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Ultrasound screening for hepatocellular carcinoma (HCC) in patients with liver cirrhosis has a poor sensitivity for small tumors. Circulating microRNAs (miRNAs) have been explored as HCC biomarkers, but results are diverging. Here, we evaluate if miRNAs up-regulated in HCC tissue can be detected in plasma and used as screening biomarkers for HCC. In this cross-sectional study, plasma, HCC tissue and surrounding non-tumorous liver tissue were collected from liver resections. Tissue miRNAs were identified and quantitated by RNA-sequencing analysis, and the fold-changes between HCC and surrounding liver tissue were calculated. The miRNAs up-regulated in HCCs were then re-analyzed in plasma from the same patients, and the miRNAs with the highest plasma levels were subsequently measured in plasma from an independent cohort of patients with cirrhosis or HCC. In tissues from 84 resected patients, RNA-sequencing detected 197 differentially expressed miRNAs, 40 of which had a raw count above 200 and were analyzed in plasma from the same cohort. Thirty-one miRNAs were selected for further analysis in 200 patients with HCC or cirrhosis. Of these, eleven miRNAs were significantly increased in HCC as compared to cirrhosis patients. Only miR-93-5p and miR-151a-3p were significantly associated with HCC, with an AUC of 0.662. In comparison, alpha-fetoprotein and des-gamma-carboxy prothrombin yielded an AUC of 0.816, which increased to 0.832 if miR-93-5p and miR-151a-3p were added. When including sex and age, the addition of miR-93-5p and miR-151a-3p did not further improve the AUC (from 0.910 to 0.911). In conclusion, micro-RNAs up-regulated in HCCs are detectable in plasma but have a poor performance as screening biomarkers of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/complicações , MicroRNAs/genética , Estudos Transversais , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/complicações , Biomarcadores Tumorais/genética , Cirrose Hepática/diagnóstico , Cirrose Hepática/genética , BiomarcadoresRESUMO
Myeloid-derived suppressor cells (MDSCs) are major regulators of T cell responses in several pathological conditions. Whether MDSCs increase and influence T cell responses in temporary inflammation, such as after vaccine administration, is unknown. Using the rhesus macaque model, which is critical for late-stage vaccine testing, we demonstrate that monocytic (M)-MDSCs and polymorphonuclear (PMN)-MDSCs can be detected using several of the markers used in humans. However, whereas rhesus M-MDSCs lacked expression of CD33, PMN-MDSCs were identified as CD33+ low-density neutrophils. Importantly, both M-MDSCs and PMN-MDSCs showed suppression of T cell proliferation in vitro. The frequency of circulating MDSCs rapidly and transiently increased 24 h after vaccine administration. M-MDSCs infiltrated the vaccine injection site, but not vaccine-draining lymph nodes. This was accompanied by upregulation of genes relevant to MDSCs such as arginase-1, IDO1, PDL1, and IL-10 at the injection site. MDSCs may therefore play a role in locally maintaining immune balance during vaccine-induced inflammation.
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Vírus da Influenza A Subtipo H10N8/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Células Supressoras Mieloides/imunologia , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Animais , Arginase/genética , Antígeno B7-H1/genética , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interleucina-10/genética , Macaca mulatta , Análise em Microsséries , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , VacinaçãoRESUMO
mRNA vaccines are rapidly emerging as a powerful platform for infectious diseases because they are well tolerated, immunogenic, and scalable and are built on precise but adaptable antigen design. We show that two immunizations of modified non-replicating mRNA encoding influenza H10 hemagglutinin (HA) and encapsulated in lipid nanoparticles (LNP) induce protective HA inhibition titers and H10-specific CD4+ T cell responses after intramuscular or intradermal delivery in rhesus macaques. Administration of LNP/mRNA induced rapid and local infiltration of neutrophils, monocytes, and dendritic cells (DCs) to the site of administration and the draining lymph nodes (LNs). While these cells efficiently internalized LNP, mainly monocytes and DCs translated the mRNA and upregulated key co-stimulatory receptors (CD80 and CD86). This coincided with upregulation of type I IFN-inducible genes, including MX1 and CXCL10. The innate immune activation was transient and resulted in priming of H10-specific CD4+ T cells exclusively in the vaccine-draining LNs. Collectively, this demonstrates that mRNA-based vaccines induce type-I IFN-polarized innate immunity and, when combined with antigen production by antigen-presenting cells, lead to generation of potent vaccine-specific responses.
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Células Apresentadoras de Antígenos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Vacinas/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Citocinas/metabolismo , Expressão Gênica , Imunização , Imunofenotipagem , Vacinas contra Influenza/imunologia , Injeções Intradérmicas , Linfonodos/imunologia , Linfonodos/metabolismo , Macaca mulatta , Fenótipo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas/administração & dosagemRESUMO
Multiple sclerosis is a chronic, often disabling, disease of the central nervous system affecting more than 1 in 1,000 people in most western countries. The inflammatory lesions typical of multiple sclerosis show autoimmune features and depend partly on genetic factors. Of these genetic factors, only the HLA gene complex has been repeatedly confirmed to be associated with multiple sclerosis, despite considerable efforts. Polymorphisms in a number of non-HLA genes have been reported to be associated with multiple sclerosis, but so far confirmation has been difficult. Here, we report compelling evidence that polymorphisms in IL7R, which encodes the interleukin 7 receptor alpha chain (IL7Ralpha), indeed contribute to the non-HLA genetic risk in multiple sclerosis, demonstrating a role for this pathway in the pathophysiology of this disease. In addition, we report altered expression of the genes encoding IL7Ralpha and its ligand, IL7, in the cerebrospinal fluid compartment of individuals with multiple sclerosis.
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Predisposição Genética para Doença , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-7/genética , Adulto , Estudos de Casos e Controles , Dinamarca , Feminino , Finlândia , Expressão Gênica , Frequência do Gene , Variação Genética , Genótipo , Humanos , Interleucina-7/genética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Desequilíbrio de Ligação , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Noruega , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , SuéciaRESUMO
Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.
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[This corrects the article DOI: 10.3389/fimmu.2017.01539.].
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High-throughput biomolecular profiling techniques such as transcriptomics, proteomics and metabolomics are increasingly being used in in vivo studies to recognize and characterize effects of xenobiotics on organs and systems. Of particular interest are biomarkers of treatment-related effects which are detectable in easily accessible biological fluids such as blood. A fundamental challenge in such biomarker studies is selecting among the plethora of biomolecular changes induced by a compound and revealed by molecular profiling, to identify biomarkers which are exclusively or predominantly due to specific processes. In this work we present a cross-compartment correlation network approach, involving no a priori supervision or design, to integrate proteomic, metabolomic and transcriptomic data for selecting circulating biomarkers. The case study we present is the identification of biomarkers of drug-induced hepatic toxicity effects in a rodent model. Biomolecular profiling of both blood plasma and liver tissue from Wistar Hannover rats administered a toxic compound yielded many hundreds of statistically significant molecular changes. We exploited drug-induced correlations between blood plasma analytes and liver tissue molecules across study animals in order to nominate selected plasma molecules as biomarkers of drug-induced hepatic alterations of lipid metabolism and urea cycle processes.
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Biologia de Sistemas , Animais , Biomarcadores , Glicosiltransferases/metabolismo , Lipídeos/sangue , Fígado/enzimologia , Masculino , Ornitina/sangue , Ratos , Ratos WistarRESUMO
Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP) in non-human primates. While both intradermal (ID) and intramuscular (IM) administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.
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We have investigated the genetic involvement of the CD4 and the LAG3 genes, two appealing candidates for MS due to their suggested role in MS pathology. We genotyped a Swedish case-control material consisting of 920 MS patients and 778 controls in an initial study of CD4, three SNPs showed a significant association with MS. An independent material consisting of 1720 Nordic MS patients and 1416 controls were used for confirmation of associated markers in CD4 and to do a confirmative study of the LAG3 gene from previous findings. The result, including a total of 2640 MS patients and 2194 controls shows no significant association with CD4 and LAG3 and MS. We conclude that these genes are of minor importance in regard of genetic predisposition to the MS.
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Antígenos CD/genética , Antígenos CD4/genética , Predisposição Genética para Doença/genética , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Antígenos CD/imunologia , Biomarcadores/metabolismo , Antígenos CD4/imunologia , Estudos de Casos e Controles , Estudos de Coortes , Análise Mutacional de DNA , Dinamarca/epidemiologia , Feminino , Finlândia/epidemiologia , Frequência do Gene/genética , Marcadores Genéticos/genética , Marcadores Genéticos/imunologia , Testes Genéticos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/epidemiologia , Esclerose Múltipla/imunologia , Noruega/epidemiologia , Valor Preditivo dos Testes , Suécia/epidemiologia , População Branca/genética , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
INTRODUCTION: The sodium channel Nav 1.9 is expressed in peripheral nociceptors and has recently been linked to human pain conditions, but the exact role of Nav 1.9 for human nociceptor excitability is still unclear. METHODS: C-nociceptors from two patients with late onset of erythromelalgia-like pain, signs of small fiber neuropathy, and rare genetic variants of Nav 1.9 (N1169S, I1293V) were assessed by microneurography. RESULTS: Compared with patients with comparable pain phenotypes (erythromelalgia-like pain without Nav-mutations and painful polyneuropathy), there was a tendency toward more activity-dependent slowing of conduction velocity in mechanoinsensitive C-nociceptors. Hyperexcitability to heating and electrical stimulation were seen in some nociceptors, and other unspecific signs of increased excitability, including spontaneous activity and mechanical sensitization, were also observed. CONCLUSIONS: Although the functional roles of these genetic variants are still unknown, the microneurography findings may be compatible with increased C-nociceptor excitability based on increased Nav 1.9 function.
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Eritromelalgia/genética , Eritromelalgia/fisiopatologia , Nociceptores , Feminino , Humanos , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Nociceptores/patologiaRESUMO
Gain-of-function mutations in the tetrodotoxin (TTX) sensitive voltage-gated sodium channel (Nav) Nav1.7 have been identified as a key mechanism underlying chronic pain in inherited erythromelalgia. Mutations in TTX resistant channels, such as Nav1.8 or Nav1.9, were recently connected with inherited chronic pain syndromes. Here, we investigated the effects of the p.M650K mutation in Nav1.8 in a 53 year old patient with erythromelalgia by microneurography and patch-clamp techniques. Recordings of the patient's peripheral nerve fibers showed increased activity dependent slowing (ADS) in CMi and less spontaneous firing compared to a control group of erythromelalgia patients without Nav mutations. To evaluate the impact of the p.M650K mutation on neuronal firing and channel gating, we performed current and voltage-clamp recordings on transfected sensory neurons (DRGs) and neuroblastoma cells. The p.M650K mutation shifted steady-state fast inactivation of Nav1.8 to more hyperpolarized potentials and did not significantly alter any other tested gating behaviors. The AP half-width was significantly broader and the stimulated action potential firing rate was reduced for M650K transfected DRGs compared to WT. We discuss the potential link between enhanced steady state fast inactivation, broader action potential width and the potential physiological consequences.
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Eritromelalgia/genética , Gânglios Espinais/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/genética , Dor/genética , Potenciais de Ação/genética , Estimulação Elétrica , Eritromelalgia/fisiopatologia , Gânglios Espinais/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Fibras Nervosas Amielínicas , Dor/fisiopatologia , Técnicas de Patch-Clamp , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Tetrodotoxina/genéticaRESUMO
Alzheimer's disease (AD) is the most common form of dementia, with no disease-modifying treatment yet available. Early detection of patients at risk of developing AD is of central importance. Blood-based genetic signatures can serve as early detection and as population-based screening tools. In this study, we aimed to identify genetic markers and gene signatures associated with cerebrospinal fluid (CSF) biomarkers levels of t-tau, p-tau181, and with the two ratios t-tau/Aß1-42 and p-tau181/Aß1-42 in the context of progression from mild cognitive impairment (MCI) to AD, and to identify a panel of genetic markers that can predict CSF biomarker p-tau181/Aß1-42 ratio with consideration of APOE ε4 stratification. We analyzed genome-wide the Alzheimer's Disease Neuroimaging Initiative dataset with up to 48 months follow-up. In the first part of the analysis, the main effect of single nucleotide polymorphisms (SNPs) under an additive genetic model was assessed for each of the four CSF biomarkers. In the second part of the analysis, we performed an integrated analysis of genome-wide association study results with pathway enrichment analysis, predictive modeling and network analysis in the subgroup of ApoE4-negative subjects. We identified a panel of five SNPs, rs6766238, rs1143960, rs1249963, rs11975968, and rs4836493, that are predictive for p-tau181/Aß1-42 ratio (high/low) with a sensitivity of 66% and a specificity of 70% (AUC 0.74). These results suggest that a panel of SNPs is a potential prognostic biomarker in ApoE4-negative MCI patients.
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Peptídeos beta-Amiloides/líquido cefalorraquidiano , Disfunção Cognitiva/líquido cefalorraquidiano , Disfunção Cognitiva/genética , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteínas tau/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Biomarcadores/líquido cefalorraquidiano , Progressão da Doença , Feminino , Seguimentos , Marcadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Polimorfismo de Nucleotídeo Único , Proteínas tau/genéticaRESUMO
The Alzheimer's Association's Research Roundtable met in May 2014 to explore recent progress in developing biomarkers to improve understanding of disease pathogenesis and expedite drug development. Although existing biomarkers have proved extremely useful for enrichment of subjects in clinical trials, there is a clear need to develop novel biomarkers that are minimally invasive and that more broadly characterize underlying pathogenic mechanisms, including neurodegeneration, neuroinflammation, and synaptic dysfunction. These may include blood-based assays and new neuropsychological testing protocols, as well as novel ligands for positron emission tomography imaging, and advanced magnetic resonance imaging methodologies. In addition, there is a need for biomarkers that can serve as theragnostic markers of response to treatment. Standardization remains a challenge, although international consortia have made substantial progress in this area and provide lessons for future standardization efforts.
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Seven patients diagnosed with erythromelalgia (EM) were investigated by microneurography to record from unmyelinated nerve fibers in the peroneal nerve. Two patients had characterized variants of sodium channel Nav1.7 (I848T, I228M), whereas no mutations of coding regions of Navs were found in 5 patients with EM. Irrespective of Nav1.7 mutations, more than 50% of the silent nociceptors in the patients with EM showed spontaneous activity. In the patient with mutation I848T, all nociceptors, but not sympathetic efferents, displayed enhanced early subnormal conduction in the velocity recovery cycles and the expected late subnormality was reversed to supranormal conduction. The larger hyperpolarizing shift of activation might explain the difference to the I228M mutation. Sympathetic fibers that lack Nav1.8 did not show supranormal conduction in the patient carrying the I848T mutation, confirming in human subjects that the presence of Nav1.8 crucially modulates conduction in cells expressing EM mutant channels. The characteristic pattern of changes in conduction velocity observed in the patient with the I848T gain-of function mutation in Nav1.7 could be explained by axonal depolarization and concomitant inactivation of Nav1.7. If this were true, activity-dependent hyperpolarization would reverse inactivation of Nav1.7 and account for the supranormal CV. This mechanism might explain normal pain thresholds under resting conditions.
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Eritromelalgia/genética , Eritromelalgia/patologia , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Condução Nervosa/fisiologia , Nociceptores/fisiologia , Estudos de Casos e Controles , Eletrofisiologia , Feminino , Humanos , Isoleucina/genética , Masculino , Fibras Nervosas Amielínicas/fisiologia , Condução Nervosa/genética , Exame Neurológico , Limiar da Dor/fisiologia , Técnicas de Patch-Clamp , Nervo Fibular/patologia , Nervo Fibular/fisiopatologia , Estimulação Física , Tempo de Reação/genética , Recuperação de Função Fisiológica/genética , Treonina/genéticaRESUMO
Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), expressed on activated T cells, binds to B7 molecules on antigen-presenting cells. Signaling via CTLA-4 results in downregulation of ongoing T-cell clonal expansion. A single-nucleotide polymorphism (SNP) in exon 1 of CTLA4 is associated with susceptibility to several autoimmune diseases, including multiple sclerosis (MS). In this two-stage study, we investigated whether haplotypes composed of exon 1-SNP alleles and alleles of a promoter-region SNP influence age at onset, disease severity and disease course in MS. In stage 1, deviations in CTLA4 haplotype frequencies were observed in patients subgrouped by course; in stage 2, none of these original associations were confirmed.
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Antígenos de Diferenciação/genética , Imunoconjugados , Esclerose Múltipla/diagnóstico , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único , Abatacepte , Idade de Início , Antígenos CD , Antígeno CTLA-4 , Progressão da Doença , Frequência do Gene , Genótipo , Haplótipos , Humanos , FenótipoRESUMO
Background and aim "Gain-of-function" mutations in voltage-gated sodium channel NaV1.7 have been linked to erythromelalgia (EM), characterized by painful hot and red hands and feet. We investigated the proportion of patients with EM that carry a mutation in NaV1.7 or in other pain-related genes and studied possible clinical differences. Methods In this study, 48 patients with EM were screened for mutations in a total of 29 candidate genes, including all sodium channel subunits, transient receptor potential channels (TRPA1, TRPV1, TRPM8), neurotrophic factors (NGF, NGFR, BDNF, GDNF, NTRK1 and WNK1) and other known pain-related genes (CACNG2, KCNS1, COMT, P2RX3, TAC1, TACR1), using a combination of next generation sequencing and classical Sanger sequencing. Results In 7/48 patients protein-modifying mutations of NaV1.7 (P187L, I228M, I848T (n = 4) and N1245S) were identified. Patients with the I848T mutation could be identified clinically based on early onset and severity of the disease. In contrast, there were no clinical characteristics that differentiated the other patients with NaV1.7 mutation from those patients without. We also found more than twenty rare protein-modifying genetic variants in the genes coding for sodium channels (NaV1.8, NaV1.9, NaV1.6, NaV1.5, NaV2.1, SCN1B, SCN3B), transient receptor potential channel (TRPA1, TRPV1), and other pain-related targets (WNK1 and NGFR). Conclusion We conclude that functionally characterized mutations of NaV1.7 (I848T) are present only in a minority of patient with EM. Albeit the majority of patients (27/48) carried rare protein-modifying mutations the vast majority of those will most probably not be causally linked to their disease. Implications The key question remaining to be solved is the possible role of rare variants of NaV1.8, NaV1.9, or beta-subunits in provoking chronic pain conditions or even EM.
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Neuronal dysfunction and degeneration are central events of a number of major diseases with significant unmet need. Neuronal dysfunction may not necessarily be the result of cell death, but may also be due to synaptic damage leading to impaired neuronal cell signaling or long-term potentiation. Once degeneration occurs, it is unclear whether axonal or synaptic loss comes first or whether this precedes neuronal cell death. In this review we summarize the pathophysiology of four major neurodegenerative diseases; Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis (Lou Gehrig's disease) For each of these diseases, we describe how biochemical biomarkers are currently understood in relation to the pathophysiology and in terms of neuronal biology, and we discuss the clinical and diagnostic utility of these potential tools, which are at present limited. We discuss how markers may be used to drive drug development and clinical practice.
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Doença de Alzheimer/diagnóstico , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Múltipla/diagnóstico , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Humanos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
Earlier publication suggested that IgG2a and IgG2c (coding for Igh-1a and Igh-1b) are organized in tandem on the same chromosome as two distinct loci in mice. Our data suggest that IgG2a and IgG2c are not physically linked on the chromosome and are allelic - single locus in majority strains of mice. In another word, IgG2b-IgG2c-IgG2a haplotype proposed by Morgado et al. (1989) may exist in some strains of mice, but IgG2b-IgG2a and IgG2b-IgG2c are likely to be most common haplotypes in mice. Therefore, inbred mice may produce different IgG2a isotypes dependent on their origin (strain); C57B/6 and SJL mice secrete IgG2c while NMRI and DBA/2 mice secrete IgG2a only. The situation is more complicated for Swiss Webster mice (outbred) and Alzheimer's disease transgenic (AD/Tg) mice with multi-genetic backgrounds; mice may secrete only IgG2a, or IgG2c, or both IgG2a and IgG2c. IgG2a and IgG2c likely have different immune profile (response, immune-decoration) in mice due to their divergence of protein sequence. If antibodies based on IgG2a (or IgG2c) are used in chronic studies for preclinical evaluation of antibody efficacy, characterization of IgG2a isotypes in advance becomes critical in the design of such biopharmaceutical projects in order to avoid immune response.
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Imunoglobulina G/genética , Alelos , Animais , Sequência de Bases , Genótipo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Prediction of developmental toxicity in vitro could be based on short-time toxicogenomic endpoints in embryo-derived cell lines. Microarray studies in P19 mouse embryocarcinoma cells and mouse embryos have indicated that valproic acid (VPA), an inducer of neural tube defects, deregulates the expression of many genes, including those critically involved in neural tube development. In this study, we exposed undifferentiated R1 mouse embryonic stem cells to VPA and VPA analogs for 6 h and used CodeLink whole-genome expression microarrays to define VPA-responsive genes correlating with teratogenicity. Compared with the nonteratogenic analog 2-ethyl-4-methylpentanoic acid, VPA and the teratogenic VPA analog (S)-2-pentyl-4-pentynoic acid deregulated a much larger number of genes. Five genes (of â¼2500 array probes correlating with the separation) were sufficient to effectively separate teratogens from nonteratogens. A large fraction of the target genes correlating with teratogenicity are functionally related to embryonic development and morphogenesis, including neural tube formation and closure. Similar responses in R1 were found for most genes previously identified as VPA responsive in P19 and embryos. A subset of target genes was evaluated as candidate markers predictive of potential teratogenicity against a range of known teratogens using TaqMan expression arrays. These marker genes showed a positive predictive value for the teratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoic acid are known histone deacetylase (HDAC) inhibitors but not for compounds that are likely to act by other mechanisms. This indicates that HDAC inhibition may be a major mechanism by which VPA induces gene deregulation and possibly teratogenicity.
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Células-Tronco Embrionárias/efeitos dos fármacos , Ácidos Graxos Insaturados/toxicidade , Regulação da Expressão Gênica no Desenvolvimento , Ácido Valproico/análogos & derivados , Anormalidades Induzidas por Medicamentos/genética , Animais , Butiratos/toxicidade , Linhagem Celular , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Estudos de Associação Genética , Marcadores Genéticos , Inibidores de Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/toxicidade , Camundongos , Análise em Microsséries , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Teratogênicos/toxicidade , Toxicogenética , Ácido Valproico/toxicidadeRESUMO
Cell-based in vitro assays would potentially reduce animal testing in preclinical drug development. Mouse embryos exposed to the teratogenic drug valproic acid (VPA) in utero for 1.5, 3 or 6h on gestational day 8 were analyzed using microarrays. Significant effects on gene expression were observed already at 1.5h, and 85 probes were deregulated across all time points. To find transcriptional markers of VPA-induced developmental toxicity, the in vivo data were compared to previous in vitro data on embryonal carcinoma P19 cells exposed to VPA for 1.5, 6 or 24h. Maximal concordance between embryos and cells was at the 6-h time points, with 163 genes showing similar deregulation. Developmentally important Gene Ontology terms, such as "organ morphogenesis" and "tube development" were overrepresented among putative VPA target genes. The genes Gja1, Hap1, Sall2, H1f0,Cyp26a1, Fgf15, Otx2, and Lin7b emerged as candidate in vitro markers of potential VPA-induced teratogenicity.