The role of the Bcl-2 family of proteins in the pathogenesis of B-cell chronic lymphocytic leukaemia

B-cell chronic lymphocytic leukaemia (B-CLL) is an acquired neoplastic disease characterised by a clonal accumulation of long-lived, functionally immature and CD5+ B-lymphocytes, which particularly accumulate in the lymphatic system, peripheral blood, bone marrow, spleen and liver. Symptoms include lymphocytosis, immune system dysfunction and autoimmune disease, but transformation to more aggressive forms of neoplastic disease occur and development of a second malignancy is not uncommon. The disease is one of later years being unusual before 50 years of age, the rates of incidence vary on a racial basis, and it has a highly variable prognosis. Some patients die within months of diagnosis despite intensive treatment, whilst others survive for 30-plus years without any form of medical intervention and die of unrelated causes. The principal causes of death in patients whose deaths are directly related to the disease are opportunistic infection due to the impairment of immune system function and bleeding disorders. No treatment has been shown to cure the disease or consistently extend life expectancy. It has been recognised for more than 30 years that the accumulation of malignant cells in B-CLL is at least as important in the pathogenesis of the disease as their neoplastic proliferation. With the discoveries that Bcl-2 extended the life of follicular lymphoma cells by conferring resistance to apoptosis and was commonly expressed in B-CLL cells, it was extrapolated that Bcl-2 might play a similar role in the development of the disease by extending the life span of B-CLL cells. Bcl-2 has frequently been shown to be over expressed in B-CLL cells and genetic translocations and/or malfunction of Bcl-2 family regulating molecular entities may play a part in this. However, since its discovery, Bcl-2 has been shown to be part of a large family of genes which is highly and evolutionarily conserved. Members of the bcl-2 family are defined by sequence homology in four Bcl-2 homology (BH) regions and a hydrophobic membrane-spanning domain, with the possession of specific BH domains determining whether individual proteins have pro- or anti-apoptotic activity. Family members such as Bcl-2 and Bcl-XL extend the life span of cells, whilst others such as Bax and Bak shorten it. Oltvai, Milliman and Korsmeyer have proposed a general model of apoptosis, in which the cell's apoptotic fate is determined by the cellular balance between pro- and anti-apoptotic bcl-2 family members. The effect of unregulated expression of Bcl-2 family members in B-CLL cells conforms to this paradigm and resistance to apoptosis appears to be conferred through a cellular imbalance of power between pro- and anti-apoptotic bcl-2 family members, particularly Bcl-2 and Bax, which is tilted in favour of cell survival. However, the apoptotic fate of B-CLL cells, and hence the neoplasm, may be influenced by other family members, with Mcl-1, Bcl-XL, Bak, with the non-family but Bcl-2-associated protein, Bag-1, also found expressed in B-CLL cells. Similarities between the structure of the more conserved family members and other pore-forming proteins, along with the ability of Bcl-2, Bcl-XL, and Bax to form pores in synthetic membranes, suggest that they may exert their influence through pore-forming activities in intracellular membranes, particularly mitochondrial membranes. Bcl-2 family members may regulate apoptosis by changing the permeability of membranes to ions and apoptosis-inducing molecules, and physical interactions between Bcl-2 family proteins mediated by the BH domains may be important in both pore-forming and pore-inhibiting activities. Research findings suggest that the levels of Bcl-2 family members in B-CLL cells may be modulated by a wide range of largely extracellular influences, including the cytokines interleukin-4 (IL-4), IL-8, IL-10, interferon-a (IFN-?), IFN-?, and basic fibroblast growth factor (bPGF). Levels of Bcl-2 family members may also be modulated by contact between B-CLL cells and bone marrow (BM) stromal cells, activation of lgM, CD95, CD40 or CD6, the p53 gene product, and co-cultivation with CDw32-transfected murine fibroblasts. Such modulation may offer some insight into the pathogenesis of the disease, an explanation for the higher level expression of Bcl-2 family members in B-CLL, and an explanation for the highly variable prognosis. Additionally, if Bcl-2 family members can be shown unequivocally to be controlled by any of these molecular entities, the existence of these influences may offer the opportunity to reduce the neoplastic cells' apoptotic threshold by manipulating the relative levels of pro- and anti-apoptotic Bcl-2 family members as a treatment regime, or prior to more conventional treatment regimes

C. elegans expressing D76N β2-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

Zoledronic acid boosts γδ T-cell activity in children receiving αβ+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients

C. elegans expressing D76N β_{2}-microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β_{2} -m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β_{2} -m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β_{2} -m expressing worms. We also demonstrated the specificity of the β_{2} -m variant in determining the pathological phenotype by rescuing the wild type phenotype when β_{2} -m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

Fenretinide-loaded mesenchymal stem cells-derived extracellular vesicles as novel drug carriers against human neuroblastoma

Fenretinide (4-HPR) is a synthetic retinoid with little adverse effects, whose antitumor efficacy has been widely demonstrated. Unfortunately, clinical responses both in adult and pediatric patients are partial, revealing a limited activity of 4-HPR against existing disease. The underlying causes of this slight therapeutic efficacy consist in its poor water solubility, low bioavailability and high first-pass hepatic effect. As a result, new formulations are required to improve clinical outcomes. To this end, we propose a new 4-HPR formulation based on mesenchymal stem cells derived extracellular vesicles (EVs) as endogenous carriers for drug delivering. After isolation from human umbilical cord, the mesenchymal stem cells were treated with high doses of 4-HPR to achieve a passive drug loading. The resulting 4-HPR-EVs were collected, purified by ultracentrifugation and characterized for size, concentration. The drug amount encapsulated into the vesicles was determined by HPLC and to estimate the analyte recovery the internal standard N-(4-ethoxyphenyl)-retinamide was synthetized by us. We evaluated the EV uptake and efficacy on neuroblastoma cell lines cultured both in 2- and 3-D

[Influenza prevention in health-care workers: enhancement of the influenza vaccination campaign at Policlinico Tor Vergata in Rome (Italy) during the 2017/2018 epidemic season]

Given the very low influenza vaccination coverage averaging 4% among health-care workers at Policlinico Tor Vergata during the previous epidemic seasons, it was decided to implement a series of interventions to promote influenza vaccination compliance in the current (2017/2018) season. The total number of vaccinated health-care workers at the end of the current season was 364, resulting in a vaccination coverage of approximately 12%. The increase took place in all professional groups, with a coverage of 25% among permanent MDs, 9% among Medical Residents, 3% among nurses and 8% among technicians. The influenza vaccination coverage of 12%, although very low compared to the minimum target of 75%, is broadly in line with the values reported by other hospitals in Italy