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Atonal homologue 8 (atoh8) is a basic helix-loop-helix transcription factor expressed in a variety of embryonic tissues. While several studies have implicated atoh8 in various developmental pathways in other species, its role in zebrafish development remains uncertain. So far, no studies have dealt with an in-depth in situ analysis of the tissue distribution of atoh8 in embryonic zebrafish. We set out to pinpoint the exact location of atoh8 expression in a detailed spatio-temporal analysis in zebrafish during the first 24 h of development (hpf). To our surprise, we observed transcription from pre-segmentation stages in the paraxial mesoderm and during the segmentation stages in the somitic sclerotome and not—as previously reported—in the myotome. With progressing maturation of the somites, the restriction of atoh8 to the sclerotomal compartment became evident. Double in situ hybridisation with atoh8 and myoD revealed that both genes are expressed in the somites at coinciding developmental stages; however, their domains do not spatially overlap. A second domain of atoh8 expression emerged in the embryonic brain in the developing cerebellum and hindbrain. Here, we observed a specific expression pattern which was again in contrast to the previously published suggestion of atoh8 transcription in neural crest cells. Our findings point towards a possible role of atoh8 in sclerotome, cerebellum and hindbrain development. More importantly, the results of this expression analysis provide new insights into early sclerotome development in zebrafish—a field of research in developmental biology which has not received much attention so far.

The enzymes can modify some effluent characteristics in order to increase the degradability, or the bioconversion of liquid effluents. The oxireductases, laccases and peroxidases have been used due their high potentiality in many environmental treatments of natural and synthetic organic compounds as dyes, phenols and polyphenolics molecules. The performance of immobilized horseradish peroxidase on aminopropyl glass beads was investigated in this work in a decolorization reaction of methylene blue colorant. The experiments were conducted in batch conditions during 3 hours, with different aqueous solutions of peroxide hydrogen (H2O2) concentration solutions (2-10 mg/L), methylene blue (ME) (5-20 mg/L) and the pH in the range from 4 to 8, according an experimental design proposed by the software STATISTICA®. After 3 hours of treatment the reduction of the color was 60% when comparing to the original color and 50% when the immobilized enzymes were reused in five sequential batch treatment cycles for a 10 mg/L of H2O2 solution, 20 mg/L of ME and pH 8.0. Working in continuous process with two microreactors in series the system showed a good performance with 97% of decolorization in the first 15 minutes. After 1 hour of continuous treatment the percentage of the color removing was around 70%.

Immunotherapy, particularly immune checkpoint blockade and chimeric antigen receptor (CAR)-T cells, holds a great promise against cancer. These treatments have markedly improved survival in solid as well as in hematologic tumors previously considered incurable. However, durable responses occur in a fraction of patients, and existing biomarkers (e.g. PD-L1) have shown limited prediction power. This scenario highlights the need to dissect the complex interplay between immune and tumor cells to identify reliable biomarkers of response to be used for patients' selection. In this context, systems immunology represents indeed the new frontier to address important clinical challenges in biomarker discovery. Through the integration of multiple layers of data obtained with several high-throughput approaches, systems immunology may give insights on the vast range of inter-individual differences and on the influences of genes and factors that cooperatively shape the individual immune response to a given treatment. In this Mini Review, we give an overview of the current high-throughput methodologies, including genomics, epigenomics, transcriptomics, metabolomics, proteomics, and multi-parametric phenotyping suitable for systems immunology as well as on the key steps of data integration and biological interpretation. Additionally, we review recent studies in which multi-omics technologies have been used to characterize mechanisms of response and to identify powerful biomarkers of response to checkpoint inhibitors, CAR-T cell therapy, dendritic cell-based and peptide-based cancer vaccines. We also highlight the need of favoring the collaboration of researchers with complementary expertise and of integrating multi-omics data into biological networks with the final goal of developing accurate markers of therapeutic response.

Highlights • Sex-based outcome of SARS-CoV-2 infection accounts for about 70% of male deaths, despite similar susceptibility to infection. • TLR7/IFN-α axis in pDCs is crucial for B-cell activation and it is at the basis of dimorphic antibody response. • Dysregulated and unbalanced IFN-driven B cell response affects COVID-19 outcome in males., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, induces severe pneumonia mainly in elderly males. Epidemiological data clearly indicate sex-based differences in disease outcomes, with men accounting for about 70% of deaths, despite similar susceptibility to infection. It is well known that females are endowed with higher capacity to produce antibodies, which correlates with viral clearance and disease resolution in the context of SARS-Cov-2 infection. Many X-linked immune genes escape X inactivation showing biallelic expression in female immune cells, particularly in plasmacytoid dendritic cells (pDCs). PDCs are more active in females and endowed with high capability to induce IFN-α-mediated B cell activation and differentiation into antibody-producing plasma cells throughout epigenetic mechanisms linked to trained immunity. Thus, we hypothesize that following SARS-CoV-2 infection, epigenetic modifications of X-linked genes involved in pDC-mediated type I IFN (IFN-I) signaling occurs more effectively in females, for inducing neutralizing antibody response as an immune correlate driving sex-biased disease outcome.

// Barbara Pascucci 1, 3 , Alessandra Fragale 2 , Veronica Marabitti 3 , Giuseppe Leuzzi 3, 4 , Angelo Salvatore Calcagnile 3 , Eleonora Parlanti 3 , Annapaola Franchitto 3 , Eugenia Dogliotti 3 and Mariarosaria D’Errico 3 1 Institute of Cristallography, Consiglio Nazionale delle Ricerche, Roma, Italy 2 Section of Tumor Immunology, Department of Oncology and Molecular Medicine, Istituto Superiore di Sanita, Roma, Italy 3 Section of Mechanisms, Biomarkers and Models, Department of Environment and Health, Istituto Superiore di Sanita, Roma, Italy 4 Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, USA Correspondence to: Eugenia Dogliotti, email: eugenia.dogliotti@iss.it Mariarosaria D’Errico, email: mariarosaria.derrico@iss.it Keywords: Cockayne syndrome; DNA damage; DNA repair; γ-H2AX Received: September 07, 2017 Accepted: January 19, 2018 Published: January 29, 2018 ABSTRACT CS proteins have been involved in the repair of a wide variety of DNA lesions. Here, we analyse the role of CS proteins in DNA break repair by studying histone H2AX phosphorylation in different cell cycle phases and DNA break repair by comet assay in CS-A and CS-B primary and transformed cells. Following methyl methane sulphate treatment a significant accumulation of unrepaired single strand breaks was detected in CS cells as compared to normal cells, leading to accumulation of double strand breaks in S and G2 phases. A delay in DSBs repair and accumulation in S and G2 phases were also observed following IR exposure. These data confirm the role of CSB in the suppression of NHEJ in S and G2 phase cells and extend this function to CSA. However, the repair kinetics of double strand breaks showed unique features for CS-A and CS-B cells suggesting that these proteins may act at different times along DNA break repair. The involvement of CS proteins in the repair of DNA breaks may play an important role in the clinical features of CS patients.

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1β, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells in vivo and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. Cancer Immunol Res; 5(7); 604–16. ©2017 AACR.

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

This review will summarize main characteristics and functions of IRF-7. IRF-7 and the highly homologous IRF-3 are two members of the IRF family of transcription factors that have emerged as crucial regulators of type I interferon (IFN) in response to pathogenic infections downstream pathogen recognition receptors. IRF-7 is also part of a positive-feedback regulatory loop essential for sustained IFN responses. Thus, tight regulation of its expression and activity is necessary to balance IFN-mediated beneficial effects and unwanted pathological consequences of IFN overproduction. Its role as an antiviral factor independent of IFN expression, and its involvement in other cellular functions beyond antiviral functions, including regulation of oncogenesis and metabolism, underscore its important role in the regulation of cellular homeostasis.

Introduction Morgana is a ubiquitously expressed protein with chaperone activity per se and HSP90 co-chaperone function. High Morgana expression correlates with high tumour grade, mitosis number, and lymph node positivity in different breast cancer subtypes. Several chaperones are overexpressed in a wide range of human cancers and are implicated in tumour progression. Moreover, it has become evident that also from the extracellular compartment, where surprisingly chaperones and co-chaperones are actively released by cancer and immune cells, they favour tumour progression. If, the cytoplasmic role of Morgana has been well characterised in tumorigenesis and metastasis formation of Triple-Negative Breast Cancer (TNBC), nothing is known about extracellular Morgana (eMorgana). Material and methods Conditioned medium from human and murine TNBCs cell lines were analysed for Morgana presence. To address the role of eMorgana, a Maltose Binding Protein (MBP) fused recombinant protein was produced in ClearColi BL21 and used to evaluate eMorgana role in migration, treating MDA-231 and BT-549. The identification of Morgana receptor was performed indirectly, through the inhibition of Toll-like 2 and Toll-like 4 receptors (TLR2, TLR4). The activity of extracellular Morgana was inhibited, in mice injected with the syngenic cancer cell line E0771, using the homemade blocking antibody 5B11. Results and discussions Morgana is secreted by TNBC cell lines and as for other chaperones and co-chaperones, Morgana reaches the outside with an unconventional mechanism. From the extracellular compartment Morgana is able to induce cell migration, through theTLR2 and TLR4. Blocking of eMorgana with 5B11 inhibits migration in vitro and proliferation in vivo. Different efforts are needed to understand if Morgana binds to TLRs alone or through HSP90 and the consequently downstream signalling pathway. Moreover since TLR are prevalently expressed by immune cells it is important to address the role of eMorgana in this context and the possible crosstalk between the tumour and microenvironment. Conclusion Since TNBCs have an high rate of recurrence and poor prognosis, the identification of innovative treatments is an urgent need. In this view, although many other studies are required, eMorgana in serum patients could represent a new biomarker and its targeting a possible clinical approach in breast cancers.

Nutrition and growth are important outcome indicators in pediatric (Ped) hemodialysis (HD) patients. We hypothesized that there is a discrepancy among traditional measures of nutrition, and that adequate nutrition may not reliably predict growth.We assessed longitudinal nutrition and growth parameters in 14 Ped HD patients over 1 year. Their age at the end of the study was 15.9 +/- 0.6 years, SEM.For the entire cohort over 1 year, serum albumin (Alb) was 4.3 +/- 0.0 g/dL, and the normalized protein catabolic rate (nPCR) was 1.0 +/- 0.0 (correlation, 0.33; P.0001). The relationship between Alb and nPCR was significant in only 4/14 (29%). The mean standard deviation and variance were higher for Alb (0.27 +/- 0.03) compared with nPCR (0.18 +/- 0.02). The body mass index percentile (BMI%) was 35.5 +/- 2.9, the percent ideal body weight (%IBW) was 96.2 +/- 1.5, the height-SDS, or standard deviation score (Ht-SDS) was -1.30 +/- 0.11, and the percent weight change (PWC) was +4.9% +/- 1.9%. The highest incidence of reaching our targets for growth was seen for the BMI% (64% of patients) and PWC (79% of patients). The target for Ht-SDS was attained in only 21%. There was a significant negative relationship between Alb and nPCR with BMI%, %IBW, and Ht-SDS, and a significant positive relationship between Alb and nPCR with PWC.We conclude that the relationship between Alb and nPCR is weak in individual patients, and that adequate nutrition does not reliably predict growth in Ped HD patients.

A new biotechnological synthetic approach to 4-hydroxybenzoic acid is reported. Thauera aromatica cells produce the phenylphosphate carboxylase enzyme that selectively carboxylates phenylphosphate in the para position, without any formation of the ortho isomer. Here we show that the enzyme also works in supercritical carbon dioxide, opening a new technological solution to the recovery of the products that is a key problem when aqueous media are used. The enzyme shows an activity comparable with that shown under CO2 pressure in aqueous media and does not require the presence of NaHCO3.

The chick embryo is widely used for the study of vertebrate development, but a general, reliable loss-of-function strategy for the analysis of gene function is currently not available. By using small inhibitory hairpin RNA (siRNA) molecules generated by the mouse U6 promoter, we have applied an RNA interference approach to achieve quantitative knockdown of the neuropilin-1 (Nrp-1) receptor in chick embryos. Functional knockdown was evident in the abolition of Sema3A-induced growth cone collapse in Nrp-1-siRNA but not Nrp-2-siRNA-expressing dorsal root ganglion (DRG) neurons. Two nervous system defects in Nrp-1 mutant mice were phenocopied in embryos treated with Nrp-1 siRNA. First, DRG axons prematurely entered the dorsal horn and projected inappropriately. Second, targeted early migrating neural crest cells destined for the sympathetic chain arrested ectopically within ventral spinal nerve roots. Localized knockdown induced by specific siRNA constructs will allow rapid functional analysis of genes regulating chick neural development whilst circumventing embryonic lethal effects often associated with global gene knockout in the mouse.

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation.

The gastrointestinal tract is lined by a layer of mucus comprised of highly glycosylated proteins called mucins. To evaluate the importance of mucin in intestinal carcinogenesis, we constructed mice genetically deficient in Muc2, the most abundant secreted gastrointestinal mucin. Muc2 −/− mice displayed aberrant intestinal crypt morphology and altered cell maturation and migration. Most notably, the mice frequently developed adenomas in the small intestine that progressed to invasive adenocarcinoma, as well as rectal tumors. Thus, Muc2 is involved in the suppression of colorectal cancer.

A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment.

Primrose syndrome and 3q13.31 microdeletion syndrome are clinically related disorders characterized by tall stature, macrocephaly, intellectual disability, disturbed behavior and unusual facial features, with diabetes, deafness, progressive muscle wasting and ectopic calcifications specifically occurring in the former. We report that missense mutations in ZBTB20, residing within the 3q13.31 microdeletion syndrome critical region, underlie Primrose syndrome. This finding establishes a genetic link between these disorders and delineates the impact of ZBTB20 dysregulation on development, growth and metabolism.

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood–testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca2+ channels are involved in the regulation of protein secretion.

Originally identified as regulators of the type I Interferon system, the nine mammalian members of the Interferon Regulatory Factor (IRF) family are transcriptional regulators with multiple biologic functions, among which the best known are those involved in initiating and regulating many aspect of host immunity, downstream pattern recognition receptors in response to cell injuries. In addition, these versatile proteins also regulate cell differentiation, cell growth and apoptosis in several cell types, and when mutated or disregulated significantly contribute to susceptibility to and progression of several cancers. IRF-1 is the most versatile member of the family, not essential for IFN gene expression and implicated in a variety of cellular functions spanning from the development and function of various immune cells to tumor suppression activity. IRF-3, IRF-7 and Irf-9 are more specifically involved in IFN induction and antiviral responses while IRF-5 is the regulator of inflammatory cytokines expression downstream pattern recognition receptors and IRF-6 is implicated in epithelial differentiation. IRF-8 as IRF-1 is considered a tumor suppressor gene, while IRF-2 and IRF-4 have generally oncogenic activity. The present review focuses principally on the current knowledge on IRF genetic characteristics, including mutations, polymorphisms and epigenetic regulation that are implicated in oncogenesis.

We report here that individuals with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) have germline mutations in PTPN11 and that somatic mutations in PTPN11 account for 34% of non-syndromic JMML. Furthermore, we found mutations in PTPN11 in a small percentage of individuals with myelodysplastic syndrome (MDS) and de novo acute myeloid leukemia (AML). Functional analyses documented that the two most common mutations in PTPN11 associated with JMML caused a gain of function.

Thirty years after the first isolation of the etiological agent of AIDS, the virus HIV-1 is still a major threat worldwide with millions of individuals currently infected. Although current combination therapies allow viral replication to be controlled, HIV-1 is not eradicated and persists in drug- and immune system-insensitive reservoirs and a cure is still lacking. Pathogens such as HIV-1 that cause chronic infections are able to adapt to the host in a manner that ensures long term residence and survival, via the evolution of numerous mechanisms that evade various aspects of the innate and adaptive immune response. One such mechanism is targeted to members of the interferon (IFN) regulatory factor (IRF) family of proteins. These transcription factors regulate a variety of biological processes including interferon induction, immune cell activation and downstream pattern recognition receptors (PRRs). HIV-1 renders IRFs harmless and hijacks them to its own advantage in order to facilitate its replication and evasion of immune responses. Type I interferon (IFN), the canonical antiviral innate response, can be induced in both acute and chronic HIV-1 infection in vivo, but in the majority of individuals this initial response is not protective and can contribute to disease progression. Type I IFN expression is largely inhibited in T cells and macrophages in order to successfully establish productive infection, whereas sustained IFN production by plasmacytoid dendritic cells is considered an important source of chronic immune activation, a hallmark to AIDS progression.

The exile experience is a trend in recent Brazilian theatre which makes use of mythical themes. Based on The Perverse Trilogy (BENDER, 1986), Ismene , Princess of Thebes (Senna, 2006) and The ‘Carioca’ Olympus (BRANDAO, 2012), this article shows that while ancient myths map questions related to the period in which they return (the immigrant pathos , the politically persecuted individuals, and even the city regarded as the ideal refuge from “Olympic” troubles), they also act as the “tiroir a double entree” mentioned by Espagne (1999, p. 78): they are not mere crossing points between cultures, but they are also triggers of “itineraries of memory” (ESPAGNE, 1999, p. 138).

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1– and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8–induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase–dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation.

Members of the IFN regulatory factors (IRFs) family are transcriptional regulators that play essential roles in the homeostasis and function of the immune system. Recent studies indicate a direct involvement of some members of the family in the development of different subsets of dendritic cells (DC). Here, we report that IRF-1 is a potent modulator of the development and functional maturation of DC. IRF-1-deficient mice (IRF-1−/−) exhibited a predominance of plasmacytoid DC and a selective reduction of conventional DC, especially the CD8α+ subset. IRF-1−/− splenic DC were markedly impaired in their ability to produce proinflammatory cytokines such as IL-12. By contrast, they expressed high levels of IL-10, TGF-β, and the tolerogenic enzyme indoleamine 2,3 dioxygenase. As a consequence, IRF-1−/− DC were unable to undergo full maturation and retained plasmacytoid and tolerogenic characteristics following virus infection ex vivo and in vivo. Accordingly, DC from IRF-1−/− mice were less efficient in stimulating the proliferation of allogeneic T cells and instead, induced an IL-10-mediated, suppressive activity in allogeneic CD4+CD25+ regulatory T cells. Together, these results indicate that IRF-1 is a key regulator of DC differentiation and maturation, exerting a variety of effects on the functional activation and tolerogenic potential of these cells.

Objectives: To determine the correlation between haemodynamic forces and the release of two mitogens for smooth muscle cells (SMC): Platelet Derived Growth Factor (PDGF) and basic Fibroblast Growth Factor (bFGF). Methodology: Bovine aortic smooth muscle cells were seeded on fibronectin coated polystyrene cylinders and allowed to reach confluence. The cells were subjected to a laminar flow of 50cd/min (3 dyne/cm 2 ), 100 cc/min (6 dyne/cm 2 ) and 150 cc/min (9 dyne/cm 2 ) in an in vitro system. Control cells were subjected to similar incubation conditions without flow. Principal results: Shear stress increased the release of mitogens by SMC. The release of mitogens was proportional to the level of shear stress and was still evident 24 hours after flow cessation. Conditioned serum-free medium from SMC subjected to shear stress increased tritiated thymidine uptake in Swiss 3T3 fibroblasts 13-fold as compared to conditioned serum-free medium from control SMC not subjected to shear stress ( p p p p

The four members of the fibroblast growth factor receptor (FGFR) family are cell-surface membrane-spanning tyrosine kinase receptors involved in a wide spectrum of biologic processes. Much evidence also indicates that mutations in FGFR genes result in several craniosynostotic disorders and chondrodysplasias, and that changes in qualitative and quantitative FGFR expression profiles are implicated in tumor induction or progression. Here, we describe a precise and reliable competitive PCR-based assay to evaluate human FGFR1-4 gene expression. A single multispecific synthetic competitive template was designed to amplify FGFR1-4 homologous stretches and constructed to contain FGFR1/FGFR2/FGFR3/FGFR4/GAPDH tandemly arranged forward and reverse primers that allow competition for cDNA-specific primer annealing. The housekeeping GAPDH transcript was utilized as a reference for comparing the expression profiles of different RNA pools. The assay herein described allows the comparison of relative FGFR expression levels, both within a single RNA pool and among multiple RNA pool samples. The major advantages of such a PCR-based approach are its ability to obtain unbiased FGFR mRNA expression patterns and to detect transcripts present in low copy number. Qualitative and semiquantitative analyses of the FGFR1-4 transcript repertoire in mesenchymal- and epithelial-derived primary cell cultures and cell lines demonstrated the utility of such a method to investigate the FGFR1-4 functional role in FGF signal transduction.

It is currently believed that the fertility level of the adult mammalian testis is related to the total number of Sertoli cells, which is established in the early prepubertal life. We have previously reported that, in an in-vitro system, terminal Sertoli cell proliferation is sustained by activin A in concert with FSH. In this paper, we have addressed the question of whether this activin A effect correlates with activin receptor II (ActRII) expression pattern during early post-natal testis development. We first determined the precise developmental interval of activin proliferative effect on Sertoli cells in vitro and then analysed the expression of ActRII in purified testicular cell populations by Northern blot and in-situ hybridization. While the 3 kb ActRII isoform was widely expressed at different ages and in several testicular cells, including Sertoli cells, germ cells and myoid cells, the canonical 6 kb ActRII isoform was specifically and transiently expressed at a high rate in Sertoli cells at 7-9 days after birth, the time when these cells respond to activin A in vitro. In the light of these results, we conclude that activin A regulates terminal Sertoli cell proliferation in the rat testis and that this effect is mediated by the 6 kb isoform of ActRII.

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Calpha and protein kinase Cepsilon isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Czeta levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype.

The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8(-/-)) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8(-/-) mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2'-deoxycytidine into melanoma-bearing IRF-8(-/-) animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness.

The action of activin-A on Sertoli and spermatogonial cell proliferation during early postnatal life was studied by using in vitro organ culture of testis fragments from 9-day-old rats. Activin significantly stimulated 3H-thymidine incorporation into testis fragments cultured for 3 days in the presence of FSH, whereas it had no effect in the absence of the hormone. This effect was dose dependent in the range 10-200 ng/ml and was specifically inhibited by the activin-binding protein, follistatin. The effect of activin upon proliferation of different testicular cells was studied in detail by 5-bromo-2'-deoxyuridine-labeling fragments at the end of in vitro culture and then determining percentages of different labeled cells on immunostained histological sections. Concomitant treatment with FSH and activin, but not with FSH or activin alone, significantly stimulated Sertoli cell proliferation but markedly depressed that of differentiating type A spermatogonia. In contrast, proliferative activity of undifferentiated type A spermatogonia was independent of activin, irrespective of the presence of FSH. The effect of donor animal age was then investigated by culturing fragments derived from 3- and 18-day-old rats for 3 days. An age-related response was evident. Sertoli cell proliferation was stimulated by FSH alone in fragments from 3-day-old rats, activin having no apparent effect at this age. In contrast, none of the hormones tested either alone or in combination was effective in 18-day-old animals. These results demonstrate that activin acts with FSH in maintaining mitotic potentiality of Sertoli cells in a defined phase of their maturation path, when their proliferative activity is approaching the final arrest. These findings suggest that activin may be an important local factor in regulating Sertoli cell number and that the mitosis of differentiating spermatogonia subsides during Sertoli cell proliferation.

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4+CD25+ Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1−/−) mice showed a selective and marked increase of highly activated and differentiated CD4+CD25+Foxp3+ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1−/− CD4+CD25− T cells showed extremely high bent to differentiate into CD4+CD25+Foxp3+ Treg cells, whereas restoring IRF-1 expression in IRF-1−/− CD4+CD25− T cells impaired their differentiation into CD25+Foxp3+ cells. Functionally, both isolated and TGF-β-induced CD4+CD25+ Treg cells from IRF-1−/− mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4+CD25+ Treg cells through direct repression of Foxp3 expression.

In this paper we report the results of our studies on Ni complexes, the chemistry of which is relevant to the carbon monoxide dehydrogenase (CODH) enzyme of acetogenic bacteria. A single Ni system is able to promote both the reduction of carbon dioxide to carbon monoxide and the subsequent coupling of bound CO with an oletin and a thio group to afford an organic thioester, mimicking the enzyme activity.