Marilena Granzotto

Oxidative stress-based cytotoxicity of delphinidin and cyanidin in colon cancer cells.

Colorectal cancer is the second most frequent cause of cancer death in the western world. Although the prognosis has improved after the introduction of newer anticancer drugs, the treatment of metastatic colorectal cancer still remains a challenge due to a high percentage of drug-resistant tumor forms. We aimed at testing whether anthocyanidins exerted cytotoxicity in primary (Caco-2) and metastatic (LoVo and LoVo/ADR) colorectal cancer cell lines. Both cyanidin and delphinidin, though neither pelargonidin nor malvidin, were cytotoxic in metastatic cells only. The cell line most sensitive to anthocyanidins was the drug-resistant LoVo/ADR. There, cellular ROS accumulation, inhibition of glutathione reductase, and depletion of glutathione could be observed. This suggests that anthocyanidins may be used as sensitizing agents in metastatic colorectal cancer therapy.

Regulatory T-Cell Function Is Impaired in Celiac Disease

Celiac disease (CD) is characterized by intolerance to gluten and high risk of developing autoimmune phenomena. Possible defects in immune tolerance could have a role in the pathogenesis of the disease. As regulatory T-cells (Tregs) are the main population involved in maintaining peripheral tolerance, we investigated the number of these cells in celiac patients as compared with healthy donors. Moreover, we analyzed the suppressive function of CD4+CD25+ T-cells from celiac disease patients and controls on autologous responder T-cells (CD4+CD25−). The percentage of CD4+CD25+FOXP3+ cells was not different in celiacs and in healthy controls, and among positive cells the level of expression of the two regulatory markers was comparable. However, the suppressor activity of Tregs was significantly impaired in CD patients. These results suggest that a defect in Tregs function could play a role in the pathogenesis of CD and in CD-associated autoimmunity.

Diagnostics and Therapeutic Insights in a Severe Case of Mevalonate Kinase Deficiency

Mevalonate kinase deficiency is a rare inborn disorder of isoprenoid and sterol biosynthesis characterized by a recurrent autoinflammatory syndrome and, in most severe cases, psychomotor delay. Clinical manifestations can be very complex and, in some cases, mimic a chronic inflammatory disease. Diagnosis is also complex and often requires immunologic, genetic, and biochemical investigations. There is no standardized therapy, but biological agents could help to control inflammatory complaints in some cases. A severe case of mevalonate kinase deficiency that was associated with nephritis and successfully treated with anakinra (interleukin 1 receptor antagonist) is reported here, and new insights into diagnosis and therapy of this complex disorder are discussed.

Toxicologic and pharmacokinetic study of low doses of verapamil combined with doxorubicin.

The effect of a chronic treatment with low oral doses of verapamil, a calcium channel blocker commonly employed in cardiovascular therapy, on doxorubicin toxicity, was evaluated in CD1 mice. Verapamil, administered at a dosage corresponding to a typical cardiovascular posology in humans, significantly increased doxorubicin toxicity. In particular the mortality was significantly higher and earlier and histological analysis revealed an increase in the severity of lesions in the liver, kidney and small bowel of verapamil pretreated animals. The pharmacokinetic profiles revealed that verapamil treated group had higher doxorubicin peak plasma and tissue levels and AUCs. This study shows that verapamil, administered at low doses, dramatically increases doxorubicin toxicity, probably through an interaction between the two drugs, both P-glycoprotein substrates, on the protein expressed in normal tissues, and suggests caution in the use of the calcium channel blocker for cardiovascular pathologies in patients who have to be treated with antineoplastic agents, substrates of P-glycoprotein.

New potential therapeutic approach for the treatment of B-Cell malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles.

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitts lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.

Effects of melatonin on doxorubicin cytotoxicity in sensitive and pleiotropically resistant tumor cells

Melatonin has been reported to attenuate the oxidative damage caused by doxorubicin on kidney, brain, heart and bone marrow, whereas the in vivo antitumor effects of doxorubicin were not attenuated. The effects of melatonin on doxorubicin cytotoxicity have, therefore, been examined on human normal mammary epithelium HBL‐100, on mammary adenocarcinoma MCF‐7, on colon carcinoma LoVo, and on mouse P388 leukemia cell lines, and on tumor cell sublines pleiotropically resistant to anthracyclines. Melatonin in the concentration range 10–2000 pg/mL causes an inhibition of the growth of the human cell lines examined which is not clearly dose‐dependent and less than 25% when significant. Melatonin similarly causes minor effects on doxorubicin cytotoxicity either on the parental human cell lines or on their resistant sublines. On the contrary, 200–1000 pg/mL melatonin cause a significant and dose‐dependent partial sensitization to doxorubicin of resistant P388 mouse leukemia (P388/ADR), which occurs also in vivo, as indicated by a significant increase in survival time of the hosts. Doxorubicin intracellular concentrations in P388/ADR cells are increased by melatonin, suggesting that melatonin might inhibit P‐glycoprotein‐mediated doxorubicin efflux from the cells. These results indicate that the use of melatonin in clinical cancer treatment should not pose the risk of an attenuation of the effectiveness of doxorubicin, and encourage the further examination of the possible reduction by melatonin of the host toxicity of antitumor chemotherapy.

Medium-term survival without haematopoietic stem cell transplantation in a case of IPEX: insights into nutritional and immunosuppressive therapy

Immunodysregulation, polyendocrinopathy, enteropathy, Xlinked (IPEX; OMIM 304930) is a severe disorder of immune development characterised by a usually fatal course in early childhood [6]. It is due to mutations in the FOXP3 gene (locus Xp11.23-q13.3) [2, 4, 9], leading to failure in immune tolerance. Haematopoietic stem cell transplantation (HSCT) is the only definitive therapy and, even if its success is inconstant, it is considered to be the treatment of choice for the disease [6, 8, 10]. On the other hand, just two cases have been reported as surviving the first decade with a severe form of the disease without HSCT [5, 7]. A 1-month-old male was referred to our department for a severe eczema (Fig. 1), intractable mucous diarrhoea and failure to thrive. Extensive investigations ruled out a severe combined immunodeficiency (SCID). Symptoms were controlled with intravenous immunoglobulin, cyclosporin A, steroids and total parenteral nutrition (TPN) (Fig. 2). During his second year of life, he developed autoimmune diabetes, autoimmune haemolytic anaemia (AHA), inflammatory interstitial pneumonia, alopecia and thyroiditis. Due to the unsatisfactory response to prednisone, oral betamethasone was prescribed, with a dramatically greater efficacy at an equipotent dosage. A bone marrow transplantation was refused by the parents. Flares of dermatitis, AHA and pneumonia occurred after every cold and required increasing doses of steroids, antibiotics and oxygen administration. Cyclosporin A (10 mg/kg/day) did not allow a reduction of steroids and the child developed a Cushing-like aspect. An experimental treatment with fludarabine followed by the infusion of autologous lymphocytes treated in vitro with vincristine and prednisone was repeated twice, allowing a prolonged reduction of steroid dosage (0.5–0.25 mg/day of betamethasone) and fairly good clinical improvement with less frequent and less intense reactivation of symptoms. At Eur J Pediatr (2007) 166:1195–1197 DOI 10.1007/s00431-006-0395-6

Neutrophils Engraftment Delay During Tigecycline Treatment in 2 Bone Marrow-transplanted Patients

Background: Tigecycline is the first available drug of glycylcycline family. Because of recent introduction, some of its adverse effects could be still unexplored. Observation: We report the cases of 2 boys who underwent an allogenic bone marrow transplantation for acute myeloid leukemia and were treated with tigecycline. Erythrocyte and platelet engraftment followed a normal course, but the neutrophil count remained low despite the increase in leukocyte count. After tigecycline interruption, the neutrophil count rapidly raised in both cases. Conclusions: Neutropenia was suspected to be secondary to tigecycline exposure. In vitro experiments were performed, which suggested tigecycline influence on myeloid cells survival.

T cells stimulated in vitro have a suppressive function but do not contain only regulatory T cells

The generation of regulatory T cells (Tregs) in vitro represents an attractive possibility to set up cellular therapies that could prevent and cure autoimmune disorders. Different methods have been proposed to generate Tregs in vitro and to evaluate their phenotype and function. Moreover, the overlap between generation of activated and regulatory cells could often be underestimated. We showed that in vitro treatment of CD4+ CD25– lymphocytes with different stimuli leads to a good expression of CD25 and forkhead box P3 (FoxP3) on most cells, but to a full Treg phenotype (including CD127 negativity) in only a minor percentage of cells, ranging from 17·38% of cells treated with phytohaemagglutinin (PHA) to 50·91% of cells treated with T cell receptor (TCR) stimulation in association with transforming growth factor (TGF)‐β. Some suppressive activity was demonstrated for T cells activated with all the different stimuli. However, while suppression mediated by TCR/TGF‐β treated T cells was associated with an inhibition of both interleukin (IL)‐2 and interferon (IFN)‐γ in the co‐culture supernatant, the suppression observed for PHA‐activated cells occurred in the presence of large amounts of these cytokines. In conclusion, also taking into account other recent publications, caution should be taken in interpretation of data in the field of regulatory T cells.

First description of Merkel Cell polyomavirus DNA detection in a patient with Stevens-Johnson syndrome.

Merkel Cell polyomavirus (MCPyV), a ubiquitous DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukaemia while other associations are still being explored. MCPyV sequences have also been detected in normal tissues of tumor patients and in the blood of healthy donors. This report documents a new MCPyV association with the Stevens–Johnson syndrome, a rare immune‐modulated mucocutaneous process particularly associated with specific drugs and infective agents. A high MCPyV viral load was detected simultaneously in fluid from skin lesions (2.0 × 104 copies/ml) and in matched blood (7.4 × 105 copies/ml) from a young adult patient after bone marrow transplant for a relapsed T‐cell acute lymphatic leukaemia. MCPyV clearance concurred with the complete resolution of skin lesions after 5 days of cidofovir treatment. DNA sequencing classified the amplicons as the European/Italian MKL‐1 strain. Given its ubiquitous nature, MCPyV could account for part of Stevens–Johnson syndrome idiopathic cases. J. Med. Virol. 85:918–923, 2013.