Induction of Therapeutic Protection in an HPV16-Associated Mouse Tumor Model Through Targeting the Human Papillomavirus-16 E5 Protein to Dendritic Cells.

HPV E5 is an oncoprotein mainly expressed in premalignant lesions, which makes it an important target for a vaccine to prevent or cure cervical cancer (CC). In this study, we evaluated whether E5 targeted to DEC-205, present in dendritic cells (DCs), could induce a therapeutic protection against HPV16-induced tumor cells in a mouse model. The HPV-16 E5 (16E5) protein was cross-linked to a monoclonal antibody (mAb) specific to mouse DEC-205 (anti-DEC-205:16E5) or to an isotype control mAb (isotype:16E5). Rotavirus VP6 was cross-linked to the mouse anti-DEC-205 mAb (anti-DEC-205:VP6) as a non-specific antigen control. BALB/c mice were inoculated subcutaneously (s.c.) with the 16E5-expressing BMK-16/myc tumor cells, and 7 and 14 days later the mice were immunized s.c. with the conjugates, free 16E5 or PBS in the presence of adjuvant. Tumor growth was monitored to evaluate protection. A strong protective immune response against the tumor cells was induced when the mice were inoculated with the anti-DEC-205:16E5 conjugate, since 70% of the mice controlled the tumor growth and survived, whereas the remaining 30% developed tumors and died by day 72. In contrast, 100% of the mice in the control groups died by day 30. The anti-DEC-205:16E5 conjugate was found to induce 16E5-specific memory T cells, with a Th1/Th17 profile. Both CD4+ and CD8+ T cells contributed to the observed protection. Finally, treating mice that had developed tumors with an anti-PD-1 mAb, delayed the tumor growth for more than 20 days. These results show that targeting 16E5 to DEC-205, alone or combined with an immune checkpoint blockade, could be a promising protocol for the treatment of the early stages of HPV-associated cancer.

Aedes aegypti Immune Response and Its Potential Impact on Dengue Virus Transmission.

Dengue virus (DENV) transmission to human populations requires infection of vector mosquitoes as an essential component of the transmission process. DENV transmission leads to infections that range from asymptomatic to life-threatening pathologies, such as dengue hemorrhagic fever and dengue shock syndrome. Aedes aegypti is the principal vector of DENV, and its vector competence consists of the intrinsic factors, genes, molecules, and pathways that allow infection, replication, and dissemination of this virus throughout the cells of mosquito tissues. In the search for mosquito molecular targets to block DENV transmission, the effect of DENV infection on mosquitoes has been an important focus of research. In this study, we review the findings of research on the effect of DENV infection on mosquito tissue cells and the immunity pathways and molecules that are involved in this infection. We emphasize the relevance of recent findings to understand the relationship between Ae. aegypti immune response, vector competence, and DENV transmission to human hosts.

Dengue virus infection induces chromatin remodeling at locus AAEL006536 in the midgut of Aedes aegypti.

OBJECTIVE: To identify and characterize Aedes aegypti's AAEL006536 gene proximal upstream cis-regulatory sequences activated by dengue virus infection. MATERIALS AND METHODS: A. aegypti Rockefeller strain mosquitoes were blood fed or infected with dengue virus 2. Open chromatinprofiling was then carried out in pools of midguts from each group of mosquitoes. RESULTS: The proximal upstream region does not contain open chromatin sites in the midguts of blood-fed mosquitoes as detected by FAIRE-qPCR. In contrast, two cis-regulatory sites were identified in the same upstream region of dengue virus-infected mosquito midguts. The distal sequence contains STAT-, REL- and C/EBP-type transcription factor binding sites. CONCLUSIONS: The activation of two proximal cis-regulatory sequences, induced by dengue virus infection, is mediated by chromatin remodeling mechanisms. Binding sites suggest a dengue virus infectioninduced participation of immunity transcription factors in the up-regulation of this gene. This suggests the participation of the AAEL006536 gene in the mosquito's antiviral innate immune response.

Dengue virus infection induces chromatin remodeling at locus AAEL006536 in the midgut of Aedes aegypti / La infección por virus dengue induce remodelación de la cromatina en el locus AAEL006536 en el intestino medio de Aedes aegypti

Abstract: Objective: To identify and characterize Aedes aegypti's AAEL006536 gene proximal upstream cis-regulatory sequences activated by dengue virus infection. Materials and methods: A. aegypti Rockefeller strain mosquitoes were blood fed or infected with dengue virus 2. Open chromatin profiling was then carried out in pools of midguts from each group of mosquitoes. Results: The proximal upstream region does not contain open chromatin sites in the midguts of blood-fed mosquitoes as detected by FAIRE-qPCR. In contrast, two cis-regulatory sites were identified in the same upstream region of dengue virus-infected mosquito midguts. The distal sequence contains STAT-, REL- and C/EBP-type transcription factor binding sites. Conclusion: The activation of two proximal cis-regulatory sequences, induced by dengue virus infection, is mediated by chromatin remodeling mechanisms. Binding sites suggest a dengue virus infection-induced participation of immunity transcription factors in the up-regulation of this gene. This suggests the participation of the AAEL006536 gene in the mosquito's antiviral innate immune response.

Resumen: Objetivo: Identificar y caracterizar las secuencias reguladoras activadas por la infección por virus dengue en la región proximal del gen AAEL006536 de Aedes aegypti. Material y métodos: Mosquitos de la cepa Rockefeller de A. aegypti se infectaron con virus dengue o se alimentaron con sangre. Se obtuvieron los perfiles de cromatina abierta del locus en los intestinos de cada uno de los grupos. Resultados: Se identificaron dos sitios reguladores solo en los intestinos de mosquitos infectados por virus dengue. El sitio distal contiene sitios de unión a factores de transcripción tipo REL, STAT y C/EBP. Conclusiones: La activación de dos sitios reguladores proximales está mediada por la remodelación de la cromatina. Los sitios de unión a factores de transcripción en el sitio regulador distal sugieren la participación de las vías de inmunidad en la regulación del gen. Esto sugiere la participación de este gen en la respuesta inmune del mosquito frente a la infección viral.

Identification of cis-regulatory sequences reveals potential participation of lola and Deaf1 transcription factors in Anopheles gambiae innate immune response.

The innate immune response of Anopheles gambiae involves the transcriptional upregulation of effector genes. Therefore, the cis-regulatory sequences and their cognate binding factors play essential roles in the mosquito's immune response. However, the genetic control of the mosquito's innate immune response is not yet fully understood. To gain further insight on the elements, the factors and the potential mechanisms involved, an open chromatin profiling was carried out on A. gambiae-derived immune-responsive cells. Here, we report the identification of cis-regulatory sites, immunity-related transcription factor binding sites, and cis-regulatory modules. A de novo motif discovery carried out on this set of cis-regulatory sequences identified immunity-related motifs and cis-regulatory modules. These modules contain motifs that are similar to binding sites for REL-, STAT-, lola- and Deaf1-type transcription factors. Sequence motifs similar to the binding sites for GAGA were found within a cis-regulatory module, together with immunity-related transcription factor binding sites. The presence of Deaf1- and lola-type binding sites, along with REL- and STAT-type binding sites, suggests that the immunity function of these two factors could have been conserved both in Drosophila and Anopheles gambiae.

Antibiotic-Resistant Acinetobacter baumannii Increasing Success Remains a Challenge as a Nosocomial Pathogen.

Antibiotic-resistant infectious bacteria currently imply a high risk and therefore constitute a strong challenge when treating patients in hospital settings. Characterization of these species and of particular strains is a priority for the establishment of diagnostic tests and preventive procedures. The relevance of Acinetobacter baumannii as a problematic microorganism in inpatient facilities, particularly intensive care units, has increased over time. This review aims to draw attention to (i) the historical emergence of carbapenem-resistant Acinetobacter baumannii, (ii) the current status of surveillance needs in Latin America, and (iii) recent data suggesting that A. baumannii continues to spread and evolve in hospital settings. First, we present synopsis of the series of events leading to the discovery and precise identification of this microorganism in hospital settings. Then key events in the acquisition of antibiotic-resistant genes by this microorganism are summarized, highlighting the race between new antibiotic generation and emergence of A. baumannii resistant strains. Here we review the historical development of this species as an infectious threat, the current state of its distribution, and antibiotic resistance characteristics, and we discuss future prospects for its control.

[Polymorphisms in gene regulatory regions and their role in the physiopathology of complex disease in the post-genomic era]. / Polimorfismos reguladores y su participación en la patogenia de enfermedades complejas en la era posgenómica.

The genomic era is imparting a new impulse to the study of the role of genetic variation in susceptibility to disease. The most common type of genetic variation between individuals is single nucleotide polymorphisms (SNP). The association of SNPs with susceptibility to disease is the current focus of intense research. Recently, the study of SNPs that alter the regulatory mechanisms of gene expression (rSNP) has emerged as a promising field for understanding disease, since this type of variation can have a profound effect on human traits related to susceptibility to disease. The finding and functional characterization of biologically significant rSNPs is advancing our knowledge of genetic determinants for multifactorial disease.

Polimorfismos reguladores y su participación en la patogenia de enfermedades complejas en la era posgenómica / Polymorphisms in gene regulatory regions and their role in the physiopathology of complex disease in the post-genomic era

El estudio de la participación de la variación genética en la predisposición a las enfermedades complejas ha cobrado nuevas dimensiones en la era genómica. Los polimorfismos de un solo nucleótido (SNP) son el tipo de variación más común entre individuos y su vinculación con enfermedades es motivo de investigación intensa. En fecha reciente, el estudio de los SNP que afectan la expresión genética (rSNP) ha suscitado mayor interés, ya que las diferencias de la expresión genética entre un sujeto y otro pueden modificar el fenotipo. El descubrimiento y caracterización funcional de los rSNP y el estudio de su frecuencia alélica representan un nuevo campo en la búsqueda de determinantes genéticos de enfermedades multifactoriales.

The genomic era is imparting a new impulse to the study of the role of genetic variation in susceptibility to disease. The most common type of genetic variation between individuals is single nucleotide polymorphisms (SNP). The association of SNPs with susceptibility to disease is the current focus of intense research. Recently, the study of SNPs that alter the regulatory mechanisms of gene expression (rSNP) has emerged as a promising field for understanding disease, since this type of variation can have a profound effect on human traits related to susceptibility to disease. The finding and functional characterization of biologically significant rSNPs is advancing our knowledge of genetic determinants for multifactorial disease.

Immunity related genes in dipterans share common enrichment of AT-rich motifs in their 5' regulatory regions that are potentially involved in nucleosome formation.

BACKGROUND: Understanding the transcriptional regulation mechanisms in response to environmental challenges is of fundamental importance in biology. Transcription factors associated to response elements and the chromatin structure had proven to play important roles in gene expression regulation. We have analyzed promoter regions of dipteran genes induced in response to immune challenge, in search for particular sequence patterns involved in their transcriptional regulation. RESULTS: 5' upstream regions of D. melanogaster and A. gambiae immunity-induced genes and their corresponding orthologous genes in 11 non-melanogaster drosophilid species and Ae. aegypti share enrichment in AT-rich short motifs. AT-rich motifs are associated with nucleosome formation as predicted by two different algorithms. In A. gambiae and D. melanogaster, many immunity genes 5' upstream sequences also showed NFkappaB response elements, located within 500 bp from the transcription start site. In A. gambiae, the frequency of ATAA motif near the NFkappaB response elements was increased, suggesting a functional link between nucleosome formation/remodelling and NFkappaB regulation of transcription. CONCLUSION: AT-rich motif enrichment in 5' upstream sequences in A. gambiae, Ae. aegypti and the Drosophila genus immunity genes suggests a particular pattern of nucleosome formation/chromatin organization. The co-occurrence of such motifs with the NFkappaB response elements suggests that these sequence signatures may be functionally involved in transcriptional activation during dipteran immune response. AT-rich motif enrichment in regulatory regions in this group of co-regulated genes could represent an evolutionary constrained signature in dipterans and perhaps other distantly species.

Characterization of a megakaryocyte-specific enhancer of the key hemopoietic transcription factor GATA1.

Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3' 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3'-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells.

GATA1 mutation and trisomy 21 are required only in haematopoietic cells for development of transient myeloproliferative disorder.

Trisomy 21 [Down's syndrome (DS)] and mutations in transcription factor GATA1 predispose neonates to a transient myeloproliferative disorder (TMD) and/or acute megakaryocytic leukaemia (AMKL). The role of trisomy 21 in their pathogenesis is unclear. We previously reported two rare neonates without DS who had TMD, one of whom progressed to AMKL. Trisomy 21 was detected only in blood cells at presentation with TMD/AMKL and disappeared with disease resolution. We now show that the blood cells at presentation of TMD harboured GATA1 genomic DNA mutations, suggesting a requirement for trisomy 21 in haematopoietic cells, rather than other cell types, for development of TMD/AMKL.

Differences in the chromatin structure and cis-element organization of the human and mouse GATA1 loci: implications for cis-element identification.

Cis-element identification is a prerequisite to understand transcriptional regulation of gene loci. From analysis of a limited number of conserved gene loci, sequence comparison has proved a robust and efficient way to locate cis-elements. Human and mouse GATA1 genes encode a critical hematopoietic transcription factor conserved in expression and function. Proper control of GATA1 transcription is critical in regulating myeloid lineage specification and maturation. Here, we compared sequence and systematically mapped position of DNase I hypersensitive sites, acetylation status of histone H3/H4, and in vivo binding of transcription factors over approximately 120 kilobases flanking the human GATA1 gene and the corresponding region in mice. Despite lying in approximately 10 megabase (Mb) conserved syntenic segment, the chromatin structures of the 2 homologous loci are strikingly different. The 2 previously unidentified hematopoietic cis-elements, one in each species, are not conserved in position and sequence and have enhancer activity in erythroid cells. In vivo, they both bind the transcription factors GATA1, SCL, LMO2, and Ldb1. More broadly, there are both species- and regulatory element-specific patterns of transcription factor binding. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. More generally, mouse human sequence comparison may fail to identify all cis-elements.

Deletion of the major GATA1 enhancer HS 1 does not affect eosinophil GATA1 expression and eosinophil differentiation.

Expression of the myeloid transcription factor GATA1 is required for early stages of eosinophil differentiation. Defining mechanisms regulating eosinophil GATA1 expression will be important to understand development of this lineage. However, the cis-elements required for eosinophil GATA1 expression are not fully characterized. Previous work identified HS 1 as a major GATA1 enhancer, but its role in eosinophil GATA1 expression is unclear. Here, we show that mouse HS 1 deletion leaves eosinophil GATA1 mRNA expression and eosinophil differentiation unaffected. Chromatin isolated from eosinophils and encompassing HS 1 is weakly enriched for acetylated histones H3/H4. HS 1 deletion does not alter eosinophil GATA1 locus histone acetylation. In eosinophils, GATA1 and CCAAT/enhancer binding protein epsilon (C/EBP epsilon) do not bind HS 1 but bind selectively a cis-element in the first GATA1 intron. Thus, HS 1 is not required for eosinophil GATA1 expression. Instead, this study suggests a previously unsuspected role for the GATA1 intron element for this function.