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1.
Proc Natl Acad Sci U S A ; 115(5): 1069-1074, 2018 01 30.
Article in English | MEDLINE | ID: mdl-29339472

ABSTRACT

Human cytomegalovirus (HCMV) impacts more than one-half of the human population owing to its capacity to manipulate the cell and create latent reservoirs in the host. Despite an extensive understanding of HCMV biology during acute infection in fibroblasts, the molecular basis for latency in myeloid cells remains incomplete. This knowledge gap is due largely to the fact that the existing genetic systems require virus rescue in fibroblasts, precluding the study of genes that are essential during acute infection, yet likely play unique roles in myeloid cells or the establishment of latency. Here we present a solution to address this restriction. Through the exploitation of a hematopoietic-specific microRNA, we demonstrate a one-step recombineering approach that enables gene silencing only in cells associated with latency. As a proof of concept, here we describe a TB40/E variant that undergoes hematopoietic targeting of the Immediate Early-2 (IE2) gene to explore its function during infection of myeloid cells. While virus replication of the hematopoietic-targeted IE2 variant was unimpaired in fibroblasts, we observed a >100-fold increase in virus titers in myeloid cells. Virus replication in myeloid cells demonstrated that IE2 has a significant transcriptional footprint on both viral and host genes. These data implicate IE2 as an essential mediator of virus biology in myeloid cells and illustrate the utility of cell-specific microRNA-based targeting.


Subject(s)
Cytomegalovirus/genetics , Immediate-Early Proteins/metabolism , MicroRNAs/metabolism , Trans-Activators/metabolism , Computational Biology , Fibroblasts/metabolism , Gene Expression Regulation, Viral , Gene Silencing , Hematopoietic Stem Cells/cytology , Humans , Macrophages/metabolism , Membrane Glycoproteins/genetics , Mutation , Myeloid Cells/metabolism , Transcriptional Activation , Transcriptome , Viral Envelope Proteins/genetics , Virus Replication
2.
Nat Commun ; 7: 13627, 2016 12 14.
Article in English | MEDLINE | ID: mdl-27966523

ABSTRACT

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Cytomegalovirus Infections/prevention & control , Cytomegalovirus/pathogenicity , Viral Envelope Proteins/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Cell Line , Cytomegalovirus Infections/immunology , Humans , Mice , Viral Envelope Proteins/chemistry , Viral Vaccines , Virus Internalization
3.
Sci Rep ; 6: 23692, 2016 Mar 30.
Article in English | MEDLINE | ID: mdl-27026399

ABSTRACT

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.


Subject(s)
Cytomegalovirus/physiology , Endoplasmic Reticulum-Associated Degradation , Viral Envelope Proteins/metabolism , Cell Line , Gene Expression , Humans , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Protein Structure, Tertiary , Protein Transport , Viral Envelope Proteins/genetics , Virus Internalization
4.
J Virol ; 88(16): 9391-405, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24920803

ABSTRACT

UNLABELLED: The ability of human cytomegalovirus (HCMV) to establish lifelong persistence and reactivate from latency is critical to its success as a pathogen. Here we describe a short-term in vitro model representing the events surrounding HCMV latency and reactivation in circulating peripheral blood monocytes that was developed in order to study the immunological consequence of latent virus carriage. Infection of human CD14(+) monocytes by HCMV resulted in the immediate establishment of latency, as evidenced by the absence of particular lytic gene expression, the transcription of latency-associated mRNAs, and the maintenance of viral genomes. Latent HCMV induced cellular differentiation to a macrophage lineage, causing production of selective proinflammatory cytokines and myeloid-cell chemoattractants that most likely play a role in virus dissemination in the host. Analysis of global cellular gene expression revealed activation of innate immune responses and the modulation of protein and lipid synthesis to accommodate latent HCMV infection. Remarkably, monocytes harboring latent virus exhibited selective responses to secondary stimuli known to induce an antiviral state. Furthermore, when challenged with type I and II interferon, latently infected cells demonstrated a blockade of signaling at the level of STAT1 phosphorylation. The data demonstrate that HCMV reprograms specific cellular pathways in monocytes, most notably innate immune responses, which may play a role in the establishment of, maintenance of, and reactivation from latency. The modulation of innate immune responses is likely a viral evasion strategy contributing to viral dissemination and pathogenesis in the host. IMPORTANCE: HCMV has the ability to establish a lifelong infection within the host, a phenomenon termed latency. We have established a short-term model system in human peripheral blood monocytes to study the immunological relevance of latent virus carriage. Infection of CD14(+) monocytes by HCMV results in the generation of latency-specific transcripts, maintenance of viral genomes, and the capacity to reenter the lytic cycle. During short-term latency in monocytes the virus initiates a program of differentiation to inflammatory macrophages that coincides with the modulation of cytokine secretion and specific cellular processes. HCMV-infected monocytes are hindered in their capacity to exert normal immunoprotective mechanisms. Additionally, latent virus disrupts type I and II interferon signaling at the level of STAT1 phosphorylation. This in vitro model system can significantly contribute to our understanding of the molecular and inflammatory factors that initiate HCMV reactivation in the host and allow the development of strategies to eradicate virus persistence.


Subject(s)
Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Immunity, Innate/immunology , Monocytes/immunology , Virus Latency/immunology , Cell Line , Cell Lineage/genetics , Cell Lineage/immunology , Cytokines/genetics , Cytokines/immunology , Cytomegalovirus/genetics , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/virology , Gene Expression/genetics , Gene Expression/immunology , Humans , Immunity, Innate/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/virology , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/immunology , Macrophages/immunology , Macrophages/virology , Monocytes/virology , Myeloid Cells/immunology , Myeloid Cells/virology , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , Transcription, Genetic/immunology , Virus Latency/genetics
5.
Viruses ; 6(3): 1202-18, 2014 Mar 12.
Article in English | MEDLINE | ID: mdl-24625810

ABSTRACT

Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.


Subject(s)
Cytomegalovirus/physiology , Receptors, Chemokine/metabolism , Viral Proteins/metabolism , Artificial Gene Fusion , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Cells, Cultured , Cytomegalovirus/genetics , Cytomegalovirus/growth & development , Cytomegalovirus/isolation & purification , Cytoplasm/chemistry , Endothelial Cells/virology , Epithelial Cells/virology , Fibroblasts/virology , Gene Deletion , Genes, Reporter , Humans , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Receptors, Chemokine/genetics , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Staining and Labeling , Viral Proteins/genetics
6.
Mol Immunol ; 51(2): 245-53, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22497807

ABSTRACT

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.


Subject(s)
Cytomegalovirus/immunology , Histocompatibility Antigens Class I/immunology , Viral Proteins/immunology , Cell Line , Cytomegalovirus/genetics , Cytomegalovirus/metabolism , Fluorescent Antibody Technique , Genes, Viral , Histocompatibility Antigens Class I/metabolism , Humans , Viral Proteins/genetics , Viral Proteins/metabolism
7.
Immunol Res ; 54(1-3): 140-51, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22454101

ABSTRACT

Members of the Herpesviridae family have the capacity to undergo both lytic and latent infection to establish a lifelong relationship with their host. Following primary infection, human cytomegalovirus (HCMV) can persist as a subclinical, recurrent infection for the lifetime of an individual. This quiescent portion of its life cycle is termed latency and is associated with periodic bouts of reactivation during times of immunosuppression, inflammation, or stress. In order to exist indefinitely and establish infection, HCMV encodes a multitude of immune modulatory mechanisms devoted to escaping the host antiviral response. HCMV has become a paradigm for studies of viral immune evasion of antigen presentation by both major histocompatibility complex (MHC) class I and II molecules. By restricting the presentation of viral antigens during both productive and latent infection, HCMV limits elimination by the human immune system. This review will focus on understanding how the virus manipulates the pathways of antigen presentation in order to modulate the host response to infection.


Subject(s)
Cytomegalovirus Infections/immunology , Cytomegalovirus/pathogenicity , Immune Evasion/immunology , Antigen Presentation , Cytomegalovirus/physiology , Cytomegalovirus Infections/virology , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Virus Latency
8.
J Clin Invest ; 120(11): 3969-78, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20978345

ABSTRACT

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3ß (GSK-3ß) function, promoted accumulation of ß-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.


Subject(s)
Intestinal Mucosa/pathology , Intestinal Neoplasms/immunology , Intestinal Neoplasms/pathology , Mice, Transgenic , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Animals , Cell Line , Cell Proliferation , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Humans , Mice , Mice, Inbred C57BL
9.
J Virol ; 83(3): 1359-67, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19004944

ABSTRACT

Throughout the course of natural evolution with its host, the human cytomegalovirus (HCMV) has developed a variety of strategies to avoid immune recognition and clearance. The major histocompatibility complex (MHC) class I antigen presentation pathway is a major target of the virus. HCMV encodes at least six gene products that modulate the processing of endoplasmic reticulum (ER)-resident MHC class I molecules. Here, we show that two virus-encoded proteins, US2 and US3, coordinate their functions toward the common goal of attenuating class I protein surface expression. In cells stably expressing both US2 and US3, class I molecules were almost completely downregulated from the cell surface. In addition, pulse-chase analysis revealed that the proteasome-dependent turnover of class I molecules occurs more rapidly in cells expressing both US2 and US3 than either US2 or US3 alone. The ability of US3 to retain class I molecules in the ER produces a target-rich environment for US2 to mediate the destruction of class I heavy chains. In fact, expression of US3 enhanced the association between US2 and class I molecules, thus encouraging their dislocation and degradation. This immune evasion strategy ensures that viral antigens are not presented on the cell surface during the early phase of HCMV infection, a critical time of replication and viral proliferation.


Subject(s)
Cytomegalovirus/physiology , Down-Regulation , Histocompatibility Antigens Class I/immunology , Astrocytoma/virology , Cell Line, Tumor , Flow Cytometry , Glycoproteins/metabolism , Glycoproteins/physiology , Humans , Hydrolysis , Immediate-Early Proteins/metabolism , Immediate-Early Proteins/physiology , Immunoprecipitation , Membrane Proteins/metabolism , Membrane Proteins/physiology , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/physiology
10.
J Biol Chem ; 283(7): 4031-43, 2008 Feb 15.
Article in English | MEDLINE | ID: mdl-18086679

ABSTRACT

Polypeptides are organized into distinct substructures, termed protein domains, that are often associated with diverse functions. These modular units can act as binding sites, areas of post-translational modification, and sites of complex multimerization. The human cytomegalovirus US2 gene product is organized into discrete domains that together catalyze the proteasome-dependent degradation of class I major histocompatibility complex heavy chains. US2 co-opts the endogenous ER quality control pathway in order to dispose of class I. The US2 endoplasmic reticulum (ER)-lumenal region is the class I binding domain, whereas the carboxyl terminus can be referred to as the degradation domain. In the present study, we examined the role of the US2 transmembrane domain in virus-mediated class I degradation. Replacement of the US2 transmembrane domain with that of the CD4 glycoprotein completely blocked the ability of US2 to induce class I destruction. A more precise mutagenesis revealed that subregions of the US2 transmembrane domain differ in their ability to trigger class I degradation. Collectively, the data support a model in which US2-mediated class I degradation occurs as a highly regulated process where the US2 transmembrane domain and cytoplasmic tail work in concert to eliminate class I molecules. Host factors, including a signal peptidase complex, probably associate with the US2 molecule in a coordinated fashion to create a predislocation complex to promote the extraction of class I out of the ER. The results imply that the ER quality control machinery may recognize and eliminate misfolded proteins using a similar multistep regulated process.


Subject(s)
Endoplasmic Reticulum/metabolism , Proteasome Endopeptidase Complex/metabolism , Viral Envelope Proteins/metabolism , Amino Acid Sequence , Cell Line, Tumor , Fluorescent Antibody Technique , Humans , Hydrolysis , Molecular Sequence Data , Viral Envelope Proteins/chemistry
11.
Nature ; 441(7095): 894-7, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16738546

ABSTRACT

Human cytomegalovirus (HCMV) prevents the display of class I major histocompatibility complex (MHC) peptide complexes at the surface of infected cells as a means of escaping immune detection. Two HCMV-encoded immunoevasins, US2 and US11, induce the dislocation of class I MHC heavy chains from the endoplasmic reticulum membrane and target them for proteasomal degradation in the cytosol. Although the outcome of the dislocation reactions catalysed is similar, US2 and US11 operate differently: Derlin-1 is a key component of the US11 but not the US2 pathway. So far, proteins essential for US2-dependent dislocation have not been identified. Here we compare interacting partners of wild-type US2 with those of a dislocation-incompetent US2 mutant, and identify signal peptide peptidase (SPP) as a partner for the active form of US2. We show that a decrease in SPP levels by RNA-mediated interference inhibits heavy-chain dislocation by US2 but not by US11. Our data implicate SPP in the US2 pathway and indicate the possibility of a previously unknown function for this intramembrane-cleaving aspartic protease in dislocation from the endoplasmic reticulum.


Subject(s)
Aspartic Acid Endopeptidases/metabolism , Endoplasmic Reticulum/metabolism , Amino Acid Sequence , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Cytomegalovirus/genetics , Cytomegalovirus/immunology , Cytomegalovirus/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Molecular Sequence Data , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Transport , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
12.
J Biol Chem ; 281(28): 19395-406, 2006 Jul 14.
Article in English | MEDLINE | ID: mdl-16687410

ABSTRACT

Human cytomegalovirus down-regulates cell surface class I major histocompatibility (MHC) molecules, thus allowing the virus to proliferate while avoiding detection by CD8+ T lymphocytes. The unique short gene product US2 is a 199-amino acid type I endoplasmic reticulum glycoprotein that modulates surface expression of class I MHC products by targeting class I heavy chains for dislocation from the endoplasmic reticulum to the cytosol, where they undergo proteasomal degradation. Although the mechanism by which this viral protein targets class I heavy chains for destruction remains unclear, the putative US2 cytoplasmic tail comprised of only 14 residues is known to play a functional role. To determine the specific residues critical for mediating class I degradation, a mutagenesis analysis of the cytoplasmic tail of US2 was performed. Using truncation mutants, the removal of only 4 residues (mutant US2(195)) from the US2 carboxyl terminus completely abolishes class I destruction. Furthermore, site-directed mutagenesis of the US2 cytoplasmic tail revealed that the most critical residues for class I-induced destruction, cysteine 187, serine 190, tryptophan 193, and phenylalanine 196, occurs every third residue. This experimental data supports a model that the US2 cytoplasmic tail is in a 3(10) helical configuration. Such a secondary structure would predict that one side of the 3(10) helical cytoplasmic tail would interact with the extraction apparatus to facilitate the dislocation and subsequent destruction of class I heavy chains.


Subject(s)
Cytomegalovirus/metabolism , Down-Regulation , Gene Expression Regulation, Neoplastic , Histocompatibility Antigens Class I/biosynthesis , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/physiology , Amino Acid Sequence , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Cell Proliferation , Genes, MHC Class I , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary
13.
Arequipa; UNSA; jun. 1995. 55 p. ilus.
Thesis in Spanish | LILACS | ID: lil-191931

ABSTRACT

OBJETIVO DEL ESTUDIO: Realizar una clasificación de las investigaciones y determinar la validez de conclusiones que provienen o no de objetivos planteados a priori en lactancia materna. DISEÑO: Transversal, de diagnóstico y estadío (valores de referencia). UBICACION: Area de Biomédica. SUJETOS: 64 objetivos reportados en las 16 tesis del listado oficial de la Biblioteca de Biomédicas. METODOS: Se realizó una clasificación de las investigaciones de acuerdo a la historia natural y de acuerdo a Bailar etal57. Luego se procedió a analizar la metodología de los trabajos partiendo de los objetivos específicos formulados a priori en cuanto a si provenían de un problema, si tenían propósito estadístico, indicadores, al cumplir esto podían considerarse verificables; luego se buscaba si eran justificados o no, si tenían análisis de datos (descriptivo o inferencial), estructura de datos, escalas, y prueba adecuada y si terminaban en una conclusión válida. En tercer lugar se procedió a la inversa con las conclusiones que cumplía con todos estos requerimientos. RESULTADOS PRINCIPALES: Menos del 11 por ciento de las conclusiones provenientes de objetivos específicos planteados a priori fueron metodológicamente válidas. Menos del 20 por ciento de las conclusiones que no provenían de objetivos específicos planteados a priori fueron metodológicamente válidas. CONCLUSIONES: La mayoría de las investigaciones se avocaron a factores de riesgo. Sólo una minoría de las conclusiones fueron metodológicamente válidas sin importar si provenian o no de objetivos específicos planteados a priori


Subject(s)
Humans , Infant , Breast Feeding , Academic Dissertations as Topic , Meta-Analysis , Infant Nutrition
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