RESUMEN

The dynamic interplay between intramammary IgG, formation of antigen-IgG complexes and effector immune cell function is essential for immune homeostasis within the bovine mammary gland. We explore how changes in the recognition and binding of anti-LPS IgG to the glycolipid "functional" core in milk from healthy or clinically diagnosed Escherichia coli (E. coli) mastitis cows' controls endotoxin function. In colostrum, we found a varied anti-LPS IgG repertoire and novel soluble LPS/IgG complexes with direct IgG binding to the LPS glycolipid core. These soluble complexes, absent in milk from healthy lactating cows, were evident in cows diagnosed with E. coli mastitis and correlated with endotoxin-driven inflammation. E. coli mastitis milk displayed a proportional reduction in anti-LPS glycolipid core IgG compared to colostrum. Milk IgG extracts showed that only colostrum IgG attenuated LPS induced endotoxin activity. Furthermore, LPS-stimulated reactive oxygen species (ROS) in milk granulocytes was only suppressed by colostrum IgG, while IgG extracts of neither colostrum nor E. coli mastitis milk influenced N-formylmethionine-leucyl-phenylalanine (fMLP)-stimulated ROS in LPS primed granulocytes. Our findings support bovine intramammary IgG diversity in health and in response to E. coli infection generate milk anti-LPS IgG repertoires that coordinate appropriate LPS innate-adaptive immune responses essential for animal health.

Asunto(s)

Calostro , Infecciones por Escherichia coli , Escherichia coli , Glucolípidos , Inmunoglobulina G , Lipopolisacáridos , Mastitis Bovina , Leche , Animales , Bovinos , Femenino , Calostro/inmunología , Calostro/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Mastitis Bovina/inmunología , Mastitis Bovina/microbiología , Escherichia coli/inmunología , Lipopolisacáridos/inmunología , Leche/inmunología , Glucolípidos/metabolismo , Glucolípidos/inmunología , Infecciones por Escherichia coli/inmunología , Endotoxinas/inmunología , Endotoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Granulocitos/inmunología , Granulocitos/metabolismo , Unión Proteica , Glándulas Mamarias Animales/inmunología , Glándulas Mamarias Animales/metabolismo

RESUMEN

The mechanisms of endotoxin tolerance (ET), which down-regulate inflammation, are well described in response to exogenous toll-like receptor ligands, but few studies have focused on ET-associated mechanisms in inflammatory disease. As blocking TNF can attenuate the development of ET, the effect of anti-TNF on the expression of key ET-associated molecules in inflammatory auto-immune disease was measured; changes in inflammatory gene expression were confirmed using an ET bioassay. The expression of immunomodulatory molecules was measured in a murine model of arthritis treated with anti-TNF and the expression of ET-associated molecules was measured in whole blood in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients, before and after therapy. The expression of ET-associated genes was also measured in RA patient monocytes before and after therapy, in anti-TNF responders and non-responders. Tnfaip3, Ptpn6 and Irak3 were differentially expressed in affected paws, spleens, lymph nodes and circulating leucocytes in experimental murine arthritis treated with anti-TNF. Prior to therapy, the expression of TNFAIP3, INPP5D, PTPN6, CD38 and SIGIRR in whole blood differed between human healthy controls and RA or AS patients. In blood monocytes from RA patients, the expression of TNFAIP3 was significantly reduced by anti-TNF therapy in non-responders. Prior to therapy, anti-TNF non-responders had higher expression of TNFAIP3 and SLPI, compared to responders. Although the expression of TNFAIP3 was significantly higher in RA non-responders prior to treatment, the post-treatment reduction to a level similar to responders did not coincide with a clinical response to therapy.

Asunto(s)

Artritis Reumatoide , Endotoxinas , Tolerancia Inmunológica , Espondilitis Anquilosante , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa , Animales , Humanos , Ratones , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Endotoxinas/inmunología , Espondilitis Anquilosante/tratamiento farmacológico , Espondilitis Anquilosante/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/genética , Femenino , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Artritis Experimental/inmunología , Artritis Experimental/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/efectos de los fármacos , Persona de Mediana Edad , Adulto , Inflamación/inmunología , Modelos Animales de Enfermedad

RESUMEN

BACKGROUND: A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. RESULTS: In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. CONCLUSION: Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.

Asunto(s)

Endotoxinas , Escherichia coli , Lípido A , Ratones Endogámicos BALB C , Parvovirus Porcino , Vacunas de Partículas Similares a Virus , Animales , Ratones , Escherichia coli/genética , Escherichia coli/metabolismo , Parvovirus Porcino/inmunología , Parvovirus Porcino/genética , Vacunas de Partículas Similares a Virus/inmunología , Endotoxinas/inmunología , Células RAW 264.7 , Lípido A/inmunología , Lípido A/análogos & derivados , Interleucina-6/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Femenino , Porcinos , Lipopolisacáridos/inmunología

RESUMEN

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 VL) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli. The HaCad-TBR-2D3 VL enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 VL over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

Asunto(s)

Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Cadherinas , Endotoxinas , Proteínas Hemolisinas , Proteínas de Insectos , Larva , Mariposas Nocturnas , Proteínas Recombinantes de Fusión , Animales , Cadherinas/genética , Cadherinas/metabolismo , Cadherinas/inmunología , Cadherinas/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/genética , Endotoxinas/inmunología , Endotoxinas/química , Endotoxinas/farmacología , Endotoxinas/metabolismo , Endotoxinas/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/farmacología , Mariposas Nocturnas/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Insectos/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Péptidos/química , Péptidos/inmunología , Péptidos/farmacología , Anticuerpos/inmunología , Anticuerpos/química , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Insecticidas/química , Insecticidas/farmacología , Control Biológico de Vectores

RESUMEN

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Asunto(s)

Anticuerpos Monoclonales , Toxinas de Bacillus thuringiensis , Endotoxinas , Ensayo de Inmunoadsorción Enzimática , Proteínas Hemolisinas , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Conejos , Ratones , Endotoxinas/análisis , Endotoxinas/inmunología , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/análisis , Proteínas Hemolisinas/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/análisis , Bacillus thuringiensis/química , Ratones Endogámicos BALB C

RESUMEN

Background: The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). Methods: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Results: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. Conclusion: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.

Asunto(s)

Proteínas de Unión al ARN , Sepsis , Animales , Humanos , Masculino , Ratones , Factores de Transcripción ARNTL/genética , Modelos Animales de Enfermedad , Endotoxinas/inmunología , Tolerancia Inmunológica , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Sepsis/inmunología , Sepsis/metabolismo , Receptor Activador Expresado en Células Mieloides 1/inmunología , Receptor Activador Expresado en Células Mieloides 1/genética , Receptor Activador Expresado en Células Mieloides 1/metabolismo

RESUMEN

For decades, innate immune cells were considered unsophisticated first responders, lacking the adaptive memory of their T and B cell counterparts. However, mounting evidence demonstrates the surprising complexity of innate immunity. Beyond quickly deploying specialized cells and initiating inflammation, two fascinating phenomena - endotoxin tolerance (ET) and trained immunity (TI) - have emerged. ET, characterized by reduced inflammatory response upon repeated exposure, protects against excessive inflammation. Conversely, TI leads to an enhanced response after initial priming, allowing the innate system to mount stronger defences against subsequent challenges. Although seemingly distinct, these phenomena may share underlying mechanisms and functional implications, blurring the lines between them. This review will delve into ET and TI, dissecting their similarities, differences, and the remaining questions that warrant further investigation.

Asunto(s)

Endotoxinas , Tolerancia Inmunológica , Inmunidad Innata , Memoria Inmunológica , Humanos , Animales , Endotoxinas/inmunología , Inflamación/inmunología , Inmunidad Adaptativa , Inmunidad Entrenada

RESUMEN

Endotoxin administration is commonly used to study the inflammatory response, and though traditionally given as a bolus injection, it can be administered as a continuous infusion over multiple hours. Several studies hypothesize that the latter better represents the prolonged and pronounced inflammation observed in conditions like sepsis. Yet very few experimental studies have administered endotoxin using both strategies, leaving significant gaps in determining the underlying mechanisms responsible for their differing immune responses. We used mathematical modelling to analyse cytokine data from two studies administering a 2 ng kg-1 dose of endotoxin, one as a bolus and the other as a continuous infusion over 4 h. Using our model, we simulated the dynamics of mean and subject-specific cytokine responses as well as the response to long-term endotoxin administration. Cytokine measurements revealed that the bolus injection led to significantly higher peaks for interleukin (IL)-8, while IL-10 reaches higher peaks during continuous administration. Moreover, the peak timing of all measured cytokines occurred later with continuous infusion. We identified three model parameters that significantly differed between the two administration methods. Monocyte activation of IL-10 was greater during the continuous infusion, while tumour necrosis factor α $ {\alpha} $ and IL-8 recovery rates were faster for the bolus injection. This suggests that a continuous infusion elicits a stronger, longer-lasting systemic reaction through increased stimulation of monocyte anti-inflammatory mediator production and decreased recovery of pro-inflammatory catalysts. Furthermore, the continuous infusion model exhibited prolonged inflammation with recurrent peaks resolving within 2 days during long-term (20-32 h) endotoxin administration.

Asunto(s)

Citocinas , Endotoxinas , Humanos , Endotoxinas/administración & dosificación , Endotoxinas/inmunología , Citocinas/metabolismo , Masculino , Inflamación/inmunología , Interleucina-10/metabolismo , Modelos Teóricos , Infusiones Intravenosas , Monocitos/inmunología , Monocitos/efectos de los fármacos , Interleucina-8/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Femenino , Lipopolisacáridos/administración & dosificación

RESUMEN

Gram-negative bacterial cell surface component lipopolysaccharide (LPS) and its active principle, lipid A, exhibit immunostimulatory effects and have the potential to act as adjuvants. However, canonical LPS acts as an endotoxin by hyperstimulating the immune response. Therefore, LPS and lipid A must be structurally modified to minimize their toxic effects while maintaining their adjuvant effect for application as vaccine adjuvants. In the field of chemical ecology research, various biological phenomena occurring among organisms are considered molecular interactions. Recently, the hypothesis has been proposed that LPS and lipid A mediate bacterial-host chemical ecology to regulate various host biological phenomena, mainly immunity. Parasitic and symbiotic bacteria inhabiting the host are predicted to possess low-toxicity immunomodulators due to the chemical structural changes of their LPS caused by co-evolution with the host. Studies on the chemical synthesis and functional evaluation of their lipid As have been developed to test this hypothesis and to apply them to low-toxicity and safe adjuvants.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Bacterias/inmunología , Endotoxinas/inmunología , Lípido A/farmacología , Lipopolisacáridos/inmunología , Adyuvantes Inmunológicos/química , Animales , Bacterias/efectos de los fármacos , Endotoxinas/metabolismo , Humanos , Lípido A/química , Lipopolisacáridos/química

RESUMEN

Increasing evidence indicates that patients or experimental animals exposure to endotoxin (lipopolysaccharides, LPS) exert deleterious cardiac functions that greatly contribute to morbidity and mortality. The pathophysiologic processes, including NLRP3 inflammasome overactivation and cardiac inflammatory injury, are complicated. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA, is a naturally occurring compound extracted from Salvia miltiorrhiza and has anti-inflammatory and cardioprotective properties. In this study we examined the effect of STS on endotoxin-induced cardiomyopathy and investigated the underlying mechanisms. An endotoxemic mouse model was established by injecting LPS (10 mg/kg). Different doses of STS were administered intraperitoneally (5, 10, or 50 mg/kg) at different time points (2/12 h, 4/12 h, and 8/12 h) after LPS challenge to assess its effect on survival of mice with endotoxemia. In parallel, cardiac function, myocardial inflammatory cytokines, cardiomyocyte pyroptosis and autophagy were evaluated to determine the extent of myocardial damage due to sepsis in the presence and absence of STS at the optimal dose (10 mg/kg) and time-point (2/12 h). The results demonstrated that STS increased the survival rates, improved the compromised cardiac function and reduced myocardial inflammatory injury associated with enhanced autophagy and mitigated NLRP3 inflammasome activation. Moreover, inhibiting of autophagy or blocking the AMPK pathway reversed STS-elicited prevention of cardiomyopathy and activated the NLRP3 inflammasome in endotoxemic mice. Collectively, our study demonstrates that STS attenuates endotoxemia-induced mortality and cardiomyopathy, which may be associated with promotion of autophagy and inhibition of NLRP3 inflammasome overactivation.

Asunto(s)

Cardiomiopatías/prevención & control , Endotoxemia/tratamiento farmacológico , Inflamasomas/antagonistas & inhibidores , Fenantrenos/farmacología , Animales , Autofagia/efectos de los fármacos , Autofagia/inmunología , Cardiomiopatías/diagnóstico , Cardiomiopatías/inmunología , Cardiomiopatías/microbiología , Modelos Animales de Enfermedad , Ecocardiografía , Endotoxemia/complicaciones , Endotoxemia/inmunología , Endotoxemia/microbiología , Endotoxinas/sangre , Endotoxinas/inmunología , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/inmunología , Ventrículos Cardíacos/patología , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Masculino , Ratones , Miocitos Cardíacos , Proteína con Dominio Pirina 3 de la Familia NLR/antagonistas & inhibidores , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fenantrenos/uso terapéutico , Piroptosis/efectos de los fármacos , Piroptosis/inmunología

RESUMEN

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Asunto(s)

Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Toxinas de Bacillus thuringiensis/química , Endotoxinas/química , Proteínas Hemolisinas/química , Región Variable de Inmunoglobulina/química , Anticuerpos de Cadena Única/química , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Toxinas de Bacillus thuringiensis/inmunología , Toxinas de Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/inmunología , Endotoxinas/metabolismo , Proteínas Hemolisinas/inmunología , Proteínas Hemolisinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Simulación del Acoplamiento Molecular , Mariposas Nocturnas , Mutación , Biblioteca de Péptidos , Conformación Proteica , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo

Asunto(s)

Asma/inmunología , Endotoxinas/inmunología , Exposición a Riesgos Ambientales/efectos adversos , Animales , Humanos , Inflamación/inmunología , Lipopolisacáridos/inmunología

RESUMEN

Anti-idiotypic antibody technique is a new approach for the rapid development of insecticidal protein. In this study, anti-Cry1A polyclonal antibodies were used as antigen to screen the anti-idiotypic antibody that can simulate Cry1A toxins from a phage display human domain antibody (DAB) library. After four rounds of panning, five positive clones that have binding activities with anti-Cry1A polyclonal antibodies were obtained. Indirect competitive ELISA (IC-ELISA) results showed that the positive clone D6 showed significant inhibition for the binding of Cry1A toxins with anti-Cry1A polyclonal antibodies, and the inhibition ratio increased with the increase of D6 content. While, B3, F4, G5, C7 and the controls showed no obvious inhibition to Cry1A toxins. The results suggest that D6 is the "ß" subtype anti-idiotypic antibody, which can simulate Cry1A toxins and competitive binding with anti-Cry1A polyclonal antibodies. Meanwhile, D6 had certain binding activity with the brush border membrane vesicles (BBMV) of p. xylostella, which was the receptor of Cry1A toxins. The results of bioassay showed that D6 had certain insecticidal activity, and the lethal concentration of 50% (LC50) was 976 ng/cm2. This study provides basic materials and experience for the development of Cry toxin simulants.

Asunto(s)

Toxinas de Bacillus thuringiensis/inmunología , Endotoxinas/inmunología , Proteínas Hemolisinas/inmunología , Biblioteca de Péptidos , Proteínas Bacterianas/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos

RESUMEN

Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses of two rodents: Peromyscus leucopus, the white-footed deermouse and reservoir for the agents of Lyme disease and other zoonoses, and the house mouse Mus musculus Four hours after injection with LPS or saline, blood, spleen, and liver samples were collected and subjected to transcriptome sequencing (RNA-seq), metabolomics, and specific reverse transcriptase quantitative PCR (RT-qPCR). Differential expression analysis was at the gene, pathway, and network levels. LPS-treated deermice showed signs of sickness similar to those of exposed mice and had similar increases in corticosterone levels and expression of interleukin 6 (IL-6), tumor necrosis factor, IL-1ß, and C-reactive protein. By network analysis, the M. musculus response to LPS was characterized as cytokine associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in the expression levels of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 responses in deermice. Analysis of metabolites in plasma and RNA in organs revealed species differences in tryptophan metabolism. Two genes in particular signified the different phenotypes of deermice and mice: the Slpi and Ibsp genes. Key RNA-seq findings for P. leucopus were replicated in older animals, in a systemic bacterial infection, and with cultivated fibroblasts. The findings indicate that P. leucopus possesses several adaptive traits to moderate inflammation in its balancing of infection resistance and tolerance.IMPORTANCE Animals that are natural carriers of pathogens that cause human diseases commonly manifest little or no sickness as a consequence of infection. Examples include the deermouse, Peromyscus leucopus, which is a reservoir for Lyme disease and several other disease agents in North America, and some types of bats, which are carriers of viruses with pathogenicity for humans. Mechanisms of this phenomenon of infection tolerance and entailed trade-off costs are poorly understood. Using a single injection of lipopolysaccharide (LPS) endotoxin as a proxy for infection, we found that deermice differed from the mouse (Mus musculus) in responses to LPS in several diverse pathways, including innate immunity, oxidative stress, and metabolism. Features distinguishing the deermice cumulatively would moderate downstream ill effects of LPS. Insights gained from the P. leucopus model in the laboratory have implications for studying infection tolerance in other important reservoir species, including bats and other types of wildlife.

Asunto(s)

Reservorios de Enfermedades/microbiología , Endotoxinas/administración & dosificación , Inflamación/genética , Peromyscus/microbiología , Zoonosis/inmunología , Zoonosis/microbiología , Animales , Susceptibilidad a Enfermedades/etiología , Susceptibilidad a Enfermedades/inmunología , Endotoxinas/inmunología , Femenino , Perfilación de la Expresión Génica , Inflamación/inmunología , Enfermedad de Lyme/microbiología , Masculino , Metabolómica , Ratones , Ratones Endogámicos BALB C , Peromyscus/inmunología , Análisis de Secuencia de ARN

RESUMEN

Despite the dramatic increase in antimicrobial resistance, there is a dearth of antibiotics in development and few pharmaceutical companies working in the field. Further, any new antibiotics are likely to have a short shelf life. Ab-based interventions offer alternatives that are not likely to be circumvented by the widely prevalent antibiotic resistance genes. Bovine colostrum (BC)-the first milk after parturition, rich in nutrients and immune components-promotes gut integrity and modulates the gut microbiome. We developed a hyperimmune BC (HBC) enriched in Abs to a highly conserved LOS core region of Gram-negative bacteria by immunizing pregnant cows with a vaccine comprised of detoxified LOS from Escherichia coli O111 Rc (J5) mutant non-covalently complexed to group B meningococcal outer membrane protein (J5dLOS/OMP). This vaccine generated robust levels of anti-J5 LOS Ab in the colostrum. When given orally to neutropenic rats challenged orally with Pseudomonas aeruginosa, administration of HBC improved survival compared to non-immune rats, while both BC preparations improved survival compared to PBS controls. Elevated circulating endotoxin levels correlated with mortality. HBC and to a lesser extent non-immune BC reduced bacterial burden from the liver, lung, and spleen. We conclude that HBC and to a lesser extent BC may be effective supplements that improve outcome from lethal gut-derived disseminated infection and may reduce transmission of Gram-negative bacilli from the gastrointestinal tract.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas/inmunología , Endotoxinas/inmunología , Escherichia coli/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulinas/metabolismo , Sepsis/inmunología , Animales , Anticuerpos Antibacterianos/metabolismo , Carga Bacteriana , Proteínas de la Membrana Bacteriana Externa/inmunología , Bovinos , Lipopolisacáridos/inmunología , Modelos Animales , Proyectos Piloto

RESUMEN

Emerging studies revealed that the Aryl hydrocarbon receptor (AhR), a receptor sensing environmental contaminants, is executing an immunomodulatory function. However, it is an open question to which extent this is achieved by its role as a transcription factor or via non-genomic signaling. We utilized a multi-post-translational modification-omics approach to examine non-genomic AhR-signaling after activation with endogenous (FICZ) or exogenous (BaP) ligand in endotoxin-activated (LPS) monocyte-derived macrophages. While AhR activation affected abundances of few proteins, regulation of ubiquitination and phosphorylation were highly pronounced. Although the number and strength of effects depended on the applied AhR-ligand, both ligands increased ubiquitination of Rac1, which participates in PI3K/AKT-pathway-dependent macrophage activation, resulting in a pro-inflammatory phenotype. In contrast, co-treatment with ligand and LPS revealed a decreased AKT activity mediating an anti-inflammatory effect. Thus, our data show an immunomodulatory effect of AhR activation through a Rac1ubiquitination-dependent mechanism that attenuated AKT-signaling, resulting in a mitigated inflammatory response.

Asunto(s)

Endotoxinas/inmunología , Ambiente , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Estrés Fisiológico , Biomarcadores , Cromatografía Liquida , Expresión Génica , Humanos , Inmunidad , Ligandos , Fosforilación , Espectrometría de Masas en Tándem , Factores de Necrosis Tumoral/metabolismo , Ubiquitinación

RESUMEN

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Asunto(s)

Linfocitos B/inmunología , Toxinas de Bacillus thuringiensis/inmunología , Bacillus thuringiensis/inmunología , Células Dendríticas/inmunología , Endotoxinas/inmunología , Proteínas Hemolisinas/inmunología , Animales , Presentación de Antígeno , Linfocitos B/metabolismo , Toxinas de Bacillus thuringiensis/administración & dosificación , Células Dendríticas/metabolismo , Endotoxinas/administración & dosificación , Femenino , Proteínas Hemolisinas/administración & dosificación , Activación de Linfocitos , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología

RESUMEN

It has been reported that patients with rheumatoid arthritis (RA) have significantly less bacteria belonging to the Bacteroides group in their microbiota. We speculate that inhibition of cytokine production is impaired in patients with RA owing to their low levels of intestinal bacteria belonging to the Bacteroidetes group. Here we investigated the effect of Bacteroides fragilis lipopolysaccharide (B-LPS) on cytokine production in vitro and on the development of collagen antibody-induced arthritis (CAIA) in DBA/1 mice, an animal model of RA. in vitro culture experiments showed that Escherichia coli LPS (E-LPS)-induced cytokine production from THP-1 monocytic cells and peripheral blood mononuclear cells was significantly suppressed by B-LPS in a dose-dependent manner. A decrease in TNF-α and IL-1ß production was also observed in LPS-tolerized macrophages induced by B-LPS at concentrations equal to and higher than that of E-LPS. Similar results were obtained when autoclaved feces were used to induce cytokine production instead of E-LPS. In in vivo experiments using CAIA models, B-LPS had no adverse effects even when administered at 10 times the concentration of E-LPS, which elicits severe arthritis. In addition, simultaneous administration of high dose B-LPS with E-LPS or administration of B-LPS prior to E-LPS significantly suppressed arthritis development in CAIA model animals when compared with administration of E-LPS alone. These results suggest that increasing certain bacterial groups such as Bacteroides is an effective strategy for preventing arthritis development in patients with RA.

Asunto(s)

Artritis Reumatoide/etiología , Artritis Reumatoide/metabolismo , Bacteroides fragilis/inmunología , Escherichia coli/inmunología , Lipopolisacáridos/inmunología , Monocitos/inmunología , Monocitos/metabolismo , Animales , Artritis Experimental , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/patología , Línea Celular , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta Inmunológica , Endotoxinas/inmunología , Humanos , Tolerancia Inmunológica , Masculino , Ratones , Monocitos/patología , Índice de Severidad de la Enfermedad , Microtomografía por Rayos X

RESUMEN

Particulate autogenous tooth roots are used for alveolar bone augmentation surgery; however, dental plaque may provoke an inflammatory response that may counteract the desired graft consolidation process. Traditional mechanical cleaning of extracted teeth may be of support to lower a possible inflammatory response of the autograft. To test this assumption, extracted porcine teeth were left either uncleaned or underwent mechanical cleaning with a toothbrush and toothpaste before being fragmented and subjected to acid lysis, termed as unclean acid dentine lysate (ucADL) and clean acid dentine lysate (cADL), respectively. The inflammatory responses of murine macrophage RAW 264.7 cells being exposed to the respective acid dentine lysates were evaluated at the level of inflammatory gene expression and IL6 immunoassays. We report here that acid lysates obtained from uncleaned teeth provoked a robust increase in IL1ß, IL6, and COX2 in RAW 264.7 cells. The mechanical removal of dental plaque significantly reduced the inflammatory response. Consistently, Limulus tests revealed that tooth cleaning lowers the presence of endotoxins in dentine lysates. To further prove the involvement of endotoxins, a toll-like receptor 4 (TLR4) inhibitor TAK242 was introduced. TAK242 abolished the inflammatory response provoked by acid lysates obtained from uncleaned teeth in RAW 264.7 cells. Moreover, nuclear translocation and phosphorylation of the TLR4 downstream NFκB-p65 were attenuated at the presence of cleaned versus uncleaned dentine lysates. Taken together, our data support the importance of dental plaque removal of teeth being extracted for alveolar bone augmentation surgery.

Asunto(s)

Dentina/metabolismo , Concentración de Iones de Hidrógeno , Macrófagos/metabolismo , Higiene Bucal , Animales , Biomarcadores , Atención Odontológica , Placa Dental , Endotoxinas/efectos adversos , Endotoxinas/inmunología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/microbiología , Ratones , Fosforilación , Células RAW 264.7 , Receptor Toll-Like 4/metabolismo , Raíz del Diente , Cepillado Dental

RESUMEN

Chorioamnionitis (CA) predisposes to preterm birth and affects the fetal mucosal surfaces (i.e., gut, lungs, and skin) via intra-amniotic (IA) inflammation, thereby accentuating the proinflammatory status in newborn preterm infants. It is not known if CA may affect more distant organs, such as the kidneys, before and after preterm birth. Using preterm pigs as a model for preterm infants, we investigated the impact of CA on fetal and neonatal renal status and underlying mechanisms. Fetal pigs received an IA dose of lipopolysaccharide (LPS), were delivered preterm by cesarean section 3 days later (90% gestation), and compared with controls (CON) at birth and at postnatal day 5. Plasma proteome and inflammatory targets in kidney tissues were evaluated. IA LPS-exposed pigs showed inflammation of fetal membranes, higher fetal plasma creatinine, and neonatal urinary microalbumin levels, indicating renal dysfunction. At birth, plasma proteomics revealed LPS effects on proteins associated with renal inflammation (up-regulated LRG1, down-regulated ICA, and ACE). Kidney tissues of LPS pigs at birth also showed increased levels of kidney injury markers (LRG1, KIM1, NGLA, HIF1A, and CASP3), elevated molecular traits related to innate immune activation (infiltrated MPO+ cells, complement molecules, oxidative stress, TLR2, TLR4, S100A9, LTF, and LYZ), and Th1 responses (CD3+ cells, ratios of IFNG/IL4, and TBET/GATA3). Unlike in plasma, innate and adaptive immune responses in kidney tissues of LPS pigs persisted to postnatal day 5. We conclude that prenatal endotoxin exposure induces fetal and postnatal renal inflammation in preterm pigs with both innate and adaptive immune activation, partly explaining the potential increased risks of kidney injury in preterm infants born with CA.

Asunto(s)

Corioamnionitis/inmunología , Endotoxinas/efectos adversos , Inflamación/inmunología , Riñón/inmunología , Nacimiento Prematuro/inmunología , Efectos Tardíos de la Exposición Prenatal/inmunología , Células TH1/inmunología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Endotoxinas/inmunología , Femenino , Humanos , Inmunidad Innata , Lipopolisacáridos/inmunología , Activación de Linfocitos , Embarazo , Porcinos