MiR-222 inhibition alleviates Staphylococcal Enterotoxin B-induced inflammatory acute lung injury by targeting Foxo3

Acute lung injury (ALI) is a common acute and severe disease in clinical practice. Staphylococcal EnterotoxinB (SEB) is a superantigen that can cause inflammatory ALI. MiR-222 has been demonstrated to be upregulatedin SEB-induced inflammatory ALI, but its exact roles and functions remain ill-defined. In this study, SEBexposure led to inflammatory ALI and high expression of miR-222 in model mice and lung infiltratingmononuclear cells, but the inflammatory response and high expression of miR-222 were restored in miR-222-/-mice. Moreover, we investigated the roles of miR-222 in vitro and observed that the concentrations ofinflammatory cytokines and the expression of miR-222 were all elevated in SEB-activated splenocytes andmiR-222 inhibition reversed the effects. Foxo3 was confirmed as a direct target of miR-222. Interestingly, SEBexposure led to a decrease of Foxo3 expression, and Foxo3 knockdown partially reversed the promotion ofFoxo3 and the inhibition of inflammatory cytokines induced by miR-222 inhibitor in SEB-activated splenocytes. Our data indicated that miR-222 inhibition could alleviate SEB-induced inflammatory ALI by directlytargeting Foxo3, shedding light on the potential therapeutic of miR-222 for SEB-induced inflammation in thelung.

Bacterial identification, somatic cell count, antimicrobial profile and toxigenic Staphylococcus strains search from mastitic cow milk samples on small farms properties / Identificação bacteriana, contagem de células somáticas, perfil antimicrobiano e pesquisa de linhagens toxigênicas de Staphylococcus em amostras de leite bovino de pequenas propriedades rurais

Bovine mastitis has a negative impact on milk production and can pose risks to public health. The present study aimed to evaluate the quality of bovine milk from small farms in the Botucatu/SP region. Somatic cell counts (SCC), identification of pathogens involved in mastitis, and sensitivity antimicrobial profile of staphylococci isolated were performed. The presence of enterotoxin encoding genes in isolates of staphylococci obtained from milk was investigated. Milk samples from individual mammary quarters of cows were submitted to the California mastitis test (CMT) and SCC. Of the 239 dairy cows from 21 dairy herds evaluated (mean = 11.4 animals/property), two cows (0.8%) presented clinical mastitis and 86 (35.9%) subclinical mastitis. Bacterial culture was performed in 177 quarter milk samples. Staphylococci were identified in 55 (31.1%), corynebacteria in 45 (25.4%), streptococci in 25 (14.1%) and coliforms in four (2.3%) milk samples. Average SCC from culture-positive samples was 1598x103 cells/mL, in case of staphylococci was 1362x103 cells/ml, streptococci was 2857x103 cells/mL, corynebacteria was 976x103 cells/mL and in the cases of coliforms 1161x103 cells/mL were obtained. Staphylococci showed a high sensitivity (>95%) to cephalothin, cotrimoxazole, enrofloxacin, and gentamicin, with a 41.2% resistance to penicillin and 11.8% to oxacillin. Both coagulase positive (CPS) and negative staphylococci (CNS) carried genes encoding enterotoxins in 21.6% of the first group and 41.9% in the second. The sea gene was the most detected 45.8% (n=24) between them, followed by seb with 29.2% and sec with 25.0%. The sed gene was not identified. We highlight the potential risk to public health in the possibility of strains of Staphylococcus spp. enterotoxin-producing genes that can cause staphylococcal food poisoning.(AU)

A mastite bovina impacta negativamente a produção leiteira e pode acarretar riscos à saúde pública. O presente estudo teve como objetivo a avaliação da qualidade do leite bovino proveniente de pequenas propriedades na região de Botucatu/SP. Foi realizada a contagem de células somáticas (CCS), identificação dos patógenos envolvidos nas mastites, e realizado o perfil de sensibilidade aos antimicrobianos dos estafilococos isolados. Pesquisou-se a presença de genes codificadores de enterotoxinas em isolados de estafilococos obtidos a partir do leite mastítico. Amostras de leite de quartos mamários individuais de vacas foram submetidas ao "California mastitis test" (CMT) e à CCS. Das 239 vacas em lactação provenientes de 21 rebanhos leiteiros avaliados (média = 11,4 animais/propriedade), dois (0,8%) animais apresentaram mastite clínica e, 86 (35,9%) mastite subclínica. 177 amostras de leite foram cultivadas em ágar sangue bovino 5% e ágar MacConckey e obteve-se 55 (31,1%) Staphylococcus spp., 25 (14,1%) Streptococcus spp., 45 (25,4%) Corynebacterium spp. e quatro (2,3%) coliformes. A média da CCS das amostras procedentes de todos os quartos mamários infectados avaliados foi de 1598x103 células/mL, enquanto que nos casos que foram isolados Staphylococcus spp. foi de 1362x103 células/mL, Streptococcus spp. 2857x103 células/mL, Corynebacterium spp. de 976x103 células/mL e nos casos de coliformes 1161x103 células/mL. Os estafilococos revelaram grande sensibilidade (>95%) à cefalotina, cotrimoxazol, enrofloxacina e gentamicina, com resistência de 41,2% à penicilina e 11,8% à oxacilina. Tanto estafilococos coagulase positivos (ECP) como negativos (ECN) revelaram genes codificadores de enterotoxinas em 21,6% do primeiro grupo e 41,9% no segundo. O gene sea foi o mais detectado 45,8% (n=24), seguido pelo seb com 29,2% e sec com 25,0%. O gene codificador da sed não foi identificado. Frente aos resultados, destaca-se o risco potencial à saúde pública pela possibilidade de veiculação de linhagens de Staphylococcus spp. carreadores de genes produtores de enterotoxinas, podendo ocasionar toxi-infecções alimentares.(AU)

Inhibition of miRNA-222-3p Relieves Staphylococcal Enterotoxin B-Induced Liver Inflammatory Injury by Upregulating Suppressors of Cytokine Signaling 1

PURPOSE: Staphylococcal enterotoxin B (SEB) has been well-documented to induce liver injury. miRNA-222-3p (miR-222-3p) was implicated in SEB-induced lung injury and several liver injuries. This study aimed to explore the role of miR-222-3p in SEB-induced liver injury. MATERIALS AND METHODS: Expression of miR-222-3p and suppressors of cytokine signaling 1 (SOCS1) was detected using real-time quantitative PCR and western blot. Liver injury was determined by levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory cytokines, numbers of infiltrating mononuclear cells using AST/ALT assay kit, enzyme-linked immunosorbent assay (ELISA), and hematoxylin-eosin staining, respectively. Target binding between miR-222-3p and SOCS1 was predicted on targetScan software, and confirmed by luciferase reporter assay. RESULTS: SEB induced liver injury in D-galactosamine (D-gal)-sensitized mice, as demonstrated by increased serum levels of AST and ALT, elevated release of interferon-gamma (INF-γ), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-2, and promoted infiltrating immune cells into liver. Expression of miR-222-3p was dramatically upregulated, and SOCS1 was downregulated in SEB-induced liver injury both in mice and splenocytes. Moreover, miR-222-3p knockout (KO) mice exhibited alleviated liver injury accompanied with SOCS1 upregulation. Besides, splenocytes under SEB challenge released less INF-γ, TNF-α, IL-6, and IL-2 during miR-222-3p knockdown. Mechanically, SOCS1 was targeted and downregulated by miR-222-3p. Upregulation of SOCS1 attenuated INF-γ, TNF-α, IL-6, and IL-2 release in SEB-induced splenocytes; downregulation of SOCS1 could block the suppressive role of miR-222-3p knockdown in SEB-induced splenocytes. CONCLUSION: Inhibition of miR-222-3p relieves SEB-induced liver inflammatory injury by upregulating SOCS1, thereby providing the first evidence of miR-222-3p in SEB-induced liver injury.

Application of flash nanoprecipitation to fabricate poorly water-soluble drug nanoparticles

Nanoparticles are considered to be a powerful approach for the delivery of poorly water-soluble drugs. One of the main challenges is developing an appropriate method for preparation of drug nanoparticles. As a simple, rapid and scalable method, the flash nanoprecipitation (FNP) has been widely used to fabricate these drug nanoparticles, including pure drug nanocrystals, polymeric micelles, polymeric nanoparticles, solid lipid nanoparticles, and polyelectrolyte complexes. This review introduces the application of FNP to produce poorly water-soluble drug nanoparticles by controllable mixing devices, such as confined impinging jets mixer (CIJM), multi-inlet vortex mixer (MIVM) and many other microfluidic mixer systems. The formation mechanisms and processes of drug nanoparticles by FNP are described in detail. Then, the controlling of supersaturation level and mixing rate during the FNP process to tailor the ultrafine drug nanoparticles as well as the influence of drugs, solvent, anti-solvent, stabilizers and temperature on the fabrication are discussed. The ultrafine and uniform nanoparticles of poorly water-soluble drug nanoparticles prepared by CIJM, MIVM and microfluidic mixer systems are reviewed briefly. We believe that the application of microfluidic mixing devices in laboratory with continuous process control and good reproducibility will be benefit for industrial formulation scale-up.

Establishment of a mouse model of IgA nephropathy induced by superantigen staphylococcal enterotoxin B / 中国比较医学杂志

Objective To establish a mouse model of IgA nephropathy and to observe its biochemical and pathological characteristics. Methods Twelve BALB/c mice were randomly divided into the normal group and model group, with 6 mice in each group. Mice in the model group received an intravenous injection of 0. 8 mg/kg superantigen staphylococcal enterotoxin B (SEB) into the tail vein once a week for three weeks. At the end of the 4th week, the mice were sacrificed, and the 24 h-urinary protein, urinary microalbumin, the renal function indicators BUN, Scr and UA were measured, levels of liver function indicators ALT, AST, ALP, and the blood lipid levels of TC, TG, and LDL were determined, the renal morphological changes were examined by pathology using HE, PAS, PASM and Masson staining, and by electron microscopy, the IgA deposition in the renal tissue was observed with immunofluorescence, and the liver and small intestine were observed by pathology using HE staining. Results Compared with the normal group, the mice of model group showed increased 24-hour urinary protein and urinary microalbumin (P<0. 01), increased CREA and UA (P<0. 05), but not significantly changed BUN, TP and ALB. The liver function indicator AST was significantly increased (P<0. 05), but ALT and ALP were not significantly changed. The blood lipid TG was significantly decreased (P<0. 05) and LDL increased (P<0. 01), while the TC was not significantly changed. The kidney tissues had moderate histological changes, and immunofluorescence observation showed granular or massive IgA deposition in the renal glomerular mesangium. The liver tissue had some inflammatory cell infiltration and hepatocyte necrosis. The small intestine showed slender and shortened villi with widened inter-villous space and sloughed off epithelial cells, dilated central lacteal, and lymphocyte infiltration. Conclusions A mouse model of IgA nephropathy can be successfully established by tail vein injection of superantigen staphylococcal entrotoxin B.

Effects of Nasal Instillation of Staphylococcal Enterotoxin B on Nasal and Bronchial Mucosa in BALB/c Mouse

BACKGROUND AND OBJECTIVES: The effect of nasal exposure to staphylococcal enterotoxin in the pathogenesis of allergic rhinitis remains controversial. We sought to determine the effect of increasing doses of intranasally applied Staphylococcus aureus enterotoxin B (SEB) on the respiratory mucosa, especially the nasal mucosa. MATERIALS AND METHODS: Nasal application of SEB was performed on four occasions (days 0-4-8-12) in unsensitized BALB/c mice. Control mice were intranasally treated with phosphate buffered saline (PBS), and 5 ng, 50 ng, 500 ng, and 5 microg of SEB was applied to the respective experimental group. The concentrations of IL-4, IL-5, and IFN-gamma in bronchoalveolar lavage fluid (BALF), nasal lavage fluid (NLF) and serum were compared among groups. Also, the counts of total inflammatory cells, macrophages, lymphocytes, neutrophils, eosinophils, and basophils in BALF and NLF were compared among the groups. Pathologic studies for inflammatory cell infiltration in the nasal mucosa and peribronchial area were conducted. RESULTS: IL-4 and IFN-gamma showed higher concentrations with increasing stimulation dose of SEB in NLF and serum. The IL-5 concentration showed a tendency to increase in NLF and serum, but these changes were not statistically significant. Total inflammatory cell count, especially macrophage count, in BALF and NLF was higher with increasing stimulation dose of SEB. Infiltration of inflammatory cells into the nasal mucosa showed a tendency to increase in a dose-dependent manner. CONCLUSION: These results suggest that nasal exposure to SEB may induce Th1 and Th2 inflammatory responses in the respiratory mucosa, especially the nasal mucosa.

The Study of SEB/OVA Allergic Rhinitis Mice Model / 医学研究杂志

Objective To establish mice modle of allergic rhinitis(AR) and to study the role of Staphylococcal enterotoxin B(SEB) and ovalbumin(OVA) in the modle.Methods Forty Balb/c mice were evenly randomized into OVA group,SEB group,OVA+SEB group and normal sodium group and AR modle was established.The symptom scores,total serum IgE concentration,IL-4 concentration were analyzed by factorial design.Meanwhile,the morphology change of nasal mucosa was observed.Results The symptom scores in OVA group,SEB group,OVA+SEB group and normal sodium group were 6.80?1.03,0.90?0.99,0.70?0.82,0.60?0.70 respectirely.The interaction of OVA and SEB had statistical significance(P

Immunity suppressive effect and possible mechanism of SEB as a superantigen / 第三军医大学学报

Objective To study the immunity suppressive effect of the staphylococcal enterotoxin as a super-antigen and investigate its mechanism.Methods BALB/c mice aged 8-12 weeks were randomly assigned to receive 0.2 ml injection of 50 ?g/ml staphylococcal enterotoxin B(SEB)(n=20) or 0.2 ml physiological saline(n=20).One day later,all mice were sacrifice to collect the splenocytes which were employed to detect the expression of TGF-?1 and to countthe cells expressing CD4 and CD25 by flow cytometry(FCM).Results FMC showed that a remarkable increase of cells that expressed CD4 and CD25 in the SEB-primed splenocytes as compared with the saline primed splenocytes.Conclusion SEB,which is used as a superantigen in vivo,can induce the regulatory cells bearing suppressive activity.This may be partial mechanism of SEB-induced hyporesponsiveness.

Immunohistochemical Characterization of Dendritic Cells after Systemic Administration of Staphylococcal Enterotoxin B / 대한해부학회지

This study was performed to investigate the effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) and other immune cells in the major lymphoid organs. A single dose of SEB (25 microgram/kg) was administered to BALB/c mice by intraperitoneal injection. After the mice were sacrificed in groups of three at 2 h, 6 h, 1 day, 2 days, 3 days, 1 week and 2 weeks, the spleen, lymph node and thymus were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. We demonstrated in this study the distribution patterns of DCs and their major costimulatory and adhesive molecules in the murine spleen, lymph node and thymus after SEB administration. We obtained the evidence for maturation of DCs in vivo in response to SEB. DCs were found in increased number in the periarterial lymphatitc sheath (PALS) of spleen, paracortex of lymph nodes and thymic medulla. CD86, ICAM-1 and MHC class II molecules were upregulated on the activated and matured DCs after SEB injection. The most salient feature of the present study was the differential expression pattern of the costimulatory and adhesive molecules on the activated DCs. In addition to DCs, T cells expressing T cell receptor Vbeta8 were increased in number after SEB treatment. In conclusion, SEB exhibited a potent and effective stimulative effect on DCs in vivo.