Emanuela Balestrieri

The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Increased caspase activation in peripheral blood mononuclear cells of patients with Alzheimer's disease

In this study, we investigated whether alterations in the pattern of caspase activation could be found at the level of peripheral blood mononuclear cells (PBMCs) in patients with Alzheimers disease (AD). The results showed that in experimental conditions resembling a physiological stimulation, there was a statistically significant increase in the enzymatic activity of caspase-3, caspase-8, and caspase-9 in PBMCs from a small, but well-characterized, cohort of sporadic AD patients compared to those from a comparable control group of aged adults (AA). This was accompanied by a parallel, early increase in the cleavage activity of the same caspases. The higher level of caspase activity in PBMCs from AD compared to AA was not associated with quantitative differences in cell subset profiles. Moreover, no increase in apoptosis level, in the same experimental conditions, was found in PBMCs from this cohort of AD patients compared to those from AA. Conversely, the higher proneness to caspase activation in PBMCs from AD patients in comparison with that from AA was associated with a higher proliferative response to PHA or CD3. These data show a new dysfunction in AD patients at the PBMCs level and suggest that increased proneness to caspase activation in lymphocytes could reflect an ongoing systemic response in neurodegenerative disease with pathogenetic implications.

Thymosin α1 and cancer: action on immune effector and tumor target cells

Since it was first identified, thymosin alpha 1 (Tα1) has been characterized to have pleiotropic effects on several pathological conditions, in particular as a modulator of immune response and inflammation. Several properties exerted by Tα1 may be attributable to a direct action on lymphoid cells. Tα1 has been shown to exert an immune modulatory activity on both T cell and natural killer cell maturation and to have an effect on functions of mature lymphocytes, including stimulating cytokine production and cytotoxic T lymphocyte–mediated cytotoxic responses. In previous studies we have shown that Tα1 increases the expression of major histocompatibility complex class I surface molecules in murine and human tumor cell lines and in primary cultures of human macrophages. In the present paper, we describe preliminary data indicating that Tα1 is also capable of increasing the expression of tumor antigens in both experimental and human tumor cell lines. This effect, which is exerted at the level of the target tumor cells, represents an additional factor increasing the antitumor activity of Tα1.

Antiviral activity of seed extract from Citrus bergamia towards human retroviruses

The effects of an extract from Citrus bergamia (BSext) and those of two products purified from the same extract, that is, nomilin and limonin, and reference compounds, towards HTLV-1 have been reported. Moreover, they were also compared with those obtained towards HIV-1. Results showed that the efficacy of both BSext and limonin in inhibiting HTLV-1 as well as HIV-1 expression in infected cells, as evaluated by comparable quantitative assays, was close to that of the effective, reference compounds, respectively. The protective effect of BSext and of the purified products was associated with the inhibition of both HTLV-1 and HIV-1 RT activities in conceptually similar, cell-free assays. The cytotoxicity of the assayed compounds of natural origin was substantially less pronounced than that of the reference compounds, thus showing a favourable selectivity index for the novel BSext product.

Effect of Phosphonated Carbocyclic 2′-Oxa-3′-Aza-Nucleoside on Human T-Cell Leukemia Virus Type 1 Infection In Vitro

ABSTRACT There is currently little research and development of new compounds with specific anti-human T-cell leukemia virus type 1 (HTLV-1) activity. The few antiretrovirals that have been tested against HTLV-1 in vitro have already been developed into anti-human immunodeficiency virus (HIV) drugs. Here, we show the effects of a newly synthesized family of phosphonated nucleoside compounds, phosphonated carbocyclic 2′-oxa-3′-aza-nucleosides (PCOANs), on HTLV-1 infection in vitro. To ascertain the anti-HTLV-1 activity of PCOANs, peripheral blood mononuclear cells from healthy donors were infected in vitro by coculture with an HTLV-1 donor cell line in the presence of three prototype PCOAN compounds. PCOANs were able to completely inhibit HTLV-1 infection in vitro at a concentration of 1 μM, similar to what has been observed for tenofovir and azidothymidine. Treatment with PCOANs was associated with inhibited growth of HTLV-1-infected cells, and their effects were 100 to 200 times more potent than that of tenofovir. The mechanisms involved in the anti-HTLV-1 effects of PCOANs can mainly be ascribed to their capacity to inhibit HTLV-1 reverse transcriptase activity, as ascertained by means of a cell-free assay. PCOANs caused little reduction in proliferation or induction of apoptotic cell death of uninfected cells, showing toxicity levels similar to tenofovir and lower than azidothymidine. Overall, these results indicate that the family of PCOANs includes potential candidate compounds for long-lasting control of HTLV-1 infection.

Susceptibility of Primary HTLV-1 Isolates from Patients with HTLV-1-Associated Myelopathy to Reverse Transcriptase Inhibitors

Since human T-lymphotropic virus type 1 (HTLV-1)-associated diseases are associated with a high HTLV-1 load, reducing this load may treat or prevent disease. However, despite in vitro evidence that certain nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs) are active against HTLV-1, in vivo results have been disappointing. We therefore assayed the sensitivity of HTLV-1 primary isolates to a panel of RT inhibitors. HTLV-1 primary isolates were obtained, pre- and post- NRTI treatment, from patients with HTLV-1-associated myelopathy. Sensitivity to azidothymidine (AZT), lamivudine (3TC), tenofovir (TDF) and three phosphonated carbocyclic 2’-oxa-3’aza nucleosides (PCOANs) was assessed in a RT inhibitor assay. With the exception of 3TC, HTLV RT from primary isolates was less sensitive to all tested inhibitors than HTLV-1 RT from MT-2 cells. HTLV-1 RT from primary isolates and from chronically infected, transformed MT-2 cells was insensitive to 3TC. Sensitivity of primary isolates to RT inhibitors was not reduced following up to 12 months of patient treatment with AZT plus 3TC. The sensitivity of HTLV-1 primary isolates to NRTIs differs from that of cell lines and may vary among patients. Failure of NRTIs to reduce HTLV-1 viral load in vivo was not due to the development of phenotypic NRTI resistance. AZT and the three PCOANs assayed all consistently inhibited primary isolate HTLV-1 RT.

Effect of Lamivudine on Transmission of Human T-Cell Lymphotropic Virus Type 1 to Adult Peripheral Blood Mononuclear Cells In Vitro

ABSTRACT The effects of lamivudine (3TC) on in vitro infection of peripheral blood mononuclear cells (PBMC) from healthy donors with human T-cell lymphotropic virus type 1 (HTLV-1) were investigated. Direct measures of viral replication (viral DNA, RNA, and protein) all gave similar, very high 50% inhibitory concentrations in comparison with those previously reported for zidovudine. Nevertheless, 3TC inhibited HTLV-1-driven long-term growth of infected PBMC in vitro at concentrations (6.25 μM) which had poor or no direct antiviral effects, suggesting that another mechanism may be playing a role.

Truncated reverse isoxazolidinyl nucleosides: a new class of allosteric HIV-1 reverse transcriptase inhibitors.

The synthesiso of HEPT-derived tru ncated reversei isoxazolidinyl nucleosides( shown)is reported. These compounds represent the first exampleso f isoxazolidinebse aringa pyrimidine scaffold at the C-3 position usinga glycoside-typeli nkage.B iological evaluation showed that some of the derivativesa ct as non-nucleosidien hibitors of HIV-1r everset ranscriptasew, ith an efficacy comparable to that of Nevirapine but with reduced toxicity.

Antitumor effects of the benzophenanthridine alkaloid sanguinarine in a rat syngeneic model of colorectal cancer.

To evaluate the in-vivo preclinical antitumor activity of sanguinarine in a rat syngeneic model of colorectal cancer. The effects of sanguinarine on DHD/K12/TRb colorectal adenocarcinoma cells were first evaluated in vitro by means of 3H-thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, and terminal transferase dUTP nick end labeling (TUNEL) microscopy. For the in-vivo studies, DHD/K12/TRb cells (1.5×106 cells/0.3 ml of sterile saline/animal) were injected subcutaneously in syngeneic BDIX rats, which were chronically treated with sanguinarine (5 mg/kg/day per os) or control diluent. Tumor growth, body weight, hematologic, and clinical chemistry measurements were monitored in individual animals at defined time intervals. After killing, subcutaneous tumors were explanted from experimental animals for histopathological examination. In vitro, micromolar concentrations of sanguinarine inhibited dose-dependently DHD/K12/TRb cell proliferation and metabolism and induced cell death by apoptosis. In vivo, oral administration of sanguinarine induced a significant inhibition of tumor growth (P<0.01 vs. untreated controls), in the absence of any toxic or side effects. Marked apoptosis and reduced peritumoral vascularization were observed in tumors from sanguinarine-treated rats as compared with the controls. Additional basic studies are needed to fully characterize the mechanism/s underlying the inhibitory effects of sanguinarine on angiogenesis and tumor growth as well as the pharmacological and safety profile of this drug in experimental tumor models. Overall, findings from this study suggest that sanguinarine is a likely candidate for further evaluation in cancer therapy.

HERVs expression in Autism Spectrum Disorders.

Background Autistic Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder, resulting from complex interactions among genetic, genomic and environmental factors. Here we have studied the expression of Human Endogenous Retroviruses (HERVs), non-coding DNA elements with potential regulatory functions, and have tested their possible implication in autism. Methods The presence of retroviral mRNAs from four HERV families (E, H, K and W), widely implicated in complex diseases, was evaluated in peripheral blood mononuclear cells (PBMCs) from ASD patients and healthy controls (HCs) by qualitative RT-PCR. We also analyzed the expression of the env sequence from HERV-H, HERV-W and HERV-K families in PBMCs at the time of sampling and after stimulation in culture, in both ASD and HC groups, by quantitative Real-time PCR. Differences between groups were evaluated using statistical methods. Results The percentage of HERV-H and HERV-W positive samples was higher among ASD patients compared to HCs, while HERV-K was similarly represented and HERV-E virtually absent in both groups. The quantitative evaluation shows that HERV-H and HERV-W are differentially expressed in the two groups, with HERV-H being more abundantly expressed and, conversely, HERV-W, having lower abundance, in PBMCs from ASDs compared to healthy controls. PMBCs from ASDs also showed an increased potential to up-regulate HERV-H expression upon stimulation in culture, unlike HCs. Furthermore we report a negative correlation between expression levels of HERV-H and age among ASD patients and a statistically significant higher expression in ASD patients with Severe score in Communication and Motor Psychoeducational Profile-3. Conclusions Specific HERV families have a distinctive expression profile in ASD patients compared to HCs. We propose that HERV-H expression be explored in larger samples of individuals with autism spectrum in order to determine its utility as a novel biological trait of this complex disorder.