The secretome from human-derived mesenchymal stem cells augments the activity of antitumor plant extracts in vitro.

Cancer is understood as a multifactorial disease that involve multiple cell types and phenotypes in the tumor microenvironment (TME). The components of the TME can interact directly or via soluble factors (cytokines, chemokines, growth factors, extracellular vesicles, etc.). Among the cells composing the TME, mesenchymal stem cells (MSCs) appear as a population with debated properties since it has been seen that they can both promote or attenuate tumor progression. For various authors, the main mechanism of interaction of MSCs is through their secretome, the set of molecules secreted into the extracellular milieu, recruiting, and influencing the behavior of other cells in inflammatory environments where they normally reside, such as wounds and tumors. Natural products have been studied as possible cancer treatments, appealing to synergisms between the molecules in their composition; thus, extracts obtained from Petiveria alliacea (Anamu-SC) and Caesalpinia spinosa (P2Et) have been produced and studied previously on different models, showing promising results. The effect of plant extracts on the MSC secretome has been poorly studied, especially in the context of the TME. Here, we studied the effect of Anamu-SC and P2Et extracts in the human adipose-derived MSC (hAMSC)-tumor cell interaction as a TME model. We also investigated the influence of the hAMSC secretome, in combination with these natural products, on tumor cell hallmarks such as viability, clonogenicity, and migration. In addition, hAMSC gene expression and protein synthesis were evaluated for some key factors in tumor progression in the presence of the extracts by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Multiplex, respectively. It was found that the presence of the hAMSC secretome did not affect the cytotoxic or clonogenicity-reducing activities of the natural extracts on cancer cells, and even this secretome can inhibit the migration of these tumor cells, in addition to the fact that the profile of molecules can be modified by natural products. Overall, our findings demonstrate that hAMSC secretome participation in TME interactions can favor the antitumor activities of natural products.

Genotoxicity and mutagenicity assessment of a standardized extract (P2Et) obtained from Caesalpinia spinosa.

The standardized P2Et extract obtained from Caesalpinia spinosa has shown antioxidant, and direct antitumor activity, but also activation of specific immune response through the induction of tumor immunogenic cell death in breast and melanoma cancer models. The present work evaluated the mutagenicity and genotoxicity profile of P2Et to continue the development of the P2Et. Genotoxicity was evaluated by OECD 1997 a guideline and mutagenicity by OECD 2016. At P2Et's doses of 500, 1000, and 2000 mg/kg body weight in mice (Mus musculus), the difference between the number of micronuclei in PCE of the groups were not statistically significant (17 (negative control), 15 (500 mg/kg), 15 (1000 mg/kg), 19 (2000 mg/kg) and 271 (positive control). Similarly, P2Et did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella Typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 at the tested range of concentrations up to 5000 µg/plate in the absence and presence of metabolic activation. Therefore, the P2Et was considered as non- mutagenic and non-genotoxic at the conditions of the tests.

Immune-system-dependent anti-tumor activity of a plant-derived polyphenol rich fraction in a melanoma mouse model.

Recent findings suggest that part of the anti-tumor effects of several chemotherapeutic agents require an intact immune system. This is in part due to the induction of immunogenic cell death. We have identified a gallotannin-rich fraction, obtained from Caesalpinia spinosa (P2Et) as an anti-tumor agent in both breast carcinoma and melanoma. Here, we report that P2Et treatment results in activation of caspase 3 and 9, mobilization of cytochrome c and externalization of annexin V in tumor cells, thus suggesting the induction of apoptosis. This was preceded by the onset of autophagy and the expression of immunogenic cell death markers. We further demonstrate that P2Et-treated tumor cells are highly immunogenic in vaccinated mice and induce immune system activation, clearly shown by the generation of interferon gamma (IFN-γ) producing tyrosine-related protein 2 antigen-specific CD8+ T cells. Moreover, the tumor protective effects of P2Et treatment were abolished in immunodeficient mice, and partially lost after CD4 and CD8 depletion, indicating that P2Et's anti-tumor activity is highly dependent on immune system and at least in part of T cells. Altogether, these results support the hypothesis that the gallotannin-rich fraction P2Et's anti-tumor effects are mediated to a great extent by the endogenous immune response following to the exposure to immunogenic dying tumor cells.

Epstein-Barr virus-specific CD8(+) T lymphocytes from diffuse large B cell lymphoma patients are functionally impaired.

Epstein-Barr virus (EBV) is a persistent virus with oncogenic capacity that has been implicated in the development of aggressive B cell lymphomas, primarily in immunosuppressed individuals, although it can be present in immunocompetent individuals. Changes in the function and clonal diversity of T lymphocytes might be implied by viral persistence and lymphoma development. The aim of the present study was to evaluate the frequency, phenotype, function and clonotypical distribution of EBV-specific T cells after peripheral blood stimulation with a virus lysate in newly diagnosed patients with diffuse large B cell lymphoma (DLBCL) aged more than 50 years without prior histories of clinical immunosuppression compared with healthy controls. Our results showed impaired EBV-specific immune responses among DLBCL patients that were associated primarily with decreased numbers of central and effector memory CD8(+) T lymphocytes. In contrast to healthy controls, only a minority of the patients showed CD4(+)/tumour necrosis factor (TNF)-α(+) T cells expressing T cell receptor (TCR)-Vß17 and CD8(+)/TNF-α(+) T cells with TCR-Vß5·2, Vß9 and Vß18 in response to EBV. Notably, the production of TNF-α was undetectable among TCR-Vß5·3(+), Vß11(+), Vß12(+), Vß16(+) and Vß23(+) CD8(+) T cells. In addition, we observed decreased numbers of CD4(+)/TNF-α(+) and CD8(+)/TNF-α(+), CD8(+)/interleukin (IL)-2(+) and CD8(+)/TNF-α(+)/IL-2(+) T lymphocytes in the absence of T cells capable of producing TNF-α, IL-2 and IFN-γ after EBV stimulation simultaneously. Moreover, DLBCL patients displayed higher IL-10 levels both under baseline conditions and after EBV stimulation. These findings were also observed in patients with positive EBV viral loads. Prospective studies including a large number of patients are needed to confirm these findings.

Topological characterization of a bacterial cellulose-acrylic acid polymeric matrix.

This paper focuses on the micro- and nano-topological organization of a hydrogel, constituted by a mixture of bacterial cellulose and acrylic acid, and intended for biomedical applications. The presence of acrylic acid promotes the formation of two interpenetrated continuous phases: the primary "pores phase" (PP) containing only water and the secondary "polymeric network phase" (PNP) constituted by the polymeric network swollen by the water. Low field Nuclear Magnetic Resonance (LF NMR), rheology, Scanning Electron Microscopy (SEM) and release tests were used to determine the characteristics of the two phases. In particular, we found that this system is a strong hydrogel constituted by 81% (v/v) of PP phase the remaining part being occupied by the PNP phase. Pores diameters span in the range 10-100 µm, the majority of them (85%) falling in the range 30-90 µm. The high PP phase tortuosity indicates that big pores are not directly connected to each other, but their connection is realized by a series of interconnected small pores that rend the drug path tortuous. The PNP is characterized by a polymer volume fraction around 0.73 while mesh size is around 3 nm. The theoretical interpretation of the experimental data coming from the techniques panel adopted, yielded to the micro- and nano-organization of our hydrogel.

Therapeutic potential of nucleic acid-based drugs in coronary hyper- proliferative vascular diseases.

The thickening of the vessel wall (intimal hyperplasia) is a pathological process which often follows revascularization approaches such as transluminal angioplasty and artery bypass graft, procedures used to re-vascularize stenotic artery. Despite the significant improvements in the treatment of intimal hyperplasia obtained in the last years, the problem has not completely solved. Nucleic acid based-drugs (NABDs) represent an emergent class of molecules with potential therapeutic value for the treatment of intimal hyperplasia. NABDs of interest in the field of intimal hyperplasia are: ribozymes, DNAzymes, antisense oligonucleotides, decoy oligonucleotides, small interfering RNAs and micro interfering RNAs. These molecules can recognize, in a sequencespecific fashion, a target which, depending on the different NABDs, can be represented by a nucleic acid or a protein. Upon binding, NABDs can down-modulate the functions of the target (mRNA/proteins) and thus they are used to impair the functions of disease-causing biological molecules.In spite of the great therapeutic potential demonstrated by NABDs in many experimental model of intima hyperplasia, their practical use is hindered by the necessity to identify optimal delivery systems to the vasculature. In the first part of this review a brief description of the clinical problem related to intima hyperplasia formation after revascularization procedures is reported. In the second part, the attention is focused on the experimental evidences of NABD therapeutic potential in the prevention of intimal hyperplasia. Finally, in the third part, we will describe the strategies developed to optimize NABD delivery to the diseased vessel.

Galactomannan from Caesalpinia spinosa induces phenotypic and functional maturation of human dendritic cells.

Plant polysaccharides present an interesting potential as immunomodulators, particularly in the induction of antitumoral responses, principally because of their molecular complexity and low in vivo toxicity. Activation of dendritic cells (DCs) could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. Herein, we investigated the immunomodulatory activity of a neutral plant polysaccharide Galactomannan on human monocyte-derived DCs (MDDC). MDDCs were stimulated with Galactomannan (GLM) from Caesalpinia spinosa and both phenotypic and functional activities were assessed by flow cytometry and real-time PCR. The phagocytic ability of MDDCs was determined by using E-coli pHrodo particles and induction of T-lymphocyte allostimulation was determined after T-cell staining with carboxyfluorescein succinimidyl ester (CFSE). In MDDCs, purified Galactomannan induced phenotypic maturation revealed by increased expression of CD83, CD86, CD206, and HLA-DR. Functional experiments showed the loss of particulate antigen uptake in Galactomannan-stimulated DCs and increased alloantigen presentation capacity. Finally, Galactomannan increased protein and mRNA levels of pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, IL-12p70, and TNF-α. These data reveal that Galactomannan obtained from Caesalpinia spinosa promotes effective activation of MDDCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting.

[Rare diseases: the model of Ospedale Maggiore Policlinico IRCCS Mangiagalli Regina Elena]. / Malattie rare: il modello della Fondazione Ospedale Mag- giore Policlinico IRCCS Mangiagalli Regina Elena.

The rare diseases are a group of diseases which diagnosis, treatement and prevention is a relevant problem all over the world. They are defined on the basis of their very low incidence also if this definition is not equal all over the world. The article offers a review of the national and regional legislation on rare diseases and it describes a survey carried out in Fondazione Ospedale Maggiore Policlinico IRCCS Mangiagalli Regina Elena, a research and teaching hospital, about the organization of the network of doctors who care for these patients.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation.

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

Altered ex vivo balance between CD28+ and CD28- cells within HIV-specific CD8+ T cells of HIV-seropositive patients.

The CD8+CD28- cell population in the blood of HIV-infected individuals is considerably expanded. Yet the cause of this expansion is not clear. The recent demonstration of identical TCR-rearranged genes in CD8+CD28+ and CD8+CD28- expanded T cells of HIV-seropositive patients supports the hypothesis that these two subpopulations are phenotypic variants of the same lineage. To further elucidate the precise relationship between them, we measured the fraction of CD28+ and CD28- T cell subsets in IFN-gamma-producing CD8+ T cells by intracellular staining and cytofluorometry as a functional test for ex vivo recognition of epitopes derived from HIV-1, Epstein-Barr virus (EBV) and influenza virus. HIV-specific CD8+ T cells were predominantly CD28 in all the eight HIV-seropositive subjects tested. In contrast, the anti-EBV and anti-influenza CD8+ T cells were mainly CD28+ in these patients as well as in HIV-seronegative individuals. This supports the notion that the CD8 CD28 hyperlymphocytosis observed in HIV infection is due mainly to chronic activation and differentiation of HIV-specific memory CD8+CD28+ T cells into terminally differentiated CD8+CD28-lymphocytes, because of intense HIV-1 replication and without any important bystander activation. This clarification of the mechanisms underlying the CD8+CD28- expansion in HIV-induced pathogenesis may have important therapeutic implications.

Tartronates: a new generation of drugs affecting bone metabolism.

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM-nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.

Delayed virus-specific CD8+ cytotoxic T lymphocyte activity in an HIV-infected individual with high CD4+ cell counts: correlations with various parameters of disease progression.

This 4-year longitudinal study monitored the temporal association between the HIV-specific cytotoxic T lymphocyte (CTL) response and the control viremia in an individual infected with human immunodeficiency virus type (HIV-1). At the beginning of the study, this asymptomatic individual with a high CD4+ cell count showed no HIV-specific cytotoxic activity after polyclonal in vitro restimulation with autologous PHA-blasts, unlike most HIV-seropositive individuals. Anti-HIV CTLs were detected only in the last year of the study, both after in vitro restimulation and directly ex vivo. This was correlated with the inversion of the CD4+/CD8+ ratio, essentially due to increased numbers of CD8+CD28- T lymphocytes. The HIV-specific cytolytic activity was mediated by this CD28+CD28- subpopulation. The amount of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) did not change during the study, but the HIV RNA in plasma increased and virus was isolated from PBMCs only at the time when HIV-specific CTL activity was detected. This suggests overall that the HIV-1 replication was low in this individual, with a transient increase that could have reached the threshold for CTL reactivation, and was perhaps controlled thereby.

Ketoprofen lysine salt inhibits disuse-induced osteopenia in a new non-traumatic immobilization model in the rat.

Immobilization and the consequent unloading can cause osteopenia both in humans and in animals due to an increased bone resorption and a parallel reduction in bone formation. Non-steroidal anti-inflammatory drugs (NSAIDs), and in particular the aryl propionic acids, are described to prevent bone loss by inhibiting the cyclo-oxygenase activity. In this study we evaluated the role of a classical aryl propionic acid, ketoprofen lysine salt (KLS), in a new model of disuse-induced osteoporosis in the rat. Tail immobilization evoked a time-dependent bone loss in the caudal vertebral bodies, measured densitometrically as a reduction of bone mineral density (BMD) and content (BMC). KLS was administered once daily for 10 days by subcutaneous route at 0.5 mg kg-1, a dose lower than that effective to elicit an anti-inflammatory response. In these conditions, KLS completely abolished BMD and BMC decrease observed in the caudal vertebrae after 10-day immobilization, without affecting bone mass in normal (non-immobilized) rats. These results suggest that KLS can exert, besides to its anti-inflammatory effect, an anti-resorptive activity on bone that could be useful in the prevention of disuse-induced osteopenia.