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1.
Theranostics ; 11(16): 7685-7699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335958

RESUMO

Rationale: Mild traumatic brain injury (mTBI), the most common type of brain trauma, frequently leads to chronic cognitive and neurobehavioral deficits. Intervening effectively is impeded by our poor understanding of its pathophysiological sequelae. Methods: To elucidate the long-term neurovascular sequelae of mTBI, we combined optogenetics, two-photon fluorescence microscopy, and intracortical electrophysiological recordings in mice to selectively stimulate peri-contusional neurons weeks following repeated closed-head injury and probe individual vessel's function and local neuronal reactivity. Results: Compared to sham-operated animals, mTBI mice showed doubled cortical venular speeds (115 ± 25%) and strongly elevated cortical venular reactivity (53 ± 17%). Concomitantly, the pericontusional neurons exhibited attenuated spontaneous activity (-57 ± 79%) and decreased reactivity (-47 ± 28%). Post-mortem immunofluorescence revealed signs of peri-contusional senescence and DNA damage, in the absence of neuronal loss or gliosis. Alteration of neuronal and vascular functioning was largely prevented by chronic, low dose, systemic administration of a GABA-A receptor inverse agonist (L-655,708), commencing 3 days following the third impact. Conclusions: Our findings indicate that repeated mTBI leads to dramatic changes in the neurovascular unit function and that attenuation of tonic inhibition can prevent these alterations. The sustained disruption of the neurovascular function may underlie the concussed brain's long-term susceptibility to injury, and calls for development of better functional assays as well as of neurovascularly targeted interventions.


Assuntos
Concussão Encefálica/metabolismo , Concussão Encefálica/fisiopatologia , Acoplamento Neurovascular/fisiologia , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência/métodos , Neurônios/fisiologia , Optogenética/métodos
2.
Theranostics ; 11(16): 8076-8091, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335981

RESUMO

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.


Assuntos
COVID-19/imunologia , COVID-19/patologia , Animais , COVID-19/metabolismo , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , SARS-CoV-2/isolamento & purificação
3.
Front Immunol ; 12: 633540, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34295325

RESUMO

Sepsis is one of the most common comorbidities observed in diabetic patients, associated with a deficient innate immune response. Recently, we have shown that glucagon possesses anti-inflammatory properties. In this study, we investigated if hyperglucagonemia triggered by diabetes might reduce the migration of neutrophils, increasing sepsis susceptibility. 21 days after diabetes induction by intravenous injection of alloxan, we induced moderate sepsis in Swiss-Webster mice through cecum ligation and puncture (CLP). The glucagon receptor (GcgR) antagonist des-his1-[Glu9]-glucagon amide was injected intraperitoneally 24h and 1h before CLP. We also tested the effect of glucagon on CXCL1/KC-induced neutrophil migration to the peritoneal cavity in mice. Neutrophil chemotaxis in vitro was tested using transwell plates, and the expression of total PKA and phospho-PKA was evaluated by western blot. GcgR antagonist restored neutrophil migration, reduced CFU numbers in the peritoneal cavity and improved survival rate of diabetic mice after CLP procedure, however, the treatment did no alter hyperglycemia, CXCL1/KC plasma levels and blood neutrophilia. In addition, glucagon inhibited CXCL1/KC-induced neutrophil migration to the peritoneal cavity of non-diabetic mice. Glucagon also decreased the chemotaxis of neutrophils triggered by CXCL1/KC, PAF, or fMLP in vitro. The inhibitory action of glucagon occurred in parallel with the reduction of CXCL1/KC-induced actin polymerization in neutrophils in vitro, but not CD11a and CD11b translocation to cell surface. The suppressor effect of glucagon on CXCL1/KC-induced neutrophil chemotaxis in vitro was reversed by pre-treatment with GcgR antagonist and adenylyl cyclase or PKA inhibitors. Glucagon also increased PKA phosphorylation directly in neutrophils in vitro. Furthermore, glucagon impaired zymosan-A-induced ROS production by neutrophils in vitro. Human neutrophil chemotaxis and adherence to endothelial cells in vitro were inhibited by glucagon treatment. According to our results, this inhibition was independent of CD11a and CD11b translocation to neutrophil surface or neutrophil release of CXCL8/IL-8. Altogether, our results suggest that glucagon may be involved in the reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. This work collaborates with better understanding of the increased susceptibility and worsening of sepsis in diabetics, which can contribute to the development of new effective therapeutic strategies for diabetic septic patients.


Assuntos
Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Suscetibilidade a Doenças/etiologia , Glucagon/administração & dosagem , Neutrófilos/efeitos dos fármacos , Sepse/etiologia , Sepse/imunologia , Adulto , Animais , Movimento Celular/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/microbiologia , Feminino , Glucagon/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Neutrófilos/imunologia
4.
Nat Genet ; 53(8): 1221-1232, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34294917

RESUMO

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.


Assuntos
Elementos Facilitadores Genéticos , Histonas/metabolismo , Células-Tronco Neurais/fisiologia , Complexo Repressor Polycomb 2/genética , Rombencéfalo/citologia , Animais , Neoplasias Encefálicas/genética , Diferenciação Celular/genética , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenoma , Regulação da Expressão Gênica no Desenvolvimento , Glioma/genética , Histonas/genética , Humanos , Lisina/metabolismo , Masculino , Camundongos Endogâmicos , Mutação , Células-Tronco Neurais/transplante , Oncogenes , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Rombencéfalo/fisiologia
5.
Nutrients ; 13(7)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206641

RESUMO

Previous studies have reported the therapeutic effects of oleuropein (OP) consumption on the early stage of type 2 diabetes. However, the efficacy of OP on the advanced stage of type 2 diabetes has not been investigated, and the relationship between OP and intestinal flora has not been studied. Therefore, in this study, to explore the relieving effects of OP intake on the advanced stage of type 2 diabetes and the regulatory effects of OP on intestinal microbes, diabetic db/db mice (17-week-old) were treated with OP at the dose of 200 mg/kg for 15 weeks. We found that OP has a significant effect in decreasing fasting blood glucose levels, improving glucose tolerance, lowering the homeostasis model assessment-insulin resistance index, restoring histopathological features of tissues, and promoting hepatic protein kinase B activation in db/db mice. Notably, OP modulates gut microbiota at phylum level, increases the relative abundance of Verrucomicrobia and Deferribacteres, and decreases the relative abundance of Bacteroidetes. OP treatment increases the relative abundance of Akkermansia, as well as decreases the relative abundance of Prevotella, Odoribacter, Ruminococcus, and Parabacteroides at genus level. In conclusion, OP may ameliorate the advanced stage of type 2 diabetes through modulating the composition and function of gut microbiota. Our findings provide a promising therapeutic approach for the treatment of advanced stage type 2 diabetes.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Microbioma Gastrointestinal/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Glucosídeos Iridoides/farmacologia , Animais , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 2/sangue , Jejum/sangue , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos
6.
Sci Rep ; 11(1): 14917, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290317

RESUMO

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.


Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Feminino , Vetores Genéticos , Hipodermóclise , Imunidade Celular/imunologia , Imunidade nas Mucosas/imunologia , Imunização Secundária , Camundongos , Camundongos Endogâmicos , Vacinação/métodos
7.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298975

RESUMO

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization-despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.


Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Ciclofosfamida/toxicidade , Neoplasias Pulmonares/secundário , Macrófagos/fisiologia , Neoplasias Mamárias Experimentais/genética , Metástase Neoplásica/fisiopatologia , Proteínas de Neoplasias/fisiologia , Estresse Fisiológico/genética , Macrófagos Associados a Tumor/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Linhagem Celular Tumoral , Ciclofosfamida/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genótipo , Humanos , Neoplasias Pulmonares/metabolismo , Ativação de Macrófagos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Terapia Neoadjuvante/efeitos adversos , Metástase Neoplásica/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transplante de Neoplasias/métodos , Células-Tronco Neoplásicas/patologia , Migração Transendotelial e Transepitelial , Microambiente Tumoral , Macrófagos Associados a Tumor/efeitos dos fármacos
8.
Molecules ; 26(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203927

RESUMO

Artemisia ludoviciana subsp. mexicana has been traditionally used for the treatment of digestive ailments such as gastritis, whose main etiological agent is Helicobacter pylori. In a previous screening study, the aqueous extract exhibited a good in vitro anti-H. pylori activity. With the aim of determining the efficacy of this species as a treatment for H. pylori related diseases and finding bioactive compounds, its aqueous extract was subjected to solvent partitioning and the fractions obtained were tested for their in vitro anti-H. pylori effect, as well as for their in vivo gastroprotective and anti-inflammatory activities. The aqueous extract showed a MIC = 250 µg/mL. No acute toxicity was induced in mice. A gastroprotection of 69.8 ± 3.8%, as well as anti-inflammatory effects of 47.6 ± 12.4% and 38.8 ± 10.2% (by oral and topical administration, respectively), were attained. Estafiatin and eupatilin were isolated and exhibited anti-H. pylori activity with MBCs of 15.6 and 31.2 µg/mL, respectively. The finding that A. ludoviciana aqueous extract has significant anti-H. pylori, gastroprotective and anti-inflammatory activities is a relevant contribution to the ethnopharmacological knowledge of this species. This work is the first report about the in vivo gastroprotective activity of A. ludoviciana and the anti-H. pylori activity of eupatilin and estafiatin.


Assuntos
Artemisia/metabolismo , Flavonoides/farmacologia , Helicobacter pylori/efeitos dos fármacos , Animais , Antiulcerosos/farmacologia , Flavonoides/metabolismo , Gastrite/tratamento farmacológico , Masculino , Medicina Tradicional , Camundongos , Camundongos Endogâmicos , Extratos Vegetais/farmacologia , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Úlcera Gástrica/tratamento farmacológico
9.
Mol Genet Genomics ; 296(5): 1103-1119, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34170407

RESUMO

In genome-wide quantitative trait locus (QTL) mapping studies, multiple quantitative traits are often measured along with the marker genotypes. Multi-trait QTL (MtQTL) analysis, which includes multiple quantitative traits together in a single model, is an efficient technique to increase the power of QTL identification. The two most widely used classical approaches for MtQTL mapping are Gaussian Mixture Model-based MtQTL (GMM-MtQTL) and Linear Regression Model-based MtQTL (LRM-MtQTL) analyses. There are two types of LRM-MtQTL approach known as least squares-based LRM-MtQTL (LS-LRM-MtQTL) and maximum likelihood-based LRM-MtQTL (ML-LRM-MtQTL). These three classical approaches are equivalent alternatives for QTL detection, but ML-LRM-MtQTL is computationally faster than GMM-MtQTL and LS-LRM-MtQTL. However, one major limitation common to all the above classical approaches is that they are very sensitive to outliers, which leads to misleading results. Therefore, in this study, we developed an LRM-based robust MtQTL approach, called LRM-RobMtQTL, for the backcross population based on the robust estimation of regression parameters by maximizing the ß-likelihood function induced from the ß-divergence with multivariate normal distribution. When ß = 0, the proposed LRM-RobMtQTL method reduces to the classical ML-LRM-MtQTL approach. Simulation studies showed that both ML-LRM-MtQTL and LRM-RobMtQTL methods identified the same QTL positions in the absence of outliers. However, in the presence of outliers, only the proposed method was able to identify all the true QTL positions. Real data analysis results revealed that in the presence of outliers only our LRM-RobMtQTL approach can identify all the QTL positions as those identified in the absence of outliers by both methods. We conclude that our proposed LRM-RobMtQTL analysis approach outperforms the classical MtQTL analysis methods.


Assuntos
Genômica/métodos , Locos de Características Quantitativas , Animais , Mapeamento Cromossômico , Simulação por Computador , Feminino , Genética Populacional/métodos , Genômica/estatística & dados numéricos , Hordeum/genética , Funções Verossimilhança , Camundongos Endogâmicos
10.
Transfusion ; 61(7): 2169-2178, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34181769

RESUMO

BACKGROUND: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses. STUDY DESIGN AND METHODS: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization. RESULTS: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG. CONCLUSION: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency.


Assuntos
Antígenos CD1d/imunologia , Preservação de Sangue , Transfusão de Sangue , Eritrócitos/imunologia , Isoanticorpos/biossíntese , Isoantígenos/imunologia , Lisofosfolipídeos/sangue , Reação Transfusional/imunologia , Alarminas/sangue , Alarminas/imunologia , Animais , Especificidade de Anticorpos , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Sistema do Grupo Sanguíneo Duffy/genética , Sistema do Grupo Sanguíneo Duffy/imunologia , Feminino , Imunização , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Isoanticorpos/imunologia , Lisofosfolipídeos/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Muramidase/imunologia , Ovalbumina/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia
11.
J Med Chem ; 64(13): 9458-9483, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34152138

RESUMO

Management of moderate to severe pain relies heavily on opioid analgesics such as morphine, oxycodone, and fentanyl in clinics. However, their prolonged use was associated with undesirable side effects. Many new strategies to reduce side effects have been proposed, but not without disadvantages. Using a hot plate model as a phenotypic screening method, our studies identified (3R,4S)-9d with a new scaffold as a potent analgesic with ED50 values of 0.54 mg/kg and 0.021 mg/kg in hot plate and antiwrithing models, respectively. Mechanistic studies showed that it elicited its analgesic effect via the active metabolite (3R,4S)-10a. The mechanism of (3R,4S)-10a-induced activation of the µ opioid receptor (MOR) was proposed by means of molecular dynamics (MD) simulation.


Assuntos
Analgésicos Opioides/farmacologia , Descoberta de Drogas , Dor/tratamento farmacológico , Receptores Opioides mu/metabolismo , Ácido Acético , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/química , Animais , Relação Dose-Resposta a Droga , Feminino , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Dor/induzido quimicamente , Relação Estrutura-Atividade
12.
Lab Anim (NY) ; 50(8): 215-223, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34155410

RESUMO

Mouse strains differ markedly in all behaviors, independently of their genetic background. We undertook this study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in the study design to ensure experimental reproducibility and comply with essential animal welfare principles.


Assuntos
Bem-Estar do Animal , Locomoção , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Reprodutibilidade dos Testes
13.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071329

RESUMO

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.


Assuntos
Hidrogéis/química , Nanopartículas/administração & dosagem , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/administração & dosagem , Testículo/transplante , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Alginatos/química , Animais , Liberação Controlada de Fármacos , Preservação da Fertilidade/métodos , Humanos , Masculino , Camundongos Endogâmicos , Nanopartículas/química , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/farmacocinética , Espermatogônias/efeitos dos fármacos , Testículo/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacocinética
14.
Toxicol Pathol ; 49(5): 1100-1108, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33942680

RESUMO

The tolerability of single daily gavage doses of 0.5% or 2.0% (wt/vol) sodium lauryl sulfate (SLS) in 11- to 12-week-old male CD-1 mice was evaluated in a study of 3 months in duration. Live-phase, gross necropsy, and histopathologic parameters were evaluated. Mortality of 14% occurred in mice administered formulations containing SLS. Clinical observations in mice administered SLS included abnormal respiration (audible, irregular, and/or labored), swollen abdomen, rough haircoat, hunched appearance, and hypoactivity. Necropsy findings in mice administered SLS consisted of enlarged intestines containing abnormal contents with gas. There were no instances of mechanical gavage-related injury. Histologic evaluation of the respiratory tract revealed injury to the nasal passages and nasopharynx, including, but not limited to, inflammation, exudate, apoptosis/necrosis of epithelium, and atrophy of epithelium or olfactory nerves. Collectively, the data indicated that under the experimental conditions of our 3-month study in male CD-1 mice, once-daily gavage administration of vehicle formulations containing SLS at 0.5% or 2.0% resulted in nasal injury and 14% mortality supportive of gastroesophageal reflux. Sponsors utilizing formulations containing SLS in toxicity studies in CD-1 mice should exclude gastroesophageal reflux as a confounding factor in studies with morbidity or mortality associated with respiratory distress or evidence of aerophagia.


Assuntos
Testes de Carcinogenicidade , Administração Oral , Animais , Masculino , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Endogâmicos F344 , Dodecilsulfato de Sódio/toxicidade
15.
J Vet Med Sci ; 83(7): 1155-1160, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34053976

RESUMO

The aim of this study is to examine 1) muscle fiber type composition, 2) myofiber diameter, and 3) aquaporin (AQP) 7 and AQP 9 mRNA expressions by quantitative PCR in muscles of obese db/db mice. The myofiber type composition of skeletal muscle was not statistically significantly different between db/db mice and control mice; while the average myofiber diameter ratio showed a decrease in db/db mice. The expression of AQP7 but not AQP9 mRNA in the skeletal and cardiac muscles was significantly upregulated in db/db mice. Thus this study revealed quantitatively that type 2 myofiber atrophy was shown in the skeletal muscles of db/db mice. AQP7 mRNA expression was upregulated in the skeletal and cardiac muscles of db/db mice.


Assuntos
Fibras Musculares Esqueléticas , Doenças dos Roedores , Animais , Camundongos , Camundongos Endogâmicos , Miocárdio , Obesidade/genética , Obesidade/veterinária , RNA Mensageiro/genética
16.
Toxicol Appl Pharmacol ; 424: 115597, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34051218

RESUMO

Trichloroethene (TCE), a widely used industrial solvent, is associated with the development of autoimmune diseases (ADs), including systemic lupus erythematosus and autoimmune hepatitis. Increasing evidence support a linkage between altered gut microbiome composition and the onset of ADs. However, it is not clear how gut microbiome contributes to TCE-mediated autoimmunity, and initial triggers for microbiome-host interactions leading to systemic autoimmune responses remain unknown. To achieve this, female MRL+/+ mice were treated with 0.5 mg/ml TCE for 52 weeks and fecal samples were subjected to 16S rRNA sequencing to determine the microbiome composition. TCE exposure resulted in distinct bacterial community revealed by ß-diversity analysis. Notably, we observed reduction in Lactobacillaceae, Rikenellaceae and Bifidobacteriaceae families, and enrichment of Akkermansiaceae and Lachnospiraceae families after TCE exposure. We also observed significantly increased colonic oxidative stress and inflammatory markers (CD14 and IL-1ß), and decreased tight junction proteins (ZO-2, occludin and claudin-3). These changes were associated with increases in serum antinuclear and anti-smooth muscle antibodies and cytokines (IL-6 and IL-12), together with increased PD1 + CD4+ T cells in TCE-exposed spleen and liver tissues. Importantly, fecal microbiota transplantation (FMT) using feces from TCE-treated mice to antibiotics-treated mice induced increased anti-dsDNA antibodies and hepatic CD4+ T cell infiltration in the recipient mice. Our studies thus delineate how imbalance in gut microbiome and mucosal redox status together with gut inflammatory response and permeability changes could be the key factors in contributing to TCE-mediated ADs. Furthermore, FMT studies provide a solid support to a causal role of microbiome in TCE-mediated autoimmunity.


Assuntos
Autoimunidade/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Microbioma Gastrointestinal/efeitos dos fármacos , Tricloroetileno/toxicidade , Animais , Esquema de Medicação , Feminino , Microbioma Gastrointestinal/fisiologia , Inflamação , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Estresse Oxidativo , Baço/efeitos dos fármacos
17.
Kidney Int ; 100(2): 364-376, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33961870

RESUMO

Dysregulation of mucosal immunity may play a role in the pathogenesis of IgA nephropathy (IgAN). However, it is unclear whether the nasal-associated lymphoid tissue (NALT) or gut-associated lymphatic tissue is the major induction site of nephritogenic IgA synthesis. To examine whether exogenous mucosal antigens exacerbate the pathogenesis of IgAN, we assessed the disease phenotypes of IgAN-onset ddY mice housed germ-free. These mice were transferred to a specific pathogen-free environment and divided into three groups: challenged with the Toll-like receptor 9 (TLR9) ligand CpG-oligodeoxynucleotide, fecal transplantation, and the untreated control group. The levels of aberrantly glycosylated IgA and IgG-IgA immune complexes were measured in the serum and supernatant of cultured cells purified from the NALT, mesenteric lymph nodes, and Peyer's patch. Although the germ-free IgAN-onset ddY mice did not develop IgAN, they showed aggravation of kidney injury with mesangial IgA deposition after transfer to the specific pathogen-free state. The NALT cells produced more aberrantly glycosylated IgA than those from the mesenteric lymph node and Peyer's patch, resulting in induction of IgG-IgA immune complexes formation. Additionally, TLR9 enhanced the production of nephritogenic IgA and IgG-IgA immune complexes by nasal-associated lymphoid but not gut-associated lymphatic cells. Furthermore, the germ-free IgAN-onset ddY mice nasally immunized with CpG-oligonucleotide showed aggravation of kidney injury with mesangial IgA deposition, whereas those that received fecal transplants did not develop IgAN. Thus, NALT is the major induction site of the production of aberrantly glycosylated IgA in murine IgAN.


Assuntos
Glomerulonefrite por IGA , Animais , Mesângio Glomerular , Imunoglobulina A , Tecido Linfoide , Camundongos , Camundongos Endogâmicos
18.
Kidney Int ; 99(6): 1262-1264, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34023024

RESUMO

In drug development, preclinical studies using laboratory animals are crucial to test efficacy and safety of drug candidates. However, there have been discrepancies between animal studies and clinical trials in human patients. Preclinical randomized controlled trials, as reported by Lei et al. in this issue of Kidney International, may reduce the gap between experimental studies and randomized controlled trials in human patients, although there remain issues to be addressed.


Assuntos
Nefrite Lúpica , Animais , Humanos , Janus Quinase 1 , Camundongos , Camundongos Endogâmicos
19.
Chem Biol Interact ; 344: 109496, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33939976

RESUMO

An increased risk of developing lung cancer has been associated with exposure to cigarette smoke carcinogens and alteration in the gut microbiota. However, there is limited understanding about the impact of exposure to NNK and BaP, the two important components of cigarette smoke carcinogens, on gut microbiota in lung cancer. The present study characterized the influence of exposure to a mixture of NNK plus BaP on lung cancer, feces metabolite composition, and gut microbiota in the A/J mice. The A/J mice were administered NNK plus BaP, and the changes in gut microbiota and feces metabolic profiles were characterized using 16S rRNA gene sequencing and metabolomics, respectively. Results presented here illustrated that a mixture of NNK plus BaP exposure triggered lung carcinogenesis as shown by light microscopy and histopathological evaluation. 16S rRNA sequencing of gut microbiota indicated that exposure to NNK plus BaP could modified fecal bacterial composition. Elevated levels of Actinobacteria, Bifidobacterium, and Intestinimonas and reduced levels of Alistipes, Odoribacter, and Acetatifactor are associated with NNK plus BaP triggered lung cancer. In addition, metabolomics profile revealed the regulation of metabolism including purine metabolism, phenylalanine metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and others. In conclusion, the results provide some guidance for using gut microbes as biomarkers to assess the progression of lung cancer, and lead to interventional targets to control the development of the disease in the future.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Benzo(a)pireno/farmacologia , Carcinógenos/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Nitrosaminas/farmacologia , Adenocarcinoma de Pulmão/complicações , Adenocarcinoma de Pulmão/patologia , Animais , Bactérias/metabolismo , Disbiose/etiologia , Disbiose/metabolismo , Fezes/química , Fezes/microbiologia , Feminino , Pulmão/patologia , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metabolômica , Camundongos Endogâmicos
20.
Am J Physiol Renal Physiol ; 320(6): F1080-F1092, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33969697

RESUMO

A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT1) receptors in the kidney, stimulating Na+ reabsorption. AT1 receptors in tubular epithelia control expression and stimulation of Na+ transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT1 receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na+ excretion in SMKO mice, we considered whether alterations in Na+ transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na+ transporters and associated proteins. Here, we show that levels of Na+-K+-2Cl- cotransporter isoform 2 (NKCC2) and Na+/H+ exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser71), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na+ channel. However, the increases in α- and γ-epithelial Na+ channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na+ excretion observed after cell-specific deletion of AT1 receptors from smooth muscle cells is associated with altered Na+ transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na+ transporters and contributing to the regulation of pressure natriuresis.NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.


Assuntos
Angiotensina II/farmacologia , Células Epiteliais/metabolismo , Rim/irrigação sanguínea , Miócitos de Músculo Liso/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Amilorida/farmacologia , Animais , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Feminino , Furosemida/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Hipertensão/induzido quimicamente , Proteínas Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Receptor Tipo 1 de Angiotensina/genética , Sódio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia
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