RESUMO
As a global problem, fine particulate matter (PM2.5) really needs local fixes. Considering the increasing epidemiological relevance to anxiety and depression but inconsistent toxicological results, the most important question is to clarify whether and how PM2.5 causally contributes to these mental disorders and which components are the most dangerous for crucial mitigation in a particular place. In the present study, we chronically subjected male mice to a real-world PM2.5 exposure system throughout the winter heating period in a coal combustion area and revealed that PM2.5 caused anxiety and depression-like behaviors in adults such as restricted activity, diminished exploratory interest, enhanced repetitive stereotypy, and elevated acquired immobility, through behavioral tests including open field, elevated plus maze, marble-burying, and forced swimming tests. Importantly, we found that dopamine signaling was perturbed using mRNA transcriptional profile and bioinformatics analysis, with Drd1 as a potential target. Subsequently, we developed the Drd1 expression-directed multifraction isolating and nontarget identifying framework and identified a total of 209 compounds in PM2.5 organic extracts capable of reducing Drd1 expression. Furthermore, by applying hierarchical characteristic fragment analysis and molecular docking and dynamics simulation, we clarified that phenyl-containing compounds competitively bound to DRD1 and interfered with dopamine signaling, thereby contributing to mental disorders. Taken together, this work provides experimental evidence for researchers and clinicians to identify hazardous factors in PM2.5 and prevent adverse health outcomes and for local governments and municipalities to control source emissions for diminishing specific disease burdens.
Assuntos
Ansiedade , Depressão , Material Particulado , Receptores de Dopamina D1 , Animais , Material Particulado/toxicidade , Camundongos , Masculino , Ansiedade/metabolismo , Depressão/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/genética , Poluentes Atmosféricos/toxicidade , Comportamento Animal/efeitos dos fármacos , Simulação de Acoplamento MolecularRESUMO
Numerous studies indicate that fine particulate matters (PM2.5) and its organic components are urgent risk factors for cardiovascular diseases (CVDs). Combining toxicological experiments, effect-directed analyses, and nontarget identification, this study aims to explore whether PM2.5 exposure in coal-combustion areas induces myocardial fibrosis and how to identify the effective organic components and their toxic structures to support regional risk control. First, we constructed an animal model of real-world PM2.5 exposure during the heating season and found that the exposure impaired cardiac systolic function and caused myocardial fibrosis, with chemokine Ccl2-mediated inflammatory response being the key cause of collagen deposition. Then, using the molecular event as target coupled with two-stage chromatographic isolation and mass spectrometry analyses, we identified a total of 171 suspect organic compounds in the PM2.5 samples. Finally, using hierarchical characteristic fragment analysis, we predicted that 40 of them belonged to active compounds with 6 alert structures, including neopentane, butyldimethylamine, 4-ethylphenol, hexanal, decane, and dimethylaniline. These findings provide evidence for risk management and prevention of CVDs in polluted areas.
Assuntos
Material Particulado , Animais , Camundongos , Masculino , Poluentes Atmosféricos , FibroseRESUMO
Emerging studies implicate fine particulate matter (PM2.5) and its organic components (OCs) as urgent hazard factors for lung cancer progression in nonsmokers. Establishing the adverse outcome pathway (AOP)-directed nontargeted identification method, this study aimed to explore whether PM2.5 exposure in coal-burning areas promoted lung tumor metastasis and how we identify its effective OCs to support traceability and control of regional PM2.5 pollution. First, we used a nude mouse model of lung cancer for PM2.5 exposure and found that the exposure significantly promoted the hematogenous metastases of A549-Luc cells in lung tissues and the adverse outcomes (AOs), with key events (KEs) including the changed expression of epithelial-mesenchymal transition (EMT) markers, such as suppression of E-cad and increased expression of Fib. Subsequently, using AOs and KEs as adverse outcome directors, we identified a total of 35 candidate chemicals based on the in vitro model and nontargeted analysis. Among them, tributyl phosphate (C12H27O4P), 2-bromotetradecane (C14H29Br), and methyl decanoate (C11H22O2) made greater contributions to the AOs. Finally, we clarified the interactions between these OCs and EMT-activating transcription factors (EMT-ATFs) as the molecular initiation event (MIE) to support the feasibility of the above identification strategy. The present study updates a new framework for identifying tumor metastasis-promoting OCs in PM2.5 and provides solid data for screening out chemicals that need priority control in polluted areas posing higher lung cancer risk.
Assuntos
Rotas de Resultados Adversos , Poluentes Atmosféricos , Neoplasias Pulmonares , Animais , Camundongos , Material Particulado , Neoplasias Pulmonares/patologia , Pulmão , Transição Epitelial-MesenquimalRESUMO
BACKGROUND: Tendinopathy is the leading sports-related injury and will cause severe weakness and tenderness. Effective therapy for tendinopathy remains limited, and extracellular vesicles (EVs) derived from adipose tissue-derived mesenchymal stem cells (ADMSCs) have demonstrated great potential in tendinopathy treatment; however, the influence of aging status on EV treatment has not been previously described. RESULTS: In this study, it was found that ADMSCs derived from old mice (ADMSCold) demonstrated remarkable cellular senescence and impaired NAD+ metabolism compared with ADMSCs derived from young mice (ADMSCyoung). Lower NAMPT contents were detected in both ADMSCold and its secreted EVs (ADMSCold-EVs). Advanced animal experiments demonstrated that ADMSCyoung-EVs, but not ADMSCold-EVs, alleviated the pathological structural, functional and biomechanical properties in tendinopathy mice. Mechanistic analyses demonstrated that ADMSCyoung-EVs improved cell viability and relieved cellular senescence of tenocytes through the NAMPT/SIRT1/PPARγ/PGC-1α pathway. ADMSCyoung-EVs, but not ADMSCold-EVs, promoted phagocytosis and M2 polarization in macrophages through the NAMPT/SIRT1/Nf-κb p65/NLRP3 pathway. The macrophage/tenocyte crosstalk in tendinopathy was influenced by ADMSCyoung-EV treatment and thus it demonstrated "One-Stone-Two-Birds" effects in tendinopathy treatment. CONCLUSIONS: This study demonstrates an effective novel therapy for tendinopathy and uncovers the influence of donor age on curative effects by clarifying the detailed biological mechanism.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Tendinopatia , Animais , Camundongos , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Sirtuína 1/metabolismo , Tendinopatia/terapiaRESUMO
As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has received much attention for its non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb. The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb. Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop the PEGylated Hb as HBOC. Here, Cys-93(ß) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K) through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate (PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hb with hydrazone hydrolysis and disulfide reduction. Reversible conjugation of PEG20K at Cys-93(ß) can protect Lys-95(ß), Val-1(α) and Lys-16(α) of Hb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramer instability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study is expected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.
Assuntos
Cisteína/química , Hemoglobina A/química , Hemoglobina A/metabolismo , Oxigênio/metabolismo , Polietilenoglicóis/química , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , UltracentrifugaçãoRESUMO
Human growth hormone (hGH) has been used to treat children with short stature, renal failure, and Turner's syndrome. However, clinical application of hGH suffers from its short plasma half-life and low bioavailability. PEGylation and albumin binding are two of the most effective approaches to prolong the plasma half-life of hGH. However, the steric shielding effects of polyethylene glycol (PEG) and albumin can drastically decrease the bioactivity of hGH, which is opposite to the increased pharmacokinetics (PK). In the present study, a long-acting hGH with markedly improved pharmacological profile was rationally designed and prepared by N-terminal modification of hGH with pseudo-bifunctional PEG-hexadecane by using PEG (3.5 kDa or 10 kDa) as the linker. PEGylation and albumin binding with hexadecane can increase the hydrodynamic volume and decrease the immunogenicity of hGH, which thereby markedly increases the PK of hGH. Since N-terminus is far from the bioactive domain of hGH, N-terminal modification of hGH can minimize the steric shielding effects on the bioactive domain of hGH. Hexadecane-bound albumin can be slowly released from hGH during the in vivo circulation, which can slowly restore the bioactivity of hGH. Thus, the high bioactivity of PEG-hexadecane modified hGH (hGH-PEG-HD) was synergistically achieved by N-terminal modification with pseudo-bifunctional PEG-hexadecane and slow-release of albumin. The high pharmacodynamics (PD) of hGH-PEG-HD was due to the synergistic effect of the high bioactivity and the overall increased PK.
Assuntos
Alcanos/química , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/farmacocinética , Polietilenoglicóis/química , Albuminas/química , Animais , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Ressonância de Plasmônio de SuperfícieRESUMO
The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn(-/-) males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn(-/-) males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn(-/-) mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.
Assuntos
Reação Acrossômica , Acrossomo/fisiologia , Proteínas de Membrana/metabolismo , Acrossomo/ultraestrutura , Animais , Feminino , Fertilização , Técnicas de Inativação de Genes , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismoRESUMO
Human growth hormone (hGH) suffers from a short plasma half-life of â¼15 min, necessitating frequent injections to maintain its physiological effect. PEGylation, conjugation of polyethylene glycol (PEG), is an effective strategy to prolong the plasma half-life of hGH. However, PEGylation can significantly decrease the bioactivity of hGH. Thus, a new PEGylation approach is desired to improve the pharmacokinetics (PK) and pharmacodynamics (PD) of the PEGylated hGH. In the present study, two N-terminally mono-PEGylated hGHs were prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The hydrodynamic volume, proteolytic sensitivity, and immunogenicity of the PEGylated hGH with phenyl amide linker (hGH-phenyl-PEG) were lower than those of the one with propyl linker (hGH-prop-PEG). In addition, hGH-phenyl-PEG showed a higher in vitro bioactivity and better PK and PD than hGH-prop-PEG. The better PK of hGH-phenyl-PEG was mainly due to its lower proteolytic sensitivity and low immunogenicity. The better PD of hGH-phenyl-PEG was mainly due to its higher in vitro bioactivity. Thus, the phenyl amide linker can improve the overall pharmacological profiles of the PEGylated hGH. Our study is expected to advance the development of long-acting protein biotherapeutics with high therapeutic efficacy.
Assuntos
Amidas/química , Hormônio do Crescimento Humano/química , Polietilenoglicóis/química , Animais , Meia-Vida , Hormônio do Crescimento Humano/farmacocinética , Hormônio do Crescimento Humano/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-DawleyRESUMO
Exposure to fine particulate matter (PM2.5) is a significant risk factor for hepatic steatosis. The N6-methyladenosine (m6A) is implicated in metabolic disturbances triggered by exogenous environmental factors. However, the role of m6A in mediating PM2.5-induced hepatic steatosis remains unclear. Herein, male C57BL/6J mice were subjected to PM2.5 exposure throughout the entire heating season utilizing a real-ambient PM2.5 whole-body inhalation exposure system. Concurrently, HepG2 cell models exposed to PM2.5 were developed to delve the role of m6A methylation modification. Following PM2.5 exposure, significant hepatic lipid accumulation and elevated global m6A level were observed both in vitro and in vivo. The downregulation of YTHDC2, an m6A-binding protein, might contribute to this alteration. In vitro studies revealed that lipid-related genes CEPT1 and YWHAH might be targeted by m6A modification. YTHDC2 could bind to CDS region of them and increase their stability. Exposure to PM2.5 shortened mRNA lifespan and suppressed the expression of CEPT1 and YWHAH, which were reversed to baseline or higher level upon the enforced expression of YTHDC2. Consequently, our findings indicate that PM2.5 induces elevated m6A methylation modification of CEPT1 and YWHAH by downregulating YTHDC2, which in turn mediates the decrease in the mRNA stabilization and expression of these genes, ultimately resulting in hepatic steatosis.
Assuntos
Adenosina , Fígado Gorduroso , Camundongos Endogâmicos C57BL , Material Particulado , Proteínas de Ligação a RNA , Animais , Humanos , Masculino , Camundongos , Adenosina/análogos & derivados , Poluentes Atmosféricos/toxicidade , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Células Hep G2 , Fígado/metabolismo , Fígado/efeitos dos fármacos , Material Particulado/toxicidade , RNA Helicases , Metilação de RNA , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Wt1 is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1 plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1(-/flox); Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1(-/flox); Amh-Cre testes. However, the mRNA level of Col4a1 and Col4a2 was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1(-/flox); Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1 and Col4a2 promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1 deletion, we conclude that the loss of Wt1 in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.
Assuntos
Colágeno Tipo IV/metabolismo , Genes do Tumor de Wilms , Cordão Espermático/embriologia , Testículo/metabolismo , Animais , Membrana Basal/fisiologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9/metabolismo , Testículo/embriologia , Ativação TranscricionalRESUMO
Scrotal hypothermia is essential for normal spermatogenesis, and temporal heat stress causes a reversible disruption of the blood-testis barrier (BTB). Previous studies have shown that AR expression in primary monkey Sertoli cells (SCs) was dramatically reduced after temporary heat treatment. However, the mechanisms underlying the heat-induced reversible disruption of the BTB, including whether it is directly regulated by the AR, remain largely unknown. In this study, we demonstrated that the AR acts upstream to regulate the heat-induced reversible change in the BTB in mice. When the AR was overexpressed in SCs using an adenovirus, the heat stress-induced down-regulation of BTB-associated proteins (Zonula occludens-1 (ZO-1), N-Cadherin, E-Cadherin, α-Catenin, and ß-Catenin) was partially rescued. AR knockdown by RNAi or treatment with flutamide (an AR antagonist) in SCs inhibited the recovery of BTB-associated protein expression after 43°C heat treatment for 30 min. The results of an in vivo AR antagonist injection experiment further showed that the recovery of BTB permeability induced by temporal heat stress was regulated by the AR. Furthermore, we observed that the co-localization and interactions of partitioning-defective protein (Par) 6-Par3-aPKC-Cdc42 polarity complex components were disrupted in both AR-knockdown and heat-induced SCs. AR overexpression in SCs prevented the disruption of these protein-protein interactions after heat treatment. AR knockdown or treatment with flutamide in SCs inhibited the restoration of these protein-protein interactions after heat treatment compared with heat treatment alone. Together, these results demonstrate that the AR plays a crucial role in the heat-induced reversible change in BTB via the Par polarity complex.
RESUMO
Aged male patients are more vulnerable to severe or critical symptoms of COVID-19, but the underlying mechanism remains elusive. In this study, we analyzed previously published scRNA-seq data from a large cohort of COVID-19 patients, castrated and regenerated mice, and bulk RNA-seq of a RNAi library of 400 genes, and revealed that both immunity and OXPHOS displayed cell-type-, sex-, and age-related variation in the severe or critical COVID-19 patients during disease progression, with a more prominent increase in immunity and decrease in OXPHOS in myeloid cells in the males relative to the females (60-69 years old). Male severe or critical patients above 70 years old were an exception in that the compromised negative correlation between OXPHOS and immunity in these patients was associated with its disordered transcriptional regulation. Finally, the expression levels of OXPHOS and androgens were revealed to be positively correlated, and the responses of macrophages to android fluctuation were more striking than other types of detected immune cells in the castrated mice model. Therefore, the interplay of OXPHOS and immunity displayed a cell-type-specific, age-related, and sex-biased pattern, and the underlying potential regulatory role of the hormonal milieu should not be neglected.
RESUMO
Nitrogen dioxide (NO2) is one of the most common indoor and outdoor air pollutants. Inhalation of NO2 is associated with an increased risk of health problems, especially cardiovascular diseases. However, the underlying pathogenic mechanisms still remain unclear. In this study, we exposed C57BL/6J mice to NO2 (2.5 ppm, 5 h/d) for 28 days and found that NO2 inhalation induced cardiac dysfunction in male mice, but not in female mice, including left ventricular dilation and cardiac systolic dysfunction. Pathological staining showed that NO2 inhalation induced eccentric hypertrophy with enlarged individual cardiomyocytes, dilated left ventricle, and thinning of the left ventricular wall in male mice. The transcriptional analysis suggested that NO2 exposure could disrupt Ca2+ homeostasis, actin cytoskeletal reorganization, myocardial contractility, and vascular dilation in male mice. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that differentially expressed genes (DEGs) were closely associated with the apoptotic signaling pathways. These findings suggested that NO2 exposure caused cardiac eccentric hypertrophy and cardiac dysfunction through apoptotic signaling pathways, and contributed to cardiotoxicity.
Assuntos
Poluentes Atmosféricos , Cardiopatias , Camundongos , Masculino , Feminino , Animais , Dióxido de Nitrogênio/análise , Actinas , Camundongos Endogâmicos C57BL , Cardiomegalia/induzido quimicamente , Poluentes Atmosféricos/análise , Apoptose , Cardiopatias/induzido quimicamente , Transdução de SinaisRESUMO
NO2 is a common indoor and outdoor air pollutant, but its health effects are still controversial. Beside respiratory injury, more epidemiological studies show that inhalation of NO2 is associated with an increased risk of anxiety and depression. However, the causal relationship at the molecular level remains unclear. In the present study, we exposed adult C57BL/6J mice to NO2 (2.5 ppm, 5 h/day) for four weeks, and found anxiety and depression-like behaviors in male mice, but not female mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that differentially expressed genes (DEGs) in the prefrontal cortex and cerebellum were closely associated with signal transduction pathways, such as axon guidance. Importantly, NO2 inhalation damaged the ultrastructure of myelin sheath and caused the abnormal expression of related genes in males, which partially contributed to mental disorders. We also found that prolactin (Prl), through its anti-inflammatory activity and remyelination, might play a major role in the sex-specific neurobehavioral disorder in male mice caused by NO2 exposure.
Assuntos
Bainha de Mielina , Dióxido de Nitrogênio , Animais , Ansiedade/induzido quimicamente , Depressão/induzido quimicamente , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Streptococcus pneumoniae is a serious Gram-positive pathogen that can lead to an invasive pneumococcal disease with high mortality rate. Pneumococcal capsular polysaccharide (PS) is a key virulence determinant and its immunogenicity can be increased by conjugation with a carrier protein. However, the PS-specific cellular and humoral immunity of pneumococcal conjugate vaccine needs further improvement. Hexadecane (HD) is an element of lipid that decorates the surface of nearly all microbial classes. Polyethylene glycol (PEG)-HD conjugate (PEG-HD) is soluble and can act as an adjuvant. In the present study, a novel pneumococcal polysaccharide conjugate vaccine was prepared by conjugation of tetanus toxoid (TT) portion of PS-TT conjugate (PS-TT) with PEG-HD. As compared with PS-TT, conjugation with PEG-HD led to an 8.0-fold increase in the PS-specific IgG titers. Conjugation with PEG-HD also gave rise to 34.9-, 3.6- and 7.7-fold increase in the IFN-γ, TNF-α and IL-5 levels, respectively. Thus, the conjugated PEG-HD has a stimulatory adjuvant activity to potentiate a robust humoral and cellular immunity. Our proposed conjugate was expected to act as an effective pneumococcal conjugate vaccine for prevention of S. pneumoniae infections.
Assuntos
Adjuvantes Imunológicos/metabolismo , Alcanos/metabolismo , Vacinas Pneumocócicas/imunologia , Polietilenoglicóis/metabolismo , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Citocinas/metabolismo , Feminino , Imunoglobulina G/sangue , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Toxoide Tetânico/metabolismo , Vacinas Conjugadas/imunologiaRESUMO
This study aimed to investigate the effect of chitosan oligosaccharide (COS) conjugation on the immunogenicity of porcine circovirus type-2 (PCV2) vaccine. Two conjugates (PCV2-COS-1 and PCV2-COS-2) were designed by covalent conjugation of an inactivated PCV2 vaccine with COS, and administered to C57BL/6 mice three times at two-week intervals. The results indicate that, as compared to PCV2 alone group, the PCV2-COS conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting T lymphocyte proliferation and initiating a mixed Th1/Th2 response, including the elevated production of PCV-2 specific antibodies and up-regulated secretion of inflammatory cytokines. Noticeably, the immunization with PCV2-COS-1 conjugate displayed similar or even better immune-stimulating effects than that by PCV2/ISA206 (a commercialized adjuvant) and showed no infection or pathological signs at injection sites of the mice. Presumably, the covalent linkage of PCV2 vaccine to COS might be a viable strategy to increase the efficacy against PCV2-associated diseases.
Assuntos
Quitosana , Infecções por Circoviridae/prevenção & controle , Imunidade Celular , Imunidade Humoral , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Infecções por Circoviridae/imunologia , Circovirus , Camundongos , Camundongos Endogâmicos C57BL , SuínosRESUMO
Meningococcal disease is a fatal illness of sudden onset caused by Neisseria meningitides. Meningococcal capsular polysaccharide (CPS) is a major virulence factor that generally does not induce immunological memory. Conjugation with a carrier protein can significantly increase the immunogenicity of CPS and induce immunological memory. However, it is highly desired to optimize the CPS-specific immunogenicity of the conjugate vaccine. Although adjuvant has been widely used to improve the immunogenicity of antigens, co-administration and conjugation of adjuvant with the conjugate vaccine has rarely been investigated. As a stimulator of humoral and cellular immunity, ß-glucan can activate macrophages and trigger intracellular processes to secrete cytokines initiating inflammatory reactions. In the present study, a conjugate vaccine (CPS-TT) was generated by conjugation of tetanus toxoid (TT) with meningococcal group Y CPS. CPS-TT was further conjugated with ß-glucan to generate CPS-TT-G. Immunization with CPS-TT-G led to an 8.2-fold increase in the CPS-specific IgG titers as compared with CPS-TT. Presumably, conjugation of ß-glucan ensured the two components to simultaneously reach the antigen presenting cells and stimulate the immune response. In contrast, co-administration of ß-glucan suppressed the CPS-specific immunogenicity of CPS-TT. Thus, conjugation of ß-glucan is an effective strategy to markedly improve the CPS-specific immunogenicity of the conjugate vaccine.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos/sangue , Vacinas Meningocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , beta-Glucanas/química , beta-Glucanas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/imunologia , Humanos , Imunoglobulina G/sangue , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Toxoide Tetânico/imunologia , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologiaRESUMO
CONTEXT: PEGylated hemoglobin (Hb) is a promising oxygen therapeutic agent for clinical application. However, it suffered from structural perturbation, functional instability and methemoglobin (metHb) formation. OBJECTIVE: To improve the structural, functional, physical and anti-oxidation properties of the PEGylated Hb. MATERIALS AND METHODS: PEGylation of Hb with CO binding (HbCO) was conducted using maleimide and acylation chemistry, respectively. Physical and chemical parameters were measured for Hb samples. The circular dichroism spectra, dynamic light scattering and analytical ultracentrifugation were used to investigate the structure and conformation of PEGylated HbCO. RESULTS: CO binding can inhibit the autoxidation of the PEGylated Hb, structurally stabilize its tetramer and improve its thermal and pH stability. Importantly, the circular dichroism spectra showed that CO binding can decrease the structural perturbation of Hb induced by PEGylation. The PEGylated HbCO with CO release showed slightly higher oxygen-delivery capacity than the PEGylated Hb. The PEGylated HbCO did not show metHb formation after 30-day storage at 4°C. DISCUSSION AND CONCLUSION: CO binding structurally stabilized the PEGylated Hb, abolished its metHb formation, and significantly increased its physical stability. In particular, it also avoided the perturbation of PEG chains on the heme microenvironment. The functional property of the PEGylated HbCO can be maintained during its long-term storage, which is of great significance for field transfusion.
Assuntos
Substitutos Sanguíneos/química , Monóxido de Carbono/química , Hemoglobinas/química , Polietilenoglicóis/química , Humanos , Concentração de Íons de Hidrogênio , Metemoglobina/antagonistas & inibidores , Oxirredução , Oxigênio/metabolismo , Estabilidade ProteicaRESUMO
Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.
Assuntos
Fatores de Transcrição Forkhead/genética , Folículo Ovariano/metabolismo , Transdução de Sinais , Proteína Wnt3A/genética , beta Catenina/genética , Animais , Apoptose/efeitos dos fármacos , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imidas/farmacologia , Cloreto de Lítio/farmacologia , Camundongos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Cultura Primária de Células , Quinolinas/farmacologia , Esteroides/biossíntese , Proteína Wnt3A/metabolismo , Proteína Wnt3A/farmacologia , beta Catenina/metabolismoRESUMO
Streptococcus pneumoniae is a serious Gram-positive pathogen responsible for several life-threatening pneumococcal diseases. Pneumococcal capsular polysaccharide (CPS) is a key virulence determinant of S. pneumoniae and its immunogenicity can be improved by conjugation with a carrier protein. Reductive amination, the most widely used approach for pneumococcal CPS conjugate vaccine (PCV), suffers from low conjugation efficiency and the problem of steric hindrance. Here, copper-catalyzed azide-alkyne cycloaddition was used for development of PCV with long spacer arm (L-PCV). Tetanus toxoid (TT) was used as the carrier protein. The long spacer arm in L-PCV can minimize the problem of steric hindrance between CPS and TT, thereby improving the CPS-specific antibody titers in the mice model. L-PCV can also induce high avidity functional antibody and elicit immunological memory in response to the native CPS.