The Variation in the Traits Ameliorated by Inhibitors of JAK1/2, TGF-ß, P-Selectin, and CXCR1/CXCR2 in the Gata1low Model Suggests That Myelofibrosis Should Be Treated by These Drugs in Combination.

Studies conducted on animal models have identified several therapeutic targets for myelofibrosis, the most severe of the myeloproliferative neoplasms. Unfortunately, many of the drugs which were effective in pre-clinical settings had modest efficacy when tested in the clinic. This discrepancy suggests that treatment for this disease requires combination therapies. To rationalize possible combinations, the efficacy in the Gata1low model of drugs currently used for these patients (the JAK1/2 inhibitor Ruxolitinib) was compared with that of drugs targeting other abnormalities, such as p27kip1 (Aplidin), TGF-ß (SB431542, inhibiting ALK5 downstream to transforming growth factor beta (TGF-ß) signaling and TGF-ß trap AVID200), P-selectin (RB40.34), and CXCL1 (Reparixin, inhibiting the CXCL1 receptors CXCR1/2). The comparison was carried out by expressing the endpoints, which had either already been published or had been retrospectively obtained for this study, as the fold change of the values in the corresponding vehicles. In this model, only Ruxolitinib was found to decrease spleen size, only Aplidin and SB431542/AVID200 increased platelet counts, and with the exception of AVID200, all the inhibitors reduced fibrosis and microvessel density. The greatest effects were exerted by Reparixin, which also reduced TGF-ß content. None of the drugs reduced osteopetrosis. These results suggest that future therapies for myelofibrosis should consider combining JAK1/2 inhibitors with drugs targeting hematopoietic stem cells (p27Kip1) or the pro-inflammatory milieu (TGF-ß or CXCL1).

Dendritic cells modulate c-kit expression on the edge between activation and death.

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI-/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.

Extracellular vesicle-based anti-HOXB7 CD8+ T cell-specific vaccination strengthens antitumor effects induced by vaccination against Her2/neu.

We previously developed an innovative strategy to induce CD8+ T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+ T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two "self" tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.

Identification of Rv1133c (MetE) as a marker of Mycobacterium tuberculosis replication and as a highly immunogenic antigen with potential immunodiagnostic power.

The immunization of mice with the sterile culture medium supernatants of Mycobacterium tuberculosis (Mtb) H37Rv permitted the production of several monoclonal antibodies (mAbs) specific for secreted and/or released antigens. Two mAbs bound and immunoprecipitated an 80-kDa protein that was identified by mass spectrometry as Rv1133c, the methionine synthase MetE. The protein MetE is ubiquitous among prokaryota and shows a significant sequence homology in many bacteria. We produced both the full-length recombinant MetE and its N-terminal fragment, whose sequence is more conserved among mycobacteria, to select mAbs recognizing an Mtb-specific region of MetE. Finally, we produced and selected eight mAbs that specifically detect the MetE protein in the supernatant and cell lysate of Mtb and BCG, but not other bacteria such as non-tuberculous mycobacteria (NTM), Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter baumanii, or Escherichia coli. Taking advantage of our mAbs, we studied (i) the vitamin B12 dependence for the synthesis of MetE in Mtb and NTM and (ii) the kinetics of MetE production and secretion in supernatants during the in vitro reproduced replicative, dormant, and resuscitation cycle of Mtb. Our data demonstrate that dormant Mtb, which are assumed to be prevalent in latent infections, as well as NTM do not produce and secrete MetE. Results indicate an unexpected specificity for Mtb of our anti-MetE mAbs and encourage the use of rMetE and our mAbs as tools for the immunodiagnosis of TB and its stages.

Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice.

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34+ cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA+TPO inhibited HSPC differentiation and preserved the CD34+ cell fraction; the self-renewal of the CD34+ TPO+VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO+VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO+VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO+VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO+VPA-treated cells in vitro.

SARS-CoV-2-Specific CD8+ T-Cells in Blood but Not in the Lungs of Vaccinated K18-hACE2 Mice after Infection.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. Then, the virus can spread towards the oropharyngeal cavity and the pulmonary tissues. The antiviral adaptive immunity is promptly induced in response to the virus's detection, with virus-specific T-lymphocytes appearing before antiviral antibodies. Both the breadth and potency of antiviral CD8+ T-cell immunity have a key role in containing viral spread and disease severity. Current anti-SARS-CoV-2 vaccines do not impede the virus's replication in the upper respiratory tract, and there is consensus on the fact that the best potency of the antiviral immune response in both blood and the upper respiratory tract can be reached upon infection in vaccinees (i.e., breakthrough infection). However, whether the antiviral CD8+ T-cells developing in response to the breakthrough infection in the upper respiratory tract diffuse to the lungs is also still largely unknown. To fill the gap, we checked the CD8+ T-cell immunity elicited after infection of K18-hACE2 transgenic mice both at 3 weeks and 3 months after anti-spike vaccination. Virus-specific CD8+ T-cell immunity was monitored in both blood and the lungs before and after infection. By investigating the de novo generation of the CD8+ T-cells specific for SARS-CoV-2 viral proteins, we found that both membrane (M) and/or nucleocapsid (N)-specific CD8+ T-cells were induced at comparable levels in the blood of both unvaccinated and vaccinated mice. Conversely, N-specific CD8+ T-cells were readily found in the lungs of the control mice but were either rare or absent in those of vaccinated mice. These results support the idea that the hybrid cell immunity developing after asymptomatic/mild breakthrough infection strengthens the antiviral cell immunity in the lungs only marginally, implying that the direct exposition of viral antigens is required for the induction of an efficient antiviral cell immunity in the lungs.

Antiviral effect of SARS-CoV-2 N-specific CD8+ T cells induced in lungs by engineered extracellular vesicles.

Induction of effective immunity in the lungs should be a requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 Nucleocapsid (N) protein induced immunity in the lungs of K18-hACE2 transgenic mice, which then can survive the lethal virus infection. However, nothing is known about the ability of the N-specific CD8+ T cell immunity in controlling viral replication in the lungs, a major pathogenic signature of severe disease in humans. To fill the gap, we investigated the immunity generated in the lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8+ T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in the lungs were evaluated. Three weeks after the second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to the control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8+ T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting, and associated with the persistence of N-specific CD8+ T-resident memory lymphocytes. In view of the quite low mutation rate of the N protein, the present vaccine strategy has the potential to control the replication of all emerging variants.

Preclinical studies on the use of a P-selectin-blocking monoclonal antibody to halt progression of myelofibrosis in the Gata1low mouse model.

The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-ß (TGF-ß) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-ß and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice.

SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization.

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Juvenile Toxicity Rodent Model to Study Toxicological Effects of Bisphenol A (BPA) at Dose Levels Derived From Italian Children Biomonitoring Study.

Bisphenol A (BPA) is a plasticizer with endocrine disrupting properties particularly relevant for children health. Recently BPA has been associated with metabolic dysfunctions but no data are yet available in specific, long-term studies. This study aimed to evaluate BPA modes of action and hazards during animal juvenile life-stage, corresponding to childhood. Immature Sprague-Dawley rats of both sexes were orally treated with 0 (vehicle only-olive oil), 2, 6, and 18 mg/kg bw per day of BPA for 28 days, from weaning to sexual maturity. Dose levels were obtained from the PERSUADED biomonitoring study in Italian children. Both no-observed-adverse-effect-level (NOAEL)/low-observed-adverse-effect-level (LOAEL) and estimated benchmark dose (BMD) approaches were applied. General toxicity, parameters of sexual development, endocrine/reproductive/functional liver and kidney biomarkers, histopathology of target tissues, and gene expression in hypothalamic-pituitary area and liver were studied. No mortality or general toxicity occurred. Sex-specific alterations were observed in liver, thyroid, spleen, leptin/adiponectin serum levels, and hypothalamic-pituitary gene expression. Thyroid homeostasis and liver were the most sensitive targets of BPA exposure in the peripubertal phase. The proposed LOAEL was 2 mg/kg bw, considering as critical effect the liver endpoints, kidney weight in male and adrenal histomorphometrical alterations and osteopontin upregulation in female rats. The BMD lower bounds were 0.05 and 1.33 mg/kg bw in males and females, considering liver and thyroid biomarkers, respectively. Overall, BPA evaluation at dose levels derived from children biomonitoring study allowed to identify sex-specific, targeted toxicological effects that may have significant impact on risk assessment for children.

Development and Preclinical Evaluation of an Integrase Defective Lentiviral Vector Vaccine Expressing the HIVACAT T Cell Immunogen in Mice.

Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.

Hazard identification of pyrogenic synthetic amorphous silica (NM-203) after sub-chronic oral exposure in rat: A multitarget approach.

Food additive E551 consists of synthetic amorphous silica (SAS), comprising agglomerates and aggregates of primary particles in the nanorange (<100 nm), which potential nanospecific risks for humans associated to dietary exposure are not yet completely assessed. In NANoREG project, aim of the study was to identify potential hazards of pyrogenic SAS nanomaterial NM-203 by a 90-day oral toxicity study (OECD test guideline 408). Adult Sprague-Dawley rats of both sexes were orally treated with 0, 2, 5, 10, 20 and 50 mg SAS/kg bw per day; dose levels were selected to be as close as possible to E551 dietary exposure. Several endpoints were investigated, the whole integrative study is presented here along with the results of dispersion characterization, tissue distribution, general toxicity, blood/serum biomarkers, histopathological and immunotoxicity endpoints. No mortality, general toxicity and limited deposition in target tissues were observed. NM-203 affected liver and spleen in both sexes. Proposed NOAEL 5 mg/kg bw per day in male rats for enlarged sinusoids in liver. In female rats, TSH and creatinine levels were affected, proposed LOAEL 2 mg/kg bw per day. Overall, these data provide new insight for a comprehensive risk assessment of SAS exposure by the oral route.

A novel oral micellar fenretinide formulation with enhanced bioavailability and antitumour activity against multiple tumours from cancer stem cells.

BACKGROUND: An increasing number of anticancer agents has been proposed in recent years with the attempt to overcome treatment-resistant cancer cells and particularly cancer stem cells (CSC), the major culprits for tumour resistance and recurrence. However, a huge obstacle to treatment success is the ineffective delivery of drugs within the tumour environment due to limited solubility, short circulation time or inconsistent stability of compounds that, together with concomitant dose-limiting systemic toxicity, contribute to hamper the achievement of therapeutic drug concentrations. The synthetic retinoid Fenretinide (4-hydroxy (phenyl)retinamide; 4-HPR) formerly emerged as a promising anticancer agent based on pre-clinical and clinical studies. However, a major limitation of fenretinide is traditionally represented by its poor aqueous solubility/bioavailability due to its hydrophobic nature, that undermined the clinical success of previous clinical trials. METHODS: Here, we developed a novel nano-micellar fenretinide formulation called bionanofenretinide (Bio-nFeR), based on drug encapsulation in an ion-pair stabilized lipid matrix, with the aim to raise fenretinide bioavailability and antitumour efficacy. RESULTS: Bio-nFeR displayed marked antitumour activity against lung, colon and melanoma CSC both in vitro and in tumour xenografts, in absence of mice toxicity. Bio-nFeR is suitable for oral administration, reaching therapeutic concentrations within tumours and an unprecedented therapeutic activity in vivo as single agent. CONCLUSION: Altogether, our results indicate Bio-nFeR as a novel anticancer agent with low toxicity and high activity against tumourigenic cells, potentially useful for the treatment of solid tumours of multiple origin.

Successful radiofrequency ablation determines atrio-ventricular remodelling and improves systo-diastolic function at tissue Doppler-imaging.

BACKGROUND: Clinical, echocardiographic results and determinants of atrial fibrillation (AF) recurrence following AF ablation during mitral valve surgery (AFAMVS) were evaluated. METHODS: Fifty-two patients undergoing radiofrequency AFAMVS between January 2003 and December 2005, underwent serial echocardiographies with tissue Doppler imaging to assess atrio-ventricular function. Recurrence of AF, hospital readmission, episodes of congestive heart failure (CHF) were recorded. Predictors for AF-recurrence were evaluated. RESULTS: At a 29.5+/-8.6 months of follow-up (100% complete), 78.8% patients were in sinus rhythm (SR). Freedom from AF-recurrence was 64.6+/-0.76%, from hospital readmission 88.9+/-0.47%, from CHF 91.6+/-0.63%. SR-patients demonstrated better freedom from hospital readmission (97.4 vs 60.6%; p=0.0003) and from CHF (100 vs 72.7%; p=0.008) during follow-up. At follow-up SR-patients demonstrated left atrial (preoperative 5.8+/-0.8 cm vs follow-up 5.1+/-0.9; p=0.013) and ventricular reverse remodelling (preoperative LVDd 5.7+/-1.1cm vs follow-up 5.2+/-1.1; p=0.048 - preoperative LVDs 4.0+/-1.4 vs follow-up 3.6+/-1.1; p=0.036). E/A ratio was normal in 73.1% (92.7% of SR-patients). TDI at the level of the left lateral annulus showed an improved left ventricular systole (Sm), and diastole (Em, E/Em) of SR-patients, compared with AF-patients (Sm 9.40+/-1.74 vs 7.72+/-1.5, p=0.0001; Em: 10.45+/-1.98 vs 7.68+/-0.72, p=0.001; E/Em: 0.07+/-0.02 vs 0.10+/-0.04, p=0.0001). Large preoperative atrial diameter (OR=5.81; p=0.002), preoperative NYHA-IV (OR=3.55; p=0.001), high diuretics at discharge (OR=1.27; p=0.03), tricuspid insufficiency at follow-up (OR=2.31; p=0.02) were independent predictors of AF-recurrence. CONCLUSIONS: Radiofrequency AFAMVS achieves 78.8% of SR recovery. Maintenance of SR improves clinic, haemodynamic and echocardiographic endpoints. Pre- and post-operative cardiac failure is the main determinant of AF-recurrence.

Unstable angina and non-ST segment elevation: surgical revascularization with different strategies.

OBJECTIVE: Unstable angina/non-ST elevation myocardial infarction (UA/NSTEMI) still causes significant hospital morbidity and mortality. We evaluated whether surgical outcome can be modified by different myocardial protection strategies. METHODS: This was a prospective clinical study conducted in the cardiac surgery units of two university hospitals. Two hundred and sixty-two consecutive patients undergoing CABG for UA/NSTEMI between January 2002 and June 2004 were prospectively divided in three groups: 126 patients underwent on-pump CABG with antegrade blood cardioplegia (Group A); 67 underwent antegrade and retrograde blood cardioplegia (Group B); 69 off-pump CABG (Group C). Hospital outcome was analysed. Differences in outcome variables were detected with ANOVA; Tukey's multiple comparison test and Tamhane's T2 test were used when appropriate. RESULTS: Group A showed higher mortality (P=.001; P=.014 vs. Group B; P=.003 vs. Group C) and perioperative myocardial infarction (P=.001; P=.016 vs. Group B; P=.05 vs. Group C). Hospital stay was shorter in Group B and Group C, compared to Group A (P=.005; P=.043 and P=.05, respectively). Group A required higher doses of inotropes compared to Group B and Group C (P=.0001; P=.0001 and P=.03, respectively), whereas Group B and Group C did not require any inotropic support at all (P=.0001; P=.002 and P=.001 vs. Group A, respectively). Total morbidity was higher in Group A (P=.006; P=.007 vs. Group B; P=.005 vs. Group C). Wall motion score index recovered only in Group B (P=.0001) and Group C (P=.001). Troponin I was higher in Group A at 12 h (P=.0001; P<.001 vs. Group B and Group C), 24 (P=.0001; P=.001 vs. Group B and Group C), 48 (P=.0001; P=.001 vs. Group B, P=.002 vs. Group C) and 72 h (P=.0001; P=.004 vs. Group B; P=.05 vs. Group C). CONCLUSIONS: Isolated antegrade cardioplegia should be questioned in UA/NSTEMI. Outcome using off-pump revascularization was as good as that of combined antegrade and retrograde warm blood cardioplegia.