Inflammatory profile of vertically HIV-1 infected adolescents receiving ART in Cameroon: a contribution toward optimal pediatric HIV control strategies.

Antiretroviral therapy (ART) has improved the lifespan of people living with HIV. However, their immune system remains in a state of sustained activation/inflammation, which favors viral replication and depletion of helper T-cells with varying profiles according to ART-response. We herein sought to ascertain the inflammatory profile of adolescents living with perinatal HIV-1 infection (ALPHI) receiving ART in an African context. In this cross-sectional and comparative study among ART-experienced ALPHI in Yaoundé-Cameroon, HIV-1 RNA was measured by Abbott Real-time PCR; CD4 cells were enumerated using flow cytometry; serum cytokines were measured by ELISA; HIV-1 proviral DNA was genotyped by Sanger-sequencing; and archived drug resistance mutations (ADRMs) were interpreted using Stanford HIVdb.v9.0.1. Overall, 73 adolescents were enrolled (60 ALPHI and 13 HIV-1 negative peers) aged 15 (13-18) years; 60.00% were female. ART median duration was 92 (46-123) months; median viral load was 3.99 (3.17-4.66) RNA Log10 (copies)/mL and median CD4 count was 326 (201-654) cells/mm3. As compared to HIV-negative adolescents, TNFα was highly expressed among ALPHI (p<0.01). Following a virological response, inflammatory cytokines (IFNγ and IL-12), anti-inflammatory cytokines (IL-4 and IL-10) and inflammation-related cytokines (IL-6 and IL-1ß) were highly expressed with viral suppression (VS) vs. virological failure (VF), while the chemokine CCL3 was highly expressed with VF (p<0.01). Regarding the immune response, the inflammatory cytokine TNFα was highly expressed in those that are immunocompetent (CD4≥500 cell/mm3) vs. immunocompromised (CD4<500 cell/mm3), p ≤ 0.01; while chemokine CCL2 was highly expressed in the immunocompromised (p<0.05). In the presence of ADRMs, IL-4 and CCL3 were highly expressed (p=0.027 and p=0.043 respectively). Among ART-experienced ALPHI in Cameroon, the TNFα cytokine was found to be an inflammatory marker of HIV infection; IFNγ, IL-1ß, IL-6, and IL-12 are potential immunological markers of VS and targeting these cytokines in addition to antiretroviral drugs may improve management. Moreover, CCL3 and CCL2 are possible predictors of VF and/or being immunocompromised and could serve as surrogates of poor ART response.

Exogenous miRNAs from Moringa oleifera Lam. recover a dysregulated lipid metabolism.

A balanced diet is critical for human health, and edible plants play an important role in providing essential micronutrients as well as specific microRNAs (miRNAs) that can regulate human gene expression. Here we present the effects of Moringa oleifera (MO) miRNAs (mol-miRs) on lipid metabolism. Through in silico studies we identified the potential genes involved in lipid metabolism targeted by mol-miRs. To this end, we tested the efficacy of an aqueous extract of MO seeds (MOES), as suggested in traditional African ethnomedicine, or its purified miRNAs. The biological properties of MO preparations were investigated using a human derived hepatoma cell line (HepG2) as a model. MOES treatment decreased intracellular lipid accumulation and induced apoptosis in HepG2. In the same cell line, transfection with mol-miRs showed similar effects to MOES. Moreover, the effect of the mol-miR pool was investigated in a pre-obese mouse model, in which treatment with mol-miRs was able to prevent dysregulation of lipid metabolism.

NH-sulfoximine: A novel pharmacological inhibitor of the mitochondrial F1 Fo -ATPase, which suppresses viability of cancerous cells.

BACKGROUND AND PURPOSE: The mitochondrial F1 Fo -ATPsynthase is pivotal for cellular homeostasis. When respiration is perturbed, its mode of action everts becoming an F1 Fo -ATPase and therefore consuming rather producing ATP. Such a reversion is an obvious target for pharmacological intervention to counteract pathologies. Despite this, tools to selectively inhibit the phases of ATP hydrolysis without affecting the production of ATP remain scarce. Here, we report on a newly synthesised chemical, the NH-sulfoximine (NHS), which achieves such a selectivity. EXPERIMENTAL APPROACH: The chemical structure of the F1 Fo -ATPase inhibitor BTB-06584 was used as a template to synthesise NHS. We assessed its pharmacology in human neuroblastoma SH-SY5Y cells in which we profiled ATP levels, redox signalling, autophagy pathways and cellular viability. NHS was given alone or in combination with either the glucose analogue 2-deoxyglucose (2-DG) or the chemotherapeutic agent etoposide. KEY RESULTS: NHS selectively blocks the consumption of ATP by mitochondria leading a subtle cytotoxicity associated via the concomitant engagement of autophagy which impairs cell viability. NHS achieves such a function independently of the F1 Fo -ATPase inhibitory factor 1 (IF1). CONCLUSION AND IMPLICATIONS: The novel sulfoximine analogue of BTB-06584, NHS, acts as a selective pharmacological inhibitor of the mitochondrial F1 Fo -ATPase. NHS, by blocking the hydrolysis of ATP perturbs the bioenergetic homoeostasis of cancer cells, leading to a non-apoptotic type of cell death.

Effect of microvesicles from Moringa oleifera containing miRNA on proliferation and apoptosis in tumor cell lines.

Human microvesicles are key mediators of cell-cell communication. Exosomes function as microRNA transporters, playing a crucial role in physiological and pathological processes. Plant microvesicles (MVs) display similar features to mammalian exosomes, and these MVs might enhance plant microRNA delivery in mammals. Considering that plant microRNAs have been newly identified as bioactive constituents in medicinal plants, and that their potential role as regulators in mammals has been underlined, in this study, we characterized MVs purified from Moringa oleifera seeds aqueous extract (MOES MVs) and used flow cytometry methods to quantify the ability to deliver their content to host cells. The microRNAs present in MOES MVs were characterized, and through a bioinformatic analysis, specific human apoptosis-related target genes of plant miRNAs were identified. In tumor cell lines, MOES MVs treatment reduced viability, increased apoptosis levels associated with a decrease in B-cell lymphoma 2 protein expression and reduced mitochondrial membrane potential. Interestingly, the effects observed with MOES MVs treatment were comparable to those observed with MOES treatment and transfection with the pool of small RNAs isolated from MOES, used as a control. These results highlight the role of microRNAs transported by MOES MVs as natural bioactive plant compounds that counteract tumorigenesis.

Adoptive Cell Therapy-Harnessing Antigen-Specific T Cells to Target Solid Tumours.

In recent years, much research has been focused on the field of adoptive cell therapies (ACT) that use native or genetically modified T cells as therapeutic tools. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) demonstrated great success in the treatment of haematologic malignancies, whereas adoptive transfer of autologous tumour infiltrating lymphocytes (TILs) proved to be highly effective in metastatic melanoma. These encouraging results initiated many studies where ACT was tested as a treatment for various solid tumours. In this review, we provide an overview of the challenges of T cell-based immunotherapies of solid tumours. We describe alternative approaches for choosing the most efficient T cells for cancer treatment in terms of their tumour-specificity and phenotype. Finally, we present strategies for improvement of anti-tumour potential of T cells, including combination therapies.

Adipocyte metabolism is improved by TNF receptor-targeting small RNAs identified from dried nuts.

There is a growing interest in therapeutically targeting the inflammatory response that underlies age-related chronic diseases including obesity and type 2 diabetes. Through integrative small RNA sequencing, we show the presence of conserved plant miR159a and miR156c in dried nuts having high complementarity with the mammalian TNF receptor superfamily member 1a (Tnfrsf1a) transcript. We detected both miR159a and miR156c in exosome-like nut nanovesicles (NVs) and demonstrated that such NVs reduce Tnfrsf1a protein and dampen TNF-α signaling pathway in adipocytes. Synthetic single-stranded microRNAs (ss-miRs) modified with 2'-O-methyl group function as miR mimics. In plants, this modification naturally occurs on nearly all small RNAs. 2'-O-methylated ss-miR mimics for miR156c and miR159a decreased Tnfrsf1a protein and inflammatory markers in hypertrophic as well as TNF-α-treated adipocytes and macrophages. miR156c and miR159a mimics effectively suppress inflammation in mice, highlighting a potential role of plant miR-based, single-stranded oligonucleotides in treating inflammatory-associated metabolic diseases.

Cytotoxic and apoptotic effects of different extracts of Moringa oleifera Lam on lymphoid and monocytoid cells.

Moringa oleifera Lam. (MO) is one of the most well-known and widely distributed species of the Moringaceae family in African communities, and various preparations of M. oleifera are used for the treatment of several diseases. Due to the extensive worldwide use of MO products, and the use of MO aqueous extract in traditional African medicine, the aim of the present study was to investigate the anti-proliferative, cytotoxic and pro-apoptotic activities of different aqueous extracts from leaves and seeds of M. oleifera (MOE), which have been prepared using different protocols, in lymphoid and monocytoid cells. The results of the present study demonstrated the anti-proliferative and pro-apoptotic effects of the aqueous extracts obtained from M. oleifera leaves and seeds on tumour cells; however, not on peripheral blood mononuclear cells (PBMCs) from healthy donors. The pro-apoptotic effect of MO seed aqueous extract (MOE-S) was correlated with decreased B-cell lymphoma 2 (BCL2) and sirtuin-1 (SIRT1) protein expression, which are involved in apoptosis. Considering the effects of plant secondary metabolites on human cells and the role of plant microRNA in cross-kingdom interactions, the presence of secondary metabolites and microRNA in MOE was characterised. In conclusion, M. oleifera aqueous extracts appeared to be able to differentially regulate proliferation and apoptosis in healthy cells and cancer cells, and this ability could be associated with the microRNA present in the extracts. These results highlighted the possible use of MOE as an adjuvant in traditional cancer therapy.

Olea europaea small RNA with functional homology to human miR34a in cross-kingdom interaction of anti-tumoral response.

Functional foods include compounds with nutritional and health properties. The human diet could play a stronger role in cancer prevention. Only a few studies have described the presence of plant small RNA, in humans who were fed with plant foods, which demonstrated the ability of these molecules to modulate consumer's genes and evidenced the existence of a plant-animal regulation. Through in silico prediction, Olea europaea small RNAs (sRs), which had been previously reported as miRNAs, were identified, each with functional homology to hsa-miR34a. According to this initial funding, we investigated the ability of oeu-sRs to regulate tumorigenesis in human cells. The transfection of these synthetic oeu-sRs reduced the protein expression of hsa-miR34a mRNA targets, increased apoptosis and decreased proliferation in different tumor cells; by contrast, no effect was observed in PBMCs from healthy donors. The introduction of oeu-small RNA in hsa-miR34a-deficient tumor cells restores its function, whereas cells with normal expression of endogenous hsa-miR34a remained unaffected. The natural oeu-small RNAs that were extracted from O. europaea drupes induce the same effects as synthetic sRs. Careful research on the small RNA sequences executed for mapping and annotation in the genome of O. europaea var. Sylvestris and var. Farga led to the hypothesis that RNA fragments with functional homology to human miRNAs could be generated from the degradation of regions of RNA transcripts. These results indicate the possibility of developing novel natural non-toxic drugs that contain active plant-derived tumor-suppressing small RNA with functional homology to hsa-miRNAs and that can support antineoplastic strategies.

Bioinformatics Prediction and Experimental Validation of MicroRNAs Involved in Cross-Kingdom Interaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as efficient post-transcriptional regulators of gene expression. In 2012, the first cross-kingdom miRNA-based interaction had been evidenced, demonstrating that exogenous miRNAs act in a manner of mammalian functional miRNAs. Starting from this evidence, we defined the concept of cross-kingdom functional homology between plant and mammalian miRNAs as a needful requirement for vegetal miRNA to explicit a regulation mechanism into the host mammalian cell, comparable to the endogenous one. Then, we proposed a new dedicated algorithm to compare plant and mammalian miRNAs, searching for functional sequence homologies between them, and we developed a web software called MirCompare. We also predicted human genes regulated by the selected plant miRNAs, and we determined the role of exogenous miRNAs in the perturbation of intracellular interaction networks. Finally, as already performed by Pirrò and coworkers, the ability of MirCompare to select plant miRNAs with functional homologies with mammalian ones has been experimentally confirmed by evaluating the ability of mol-miR168a to downregulate the protein expression of SIRT1, when its mimic is transfected into human hepatoma cell line G2 (HEPG2) cells. This tool is implemented into a user-friendly web interface, and the access is free to public through the website http://160.80.35.140/MirCompare.

MicroRNA from Moringa oleifera: Identification by High Throughput Sequencing and Their Potential Contribution to Plant Medicinal Value.

Moringa oleifera is a widespread plant with substantial nutritional and medicinal value. We postulated that microRNAs (miRNAs), which are endogenous, noncoding small RNAs regulating gene expression at the post-transcriptional level, might contribute to the medicinal properties of plants of this species after ingestion into human body, regulating human gene expression. However, the knowledge is scarce about miRNA in Moringa. Furthermore, in order to test the hypothesis on the pharmacological potential properties of miRNA, we conducted a high-throughput sequencing analysis using the Illumina platform. A total of 31,290,964 raw reads were produced from a library of small RNA isolated from M. oleifera seeds. We identified 94 conserved and two novel miRNAs that were validated by qRT-PCR assays. Results from qRT-PCR trials conducted on the expression of 20 Moringa miRNA showed that are conserved across multiple plant species as determined by their detection in tissue of other common crop plants. In silico analyses predicted target genes for the conserved miRNA that in turn allowed to relate the miRNAs to the regulation of physiological processes. Some of the predicted plant miRNAs have functional homology to their mammalian counterparts and regulated human genes when they were transfected into cell lines. To our knowledge, this is the first report of discovering M. oleifera miRNAs based on high-throughput sequencing and bioinformatics analysis and we provided new insight into a potential cross-species control of human gene expression. The widespread cultivation and consumption of M. oleifera, for nutritional and medicinal purposes, brings humans into close contact with products and extracts of this plant species. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for beneficial properties of this valuable species.

Amino acid mutations in Ebola virus glycoprotein of the 2014 epidemic.

Zaire Ebola virus (EBOV) is an enveloped non-segmented negative strand RNA virus of 19 kb in length belonging to the family Filoviridae. The virus was isolated and identified in 1976 during the epidemic of hemorrhagic fever in Zaire. The most recent outbreak of EBOV among humans, was that occurred in the forested areas of south eastern Guinea, that began in February 2014 and is still ongoing. The recent Ebola outbreak, is affecting other countries in West Africa, in addiction to Guinea: Liberia, Nigeria, and Sierra Leone. In this article, a selective pressure analysis and homology modeling based on the G Glycoprotein (GP) sequences retrieved from public databases were used to investigate the genetic diversity and modification of antibody response in the recent outbreak of Ebola Virus. Structural and the evolutionary analysis underline the 2014 epidemic virus being under negative selective pressure does not change with respect to the old epidemic in terms of genome adaptation.

Antioxidant and anti-inflammatory activities of extracts from Cassia alata, Eleusine indica, Eremomastax speciosa, Carica papaya and Polyscias fulva medicinal plants collected in Cameroon.

BACKGROUND: The vast majority of the population around the world has always used medicinal plants as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice. OBJECTIVE: The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of ethanol extracts of leaves of Cassia alata, Eleusine indica, Carica papaya, Eremomastax speciosa and the stem bark of Polyscias fulva, collected in Cameroon. METHODS: Chemiluminescence was used to analyze the antioxidant activities of plant extracts against hydrogen peroxide or superoxide anion. Comet assays were used to analyze the protection against antioxidant-induced DNA damage induced in white blood cells after treating with hydrogen peroxide. Flow cytometry was used to measure γδ T cells proliferation and anti-inflammatory activity of γδ T cells and of immature dendritic cells (imDC) in the presence of different concentrations of plant extracts. RESULTS: Ethanol extracts showed strong antioxidant properties against both hydrogen peroxide and superoxide anion. Cassia alata showed the highest antioxidant activity. The effect of plant extracts on γδ T cells and imDC was evidenced by the dose dependent reduction in TNF-α production in the presence of Cassia alata, Carica papaya, Eremomastax speciosa Eleusine indica, and Polyscias fulva. γδ T cells proliferation was affected to the greatest extent by Polyscias fulva. CONCLUSION: These results clearly show the antioxidant capacity and anti-inflammatory activities of plant extracts collected in Cameroon. These properties of leaves and stem bark extracts may contribute to the value for these plants in traditional medicine and in general medical practice.

Immunotherapy with an HIV-DNA Vaccine in Children and Adults.

Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals' immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children's and adults' responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4-16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation and late CD8+ T-cell responses. In the adult cohort, primarily CD8+ T-cell responses related to MHC class I alleles were noted. However, no clinical benefits with respect to viral load reduction were ascribable to the vaccinations alone. No severe adverse effects related to the vaccine were found in either cohort, and no virological failures or drug resistances were detected.

Therapeutic DNA vaccination of vertically HIV-infected children: report of the first pediatric randomised trial (PEDVAC).

SUBJECTS: Twenty vertically HIV-infected children, 6-16 years of age, with stable viral load control and CD4+ values above 400 cells/mm(3). INTERVENTION: Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. RESULTS: Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. CONCLUSIONS: The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. TRIAL REGISTRATION: clinicaltrialsregister.eu _2007-002359-18IT.

An abnormal phenotype of lung Vγ9Vδ2 T cells impairs their responsiveness in tuberculosis patients.

Antigen-specific γδ T cells represent an early innate defense known to play an important role in anti-mycobacterial immunity. We have investigated the immune functions of Vγ9Vδ2 T cells from Broncho-Alveolar lavages (BAC) samples of active TB patients. We observed that BAC Vγ9Vδ2 T cells presented a strong down-modulation of CD3 expression compared with Vγ9Vδ2 T cells from peripheral blood. Furthermore, Vγ9Vδ2 T cells mainly showed a central memory phenotype, expressed high levels of NK inhibitory receptors and TEMRA cells showed low expression of CD16 compared to circulating Vγ9Vδ2 T cells. Interestingly, the ability of BAC Vγ9Vδ2 T cells to respond to antigen stimulation was dramatically reduced, differently from blood counterpart. These observations indicate that γδ T cell functions are specifically impaired in situ by active TB, suggesting that the alveolar ambient during tuberculosis may affect resident γδ T cells in comparison to circulating cells.

Aloe-emodin as antiproliferative and differentiating agent on human U937 monoblastic leukemia cells.

AIMS: Aloe-emodin (AE), a plant derived anthraquinone, has been shown to have anticancer activity in various human cancer cell lines. We have recently reported that AE possesses a differentiative potential on melanoma cells. The purpose of this study was to investigate the possible modulation of defined markers of monocytic differentiation of AE on human U937 cell line. MAIN METHODS: U937 cells differentiation has been confirmed unequivocally by Griess and nitroblue tetrazolium reduction assays, protoporphyrin IX accumulation, expression of CD14 and CD11b surface antigens, phagocytic activity, migration and attachment ability. The effect on polyamine metabolism, apoptosis and cytokine production was also investigated. KEY FINDINGS: We showed that AE-treated U937 cells exhibit a noticeably rise in transglutaminase activity. This enhanced enzyme activity correlates with AE-induced growth arrest and differentiation to functionally mature monocytes. SIGNIFICANCE: Taken together, the results reported here show that AE can promote the macrophage differentiation of U937 cells, suggesting that this anthraquinone could be a potential candidate as a differentiation-inducing selective agent for therapeutic treatment of leukemia.

The PEDVAC trial: preliminary data from the first therapeutic DNA vaccination in HIV-infected children.

The PEDVAC study is the first trial designed to analyze safety and immunogenicity of a therapeutic vaccination with a multiclade multigene HIV DNA vaccine (HIVIS) in infected children. Twenty HIV-1 vertically infected children (6-16 years of age), on stable antiretroviral treatment for at least 6 months with HIV-1 RNA<50 copies/ml and stable CD4 counts (> 400 cells/mm³ or 25%) over 12 months of follow-up, were recruited into the study. Enrolled patients have been randomized into two arms: a control group of 10 children who continued previous antiretroviral treatment (HAART) (arm A) and a group of 10 children immunized intramuscularly with the HIVIS DNA vaccine in addition to previous HAART (arm B). Immunizations took place at week 0, 4, 12 and the boosting dose is planned at week 36. The 10 children in the vaccine group have received the first 3 priming doses of the HIVIS vaccine. Safety data showed good tolerance to the vaccination schedule. Mild cutaneous self-limeted reactions consisted of local irritation, usually itching or erythema +/- swelling at the injection site, were reported. No severe systemic adverse events have been observed. No vaccinated children had a decrease of CD4 T-cell counts from baseline. None experienced virological failure. Analysis of cellular immune responses was scheduled at week 0, 4, 12, 16, 20, 40, 60, 72 and 96 by standard lymphoproliferation assay, intracellular cytokine staining and cell-ELISA, a miniaturized assay to measure antigen-induced IFNγ secretion. Evaluation of these results is in progress and will provide key information on the status and changes of antigen specific immunity during HIV DNA immunization.

Delayed early antiretroviral treatment is associated with an HIV-specific long-term cellular response in HIV-1 vertically infected infants.

Antiviral T-cell immune responses appear to be crucial to control HIV replication. Infants treated before the third month of life with highly active antiretroviral treatment (HAART) did not develop a persistent HIV-specific immune response. We evaluated how delayed initiation of HAART after 3 months of age influences the development of HIV-1-specific T-cell responses during long-term follow-up in 9 HIV-1 vertically infected infants. These data suggest that a longer antigenic stimulation, due to a larger window for therapeutic intervention with HAART, is associated with the establishment of a persistent specific HIV immune response resulting in a long-term viral control of vertically infected infants.

Cell cycle arrest and differentiation induction by 5,7-dimethoxycoumarin in melanoma cell lines.

In the present study we investigated the antiproliferative activity of 5,7-dimethoxycoumarin on the murine B16 and human A375 melanoma cell lines. The inhibitory concentration 50 (IC50) was estimated for each cell line by preliminary assay of tetrazolium salt reduction (MTT). With Trypan blue exclusion test we detected a cytostatic but not cytotoxic effect of the treatment in melanoma cells: 5,7-dimethoxycoumarin significantly reduced cell proliferation in a time- and dose-dependent manner, blocking the cell cycle in the G0/G1 phase both in B16 and A375 cells. Melanoma growth reduction was coupled to a differentiation process detected by monitoring some specific markers: i) morphological changes with development of dendrite-like projections from the cell surface; ii) melanin synthesis; and iii) PpIX accumulation. Induction of the differentiation process was more significant in murine melanoma cells, where the treatment irreversibly reduced cell growth. Consistent with G0/G1 arrest and melanogenesis in B16 cells, 5,7-dimethoxycoumarin strongly decreased activation of the mitogen-activated protein kinase extracellular signal-related kinase 1/2, which is upregulated in many types of cancer. These findings suggest that 5,7-dimethoxycoumarin should be further investigated through studies both in vitro, to identify the binding partners for this compound, and in preclinical animal models.