ETS1 loss in mice impairs cardiac outflow tract septation via a cell migration defect autonomous to the neural crest.

Ets1 deletion in some mouse strains causes septal defects and has been implicated in human congenital heart defects in Jacobsen syndrome, in which one copy of the Ets1 gene is missing. Here, we demonstrate that loss of Ets1 in mice results in a decrease in neural crest (NC) cells migrating into the proximal outflow tract cushions during early heart development, with subsequent malalignment of the cushions relative to the muscular ventricular septum, resembling double outlet right ventricle (DORV) defects in humans. Consistent with this, we find that cultured cardiac NC cells from Ets1 mutant mice or derived from iPS cells from Jacobsen patients exhibit decreased migration speed and impaired cell-to-cell interactions. Together, our studies demonstrate a critical role for ETS1 for cell migration in cardiac NC cells that are required for proper formation of the proximal outflow tracts. These data provide further insights into the molecular and cellular basis for development of the outflow tracts, and how perturbation of NC cells can lead to DORV.

Role of androgens in dhea-induced rack1 expression and cytokine modulation in monocytes.

BACKGROUND: Over the past fifteen years, we have demonstrated that cortisol and dehydroepiandrosterone (DHEA) have opposite effects on the regulation of protein kinase C (PKC) activity in the context of the immune system. The anti-glucocorticoid effect of DHEA is also related to the regulation of splicing of the glucocorticoid receptor (GR), promoting the expression of GRß isoform, which acts as a negative dominant form on GRα activity. Moreover, it is very well known that DHEA can be metabolized to androgens like testosterone, dihydrotestosterone (DHT), and its metabolites 3α-diol and 3ß-diol, which exert their function through the binding of the androgen receptor (AR). Based on this knowledge, and on early observation that castrated animals show results similar to those observed in old animals, the purpose of this study is to investigate the role of androgens and the androgen receptor (AR) in DHEA-induced expression of the PKC signaling molecule RACK1 (Receptor for Activated C Kinase 1) and cytokine production in monocytes. RESULTS: Here we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRß expression, and cytokine production. In both THP-1 cells and human peripheral blood mononuclear cells (PBMC), flutamide blocked the effects of DHEA, suggesting a role of the AR in these effects. As DHEA is not considered a direct AR agonist, we investigated the metabolism of DHEA in THP-1 cells. We evaluated the ability of testosterone, DHT, and androstenedione to induce RACK1 expression and cytokine production. In analogy to DHEA, an increase in RACK1 expression and in LPS-induced IL-8 and TNF-α production was observed after treatment with these selected androgens. Finally, the silencing of AR with siRNA completely prevented DHEA-induced RACK1 mRNA expression, supporting the idea that AR is involved in DHEA effects. CONCLUSIONS: We demonstrated that the conversion of DHEA to active androgens, which act via AR, is a key mechanism in the effect of DHEA on RACK1 expression and monocyte activation. This data supports the existence of a complex hormonal balance in the control of immune modulation, which can be further studied in the context of immunosenescence and endocrinosenescence.

Corticosteroids modulate the expression of the PKC-anchoring protein RACK-1 and cytokine release in THP-1 cells.

We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.

Role of PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-ß. The purpose of this study was to investigate the role of RACK-1 and PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-ß and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-ß, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-ß and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-ß in the initiation of chemical allergen-induced DC activation.

Zyxin is a novel target for ß-amyloid peptide: characterization of its role in Alzheimer's pathogenesis.

Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer 's disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aß) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Aß activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities.

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer's disease.

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aß aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer's disease.

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.

The Rise of Induced Pluripotent Stem Cell Approach to Hair Restoration.

SUMMARY: The advent of pluripotent stem cells following the discovery of Shinya Yamanaka (2012 Nobel prize in Medicine) brought about a regenerative medicine approach to virtually every human condition including hair loss. It is now possible to reprogram somatic cells (eg, blood or skin cells) from a person experiencing hair loss to generate autologous induced pluripotent stem cells (iPSCs), which could be amplified and cryopreserved. Subsequently, these iPSCs could be differentiated into various cell types such as dermal papilla cells, epithelial cells, melanocytes, and other cell types constituting functional hair follicle. Transplantation of human iPSC-derived folliculogenic cells into the nude mice has successfully generated xenografts with hair outgrowth. Because iPSCs provide a virtually unlimited source of folliculogenic cells for de novo formation of hair follicles, this approach has major advantages over current surgical hair restoration procedures, which merely redistribute existing hair follicles from one part of the sculp to another. Combined with robotics and automation of the transplantation process, this novel regenerative medicine approach is well poised to make hair restoration a routine procedure affordable for everybody who can benefit from it.

Zika Virus: Origins, Pathological Action, and Treatment Strategies.

The Zika virus (ZIKV) global epidemic prompted the World Health Organization to declare it a 2016 Public Health Emergency of International Concern. The overwhelming experience over the past several years teaches us that ZIKV and the associated neurological complications represent a long-term world-wide challenge to public health. Although the number of ZIKV cases in the Western Hemisphere has dropped since 2016, the need for basic research and anti-ZIKV drug development remains strong. Re-emerging viruses like ZIKV are an ever-present threat in the 21st century where fast transcontinental travel lends itself to viral epidemics. Here, we first present the origin story for ZIKV and review the rapid progress researchers have made toward understanding of the ZIKV pathology and in the design, re-purposing, and testing-particularly in vivo-drug candidates for ZIKV prophylaxis and therapy ZIKV. Quite remarkably, a short, but intensive, drug-repurposing effort has already resulted in several readily available FDA-approved drugs that are capable of effectively combating the virus in infected adult mouse models and, most importantly, in both preventing maternal-fetal transmission and severe microcephaly in newborns in pregnant mouse models.

Tetanus-diphtheria vaccination in adults: the long-term persistence of antibodies is not dependent on polyclonal B-cell activation and the defective response to diphtheria toxoid re-vaccination is associated to HLADRB1∗01.

Cellular and humoral immune responses to tetanus-diphtheria vaccine (Td) were assessed in human leukocyte antigen (HLA)-typed Italian military personnel who received multiple concomitant vaccines. Td-specific antibodies and T-lymphocytes were measured in individuals with one (group-1) and more than one (group-2) Td boosters. A third group (group-3), who received several vaccines, but not Td, was studied to verify the hypothesis of the polyclonal B-cell activation as mechanism for antibody persistence. The antibody response to Td toxoids was higher in group-1, who showed lower baseline antibody levels, than in group-2 subjects. The antibody response to tetanus was higher than to diphtheria toxoid in both groups. No correlation between antibody and cellular response, and no interference in the response to Td by co-administration of different vaccines were observed. HLA-DRB1∗01 allele was detected at significant higher frequency in subjects unable to double the baseline anti-diphtheria antibody levels after the vaccination. Anti-tetanus and diphtheria antibodies half-lives were assessed and the long-lasting persistence above the threshold for protection (0.1 IU/ml) was estimated in over 65 and 20 years, respectively. No significant increase of anti-diphtheria antibodies was observed in consequence of polyclonal B-cell activation. This study emphasizes the duration of Td vaccination-induced seroprotection, suggesting that re-vaccination should probably be performed at intervals longer than 10 years. No reciprocal interference by concomitantly administered vaccines has been observed. HLA-DRB1∗01 allele was significantly associated with anti-diphtheria defective response. Finally, this study does not confirm that anti-diphtheria antibody levels are maintained by polyclonal B-cell activation. Clinical trial registry: The study was registered with NCT01807780.

Author Correction: Blocking Zika virus vertical transmission.

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Blocking Zika virus vertical transmission.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry.

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis.

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

Current knowledge of rickettsial diseases in Italy.

Rickettsial diseases continue to be the cause of serious health problems in Italy. From 1998 to 2002, 4,604 clinical cases were reported, with 33 deaths in the period from 1998 to 2001. Almost all the cases reported in Italy are cases of Mediterranean spotted fever (MSF). Other rickettsioses that have been historically documented are murine typhus and epidemic typhus. Since 1950, only sporadic cases of murine typhus have been reported, and Italy currently appears to be free of epidemic typhus. As in other European countries, imported cases of rickettsialpox, African tick-bite fever (ATBF), and scrub typhus have been reported. In 2004, three cases of a mild form of rickettsiosis were serologically attributed to Rickettsia helvetica.

The role of HSP27 in RACK1-mediated PKC activation in THP-1 cells.

Receptor for Activated C Kinase 1 (RACK1) pseudosubstrate is a commercially available peptide that directly activates protein kinase C-ß (PKCß). We have recently shown that RACK1 pseudosubstrate, alone or in combination with classical immune activators, results in increased cytokine production and CD86 upregulation in primary leukocytes. Furthermore, we demonstrated a role of PKCß and RACK1 in chemical allergen-induced CD86 expression and IL-8 production in both THP-1 cells and primary human dendritic cells. Aim of this study was to shed light on the mechanisms underlying RACK1 pseudosubstrate-induced immune activation and to compare it to lipopolysaccharide (LPS). The human promyelocytic cell line THP-1 was used throughout the study. RACK1 pseudosubstrate induced rapid (5 min) and dose-related PKCß activation as assessed by its membrane translocation. Among the proteins phosphorylated, we identified Hsp27. Both RACK1 pseudosubstrate and LPS induce its phosphorylation and release in culture medium. The release of Hsp27 induced by RACK1 pseudosubstrate was also confirmed in peripheral blood mononuclear cells. To evaluate the role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, we conducted Hsp27 silencing and neutralization experiments. Both strategies confirmed the central role of Hsp27 in RACK1 pseudosubstrate or LPS-induced cell activation, as assessed by IL-8 production and upregulation of CD86.

Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease.

The amyloidogenic pathway is a prominent feature of Alzheimer's disease (AD). However, growing evidence suggests that a linear disease model based on ß-amyloid peptide (Aß) alone is not likely to be realistic, which therefore calls for further investigations on the other actors involved in the play. The pro-oxidant environment induced by Aß in AD pathology is well established, and a correlation among Aß, oxidative stress, and conformational changes in p53 has been suggested. In this study, we applied a multifunctional approach to identify allyl thioesters of variously substituted trans-cinnamic acids for which the pharmacological profile was strategically tuned by hydroxy substituents on the aromatic moiety. Indeed, only catechol derivative 3 [(S)-allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enethioate] inhibited Aß fibrilization. Conversely, albeit to different extents, all compounds were able to decrease the formation of reactive oxygen species in SH-SY5Y neuroblastoma cells and to prevent alterations in the conformation of p53 and its activity mediated by soluble sub-lethal concentrations of Aß. This may support an involvement of oxidative stress in Aß function, with p53 emerging as a potential mediator of their functional interplay.

Rapid identification of Bartonella henselae by real-time polymerase chain reaction in a patient with cat scratch disease.

We report a localized submandibular lymph node infection in a patient with cat scratch disease. Directly performing real-time polymerase chain reaction assay on the biopsy sample, Bartonella henselae DNA was simultaneously detected and identified.

Immunostimulatory effects of RACK1 pseudosubstrate in human leukocytes obtained from young and old donors.

Aims of this study were to investigate the ability of RACK1 pseudosubstrate alone or in combination with classical immune stimuli to activate human leukocytes, and to restore age-associated immune defects.A total of 25 donors (17 old donors, 77-79 yrs; 8 young donors, 25-34 yrs) were enrolled. To evaluate the effect of RACK1 pseudosubstrate on cytokine production and CD86 expression the whole blood assay was used. Cultures were treated with RACK1 pseudosubstrate in the presence or absence of lipopolysaccharide (LPS) or phytohaemagglutinin (PHA) and incubated for 24 h or 48 h for LPS-induced CD86 expression, TNF-α, IL-6, IL-8, IL-10 production, and PHA-induced IL-4, IL-10, IFN-γ, respectively. RACK1 pseudosubstrate alone induced IL-6, IL-8, and CD86 expression in both young and old donors, and IFN-γ in old donors. In combination with LPS an increase in IL-8, IL-10 and TNF-α was observed, also resulting in restoration of age-associated defective production, while no changes in the other parameters investigated were found.Even if based on a small sample size, these results suggest the possibility to by-pass some of age-associated immune alterations, which may be beneficial in situations were natural immune stimulation is required, and highlight a different role of PKCß in immune cells activation.