. In Group 2, there was an increased prevalence of H pylori strains resistant to multiple antimicrobials. Conclusions: This study confirms the high prevalence of H pylori strains resistant to CLA and MET, and indicates that unsuccessful treatments significantly increase resistance. Choosing eradication regimens other than standard triple therapy as a first-line therapy should be advisable in areas with high primary antimicrobial resistance prevalence.

Quercetin and anti-CD95(Fas/Apo1) enhance apoptosis in HPB-ALL cell line

Several malignant cell lines are resistant to CD95(Apo1/Fas)‐mediated apoptosis, even when the CD95 receptor is highly expressed. Sensitivity to CD95‐induced apoptosis can be restored using different molecules. In this study, we showed that quercetin, a naturally occurring flavonoid, in association with the agonistic anti‐CD95 monoclonal antibody, increases DNA fragmentation and caspase‐3 activity in HPB‐ALL cells. These cells have been selected for their known resistance to CD95‐induced apoptosis. At molecular level, quercetin lowers the level of intracellular reactive oxygen species, reduces mitochondrial transmembrane potential, thereby leaving the expression of CD95 receptor unchanged.

Flavonoid quercetin sensitizes a CD95-resistant cell line to apoptosis by activating protein kinase Cα

We previously demonstrated that quercetin, a naturally occurring flavonoid with strong antioxidant properties, was able to enhance programmed cell death in HPB-acute lymphoblastic leukemia (ALL) cell line, derived from a human tymoma, when associated with the agonistic anti-CD95 monoclonal antibody. Here, we report that HPB-ALL cells are normally resistant to CD95-mediated apoptosis, and quercetin is able to sensitize this cell line through a mechanism independent of its antioxidant properties. In fact, other compounds structurally and functionally similar to quercetin, when associated with anti-CD95 antibody did not induce any CD95-mediated apoptosis, still maintaining their antioxidant capacity. We found that quercetin effects are mediated by the activation of PKCα. Treatment of HPB-ALL cells with quercetin slightly decreased PKCα activity, but when the flavonoid was associated with anti-CD95, the kinase activity increased by 12-fold with respect to the treatment with quercetin. In addition, overexpression of PKCα induced programmed cell death in the absence of any additional stimulus, while a kinase-defective mutant of PKCα was ineffective. Our data confirm the involvement of specific PKC isoforms in CD95 signaling and suggest, for the first time, that quercetin targets this pathway increasing apoptogenic response in a cell line resistant to CD95-mediated apoptosis.

Ovothiol Isolated from Sea Urchin Oocytes Induces Autophagy in the Hep-G2 Cell Line

Ovothiols are histidine-derived thiols isolated from sea urchin eggs, where they play a key role in the protection of cells toward the oxidative burst associated with fertilization by controlling the cellular redox balance and recycling oxidized glutathione. In this study, we show that treatment of a human liver carcinoma cell line, Hep-G2, with ovothiol A, isolated from Paracentrotus lividus oocytes, results in a decrease of cell proliferation in a dose-dependent manner. The activation of an autophagic process is revealed by phase contrast and fluorescence microscopy, together with the expression of the specific autophagic molecular markers, LC3 II and Beclin-1. The effect of ovothiol is not due to its antioxidant capacity or to hydrogen peroxide generation. The concentration of ovothiol A in the culture media, as monitored by HPLC analysis, decreased by about 24% within 30 min from treatment. The proliferation of normal human embryonic lung cells is not affected by ovothiol A. These results hint at ovothiol as a promising bioactive molecule from marine organisms able to inhibit cell proliferation in cancer cells.

Design and Synthesis of Pro-Apoptotic Compounds Inspired by Diatom Oxylipins

Oxylipins are a large and diverse family of fatty acid derivatives exhibiting different levels of oxidation of the carbon chain. They are involved in many biological functions in mammals, plants and diatoms. In this last group of organisms, they are suggested to play a role in the reproductive failure of copepod predators, showing clear pro-apoptotic effects on newborn nauplii. In this work, these compounds were tested for the ability to induce mitotic arrest in sea urchin embryos. We show for the first time that oxylipins have an increased efficacy in their corresponding methylated form. Natural oxylipins were also used as an inspiration for the rational design and synthesis of stable chemical analogs with apoptotic activity against tumor cell lines. This approach led to the synthesis of the linear C15-ketol (22) that was shown to induce apoptosis in human leukemia U-937 cells. These results are proof of the concept of the use of eco-physiological considerations as a platform to guide the search for novel drug candidates.

CK2 and PI 3 K are direct molecular targets of quercetin in chronic lymphocytic leukaemia

Despite the encouraging results of the innovative therapeutic treatments, complete remission is uncommon in patients affected by chronic lymphocytic leukaemia, which remains an essentially incurable disease. Recently, clinical trials based on BH3-mimetic drugs showed positive outcomes in subjects with poor prognostic features. However, resistance to treatments occurs in a significant number of patients. We previously reported that the multi-kinase inhibitor quercetin, a natural flavonol, restores sensitivity to ABT-737, a BH3-mimetic compound, in both leukemic cell lines and B-cells isolated from patients. To identify the molecular target of quercetin, we employed a new cell line, HG3, obtained by immortalization of B-cells from a chronic lymphocytic leukaemia patient at the later stage of disease. We confirmed that quercetin in association with ABT-737 synergistically enhances apoptosis in HG3 (combination index < 1 for all fractions affected). We also reported that the cellular uptake of quercetin is extremely rapid, with an intracellular concentration of about 38.5 ng/106 cells, after treatment with 25 μM for 5 min. We demonstrated that the activity of protein kinase CK2, which positively triggers PI3K/Akt pathway by inactivating PTEN phosphatase, is inhibited by quercetin immediately after its addition to HG3 cells (0–2 min). PI3K activity was also inhibited by quercetin within 60 min from the treatment. The combined inhibition of CK2 and PI3K kinase activities by quercetin restored ABT-737 sensitivity and increased lethality in human leukemia cells.

Peptic ulcer occurrence in follow-up of chronic gastritis in patients with treated and not eradicated CagA-positive Helicobacter pylori infection.

The aim of the present prospective investigation was to study 49 dyspeptic Helicobacter pylori (HP)-positive (HP+) or -negative (HP−), CagA+ and CagA− patients with a normal pattern or pure chronic gastritis at initial histology as well as normal features or hyperemic gastropathy at initial endoscopy in a two-year follow up. All the HP+ patients were treated with omeprazole 20 mg twice a day plus amoxicillin 1 g twice a day for two weeks. No substantial change was seen in gastritis in CagA+ patients in whom the infection was not eradicated, and, in contrast, a progressive improvement in 13/14 successfully treated patients was found. At endoscopy, a progressive change to a normal picture was seen in 8 and no change in 6 of 14 patients whose HP infection was eradicated, in contrast a worsening in the 9 HP+ patients who were still infected was observed. In particular, peptic lesions arose in 6 of 21 CagA+ patients in whom the infection was not eradicated. In conclusions, the lack of change in chronic gastritis at histology and the progressive worsening of endoscopic hyperemic gastropathy (with peptic lesions arising in 28,6%) when HP+ CagA+ infection is not eradicated, unlike the progressive improvement of the anatomoclinical condition in the patients whose infection was eradicated, draws attention to the relevance of eradicating HP in CagA+ patients even when no peptic lesion is found at initial endoscopy.