PEGylated nano-Rehmannia glutinosa polysaccharide induces potent adaptive immunity against Bordetella bronchiseptica.

Vaccines, in many cases, stimulate only too weak immunogenicity to prevent infection. Therefore, adjuvants are required during their preparation to boost the immune response. We herein developed a PEGylated nano-adjuvant based on Rehmannia glutinosa polysaccharide (RGP). The addition of PEG layer exhibits enhanced immune performance of the nano-RGP. Stimulation of dendritic cells (DCs) with PEGylated nano-RGP (pRL) led to increased proliferation and cytokine production (IL-6, IL-12, IL-1ß and TNF-α). The pRL was internalized into DCs via a rapid and efficient method. The mice immunized with pRL exhibited enhanced antigen-specific serum IgG and Th1-(IFN-γ), Th2-(IL-4), and Th17-(IL-17, IL-6) cytokine production, contributing to a good anti-infection performance. Furthermore, the pRL could effectively deliver the antigen to the lymph nodes (LNs), activate DC in the LN and produce enhanced CD4+and CD8+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) as well as functional phenotypes. Our results revealed that pRL can act as a promising adjuvant with targeted delivery of antigen due to its effective activation and robust adaptive immunity induction of DCs.

Inhibitory Effects of Berberine Hydrochloride on Trichophyton mentagrophytes and the Underlying Mechanisms.

BACKGROUND: T. mentagrophytes can infect all mammals, including rabbits, causing serious infections with remarkable economic losses for rabbit farmers. Berberine is an alkaloid that is effective against a variety of microbial infections such as T. mentagrophytes. Growth curve by dry weight determination and in-vivo antifungal assay were carried out to clarify the inhibitory effect of berberine hydrochloride against T. mentagrophytes. Transcriptomics analyses were also carried out for better understanding of the underlying mechanisms. RESULTS: The growth rate of T. mentagrophytes was significantly higher in control condition than under berberine hydrochloride or clotrimazole for 60 h. The growth rate of T. mentagrophytes was significantly slighter higher in berberine condition (1 mg) than under clotrimazole for 46 h. T. mentagrophytes seriously shrunk after berberine or clotrimazole treatment, as observed by TEM and in SEM. Significant recovery was evident in three berberine groups on day 6 compared with the DMSO group. Results from transcriptomics analyses showed 18,881 identified unigenes, including 18,754 and 12,127 in the NT and SwissProt databases. Among these, 12,011, 9174, and 11,679 unigenes belonged to 3 Gene Ontology (GO), 43 KEGG, and 25 KOG categories, respectively. Interestingly, we found that down-regulation of 14α-demethylase exposed to various medicines was slightly different, i.e., berberine hydrochloride (fold change -3.4956) and clotrimazole (fold change -2.1283) caused various degrees of alteration. CONCLUSIONS: Berberine hydrochloride could inhibit the growth of T. mentagrophytes. Berberine hydrochloride could also cure dermatosis induced by T. mentagrophytes. Down-regulation of 14α-demethylase exposed to various medicines was slightly different and might be one of the anti-resistance mechanisms of berberine hydrochloride in T. mentagrophytes. The present investigation provides considerable transcript sequence data that would help further assess the antifungal mechanisms against T. mentagrophytes, for antifungal medicine development.

Application of the red fluorescent protein mCherry in mycelial labeling and organelle tracing in the dermatophyte Trichophyton mentagrophytes.

Trichophyton mentagrophytes is a fungus that causes skin disease in humans and other animals worldwide. Studies on molecular biology and fluorescent labeling of the fungus are limited. Here, we applied mCherry for the first time in T. mentagrophytes to label the fungus and its organelles. We constructed four expression vectors of mCherry or mCherry fusions containing a variety of resistance markers and promoters, which were then integrated, together with two previous mCherry expression vectors, in T. mentagrophytes via Agrobacterium tumefaciens-mediated transformation (AtMT). The resulting transformants emitted bright red fluorescence. We used the histone protein H2B and the peroxisome targeting signal 1 (PTS1) peptide to target the nucleus and peroxisomes, respectively, in T. mentagrophytes. In the transformants expressing mCherry-fused H2B, the fluorescence was distinctly localized to the nuclei in hyphae, spores and the fungal cells in infected animal tissue. In the T. mentagrophytes transformants where the peroxisome was targeted, the mCherry was present as small dots (0.2-1 µm diameter) throughout the spores and the hyphae. We also constructed a T. mentagrophytes AtMT library containing more than 1000 hygromycin-resistant transformants that were genetically stable. Our results provide useful tools for further investigations on molecular pathogenesis of T. mentagrophytes.

Antifungal activity of berberine hydrochloride and palmatine hydrochloride against Microsporum canis -induced dermatitis in rabbits and underlying mechanism.

BACKGROUND: Phellodendron amurense, exhibits antifungal activity mainly by bioactive components including berberine hydrochloride and palmatine hydrochloride. This study was conducted to evaluate the antifungal effects of berberine hydrochloride, palmatine hydrochloride, and a mixture of both substances against Microsporum canis in vivo and in vitro. METHODS: The minimal inhibitory concentrations (MICs) of monomers and clotrimazole were determined using 1.5 % tryptic soy agar. The effects of these drugs on Microsporum canis growth was detected by determining dry weight. Transmission electron microscopy (TEM) was performed to observe the effect of chemicals on cell ultrastructure. Differential mRNA expressions of eight genes of M. canis treated with berberine or palmatine or their combination at different time points were determined by real-time PCR. NADH enzyme concentration was also detected. Clinical evaluation via in-vivo antifungal assay was also performed. Skin histology PAS staining was also carried out. RESULTS: Results showed that MICs of berberine, palmatine and clotrimazole were 1, 1, and 0.015 mg/mL, respectively. No significant difference was observed among the growth curves of the three groups before 18 h was reached. TEM showed that these drugs could destroy the cell membrane and organelles of M. canis at different time points. After 30 h of incubation, relative mRNA expressions of the genes in the combined group were significantly higher than those in the other groups including the clotrimazole group (P < 0.05); Palmatine initially induced the mRNA up-regulation of PGAL4, FSH1, PQ-LRP, NADH1 and NDR in M. canis; by contrast, berberine maintained a high expression level of these genes to shorten fungal life cycle and eradicate M. canis. Clinical results showed that combined treatment was more effective than single administration of each monomer or clotrimazole. Hence, berberine mixed with palmatine significantly elicited antifungal activities and could be used to treat M. canis in rabbits. CONCLUSION: These results provide a comprehensive view of the mechanism of berberine and palmatine in anti-M. canis activity.

Digital gene expression analysis of Microsporum canis exposed to berberine chloride.

Berberine, a natural isoquinoline alkaloid of many medicinal herbs, has an active function against a variety of microbial infections including Microsporum canis (M. canis). However, the underlying mechanisms are poorly understood. To study the effect of berberine chloride on M. canis infection, a Digital Gene Expression (DGE) tag profiling was constructed and a transcriptome analysis of the M. canis cellular responses upon berberine treatment was performed. Illumina/Hisseq sequencing technique was used to generate the data of gene expression profile, and the following enrichment analysis of Gene Ontology (GO) and Pathway function were conducted based on the data of transcriptome. The results of DGE showed that there were 8476945, 14256722, 7708575, 5669955, 6565513 and 9303468 tags respectively, which was obtained from M. canis incubated with berberine or control DMSO. 8,783 genes were totally mapped, and 1,890 genes have shown significant changes between the two groups. 1,030 genes were up-regulated and 860 genes were down-regulated (P<0.05) in berberine treated group compared to the control group. Besides, twenty-three GO terms were identified by Gene Ontology functional enrichment analysis, such as calcium-transporting ATPase activity, 2-oxoglutarate metabolic process, valine catabolic process, peroxisome and unfolded protein binding. Pathway significant enrichment analysis indicated 6 signaling pathways that are significant, including steroid biosynthesis, steroid hormone biosynthesis, Parkinson's disease, 2,4-Dichlorobenzoate degradation, and tropane, piperidine and Isoquinoline alkaloid biosynthesis. Among these, eleven selected genes were further verified by qRT-PCR. Our findings provide a comprehensive view on the gene expression profile of M. canis upon berberine treatment, and shed light on its complicated effects on M. canis.

[Analysis of outer membrane proteins of Riemerella antipestifer].

An isolated virulence Riemerella anatipestifer strain passaged 200 times on TSB agar were used for the virulent to avirulent conversion. The effects of passage on biological properties of outer membrane proteins (OMPs) were investigated using the virulent and avirulent strains. Transmission electron microscopy demonstrated that the avirulent strain produced lower amounts of outer membrane vesicles and the outer membrane decreased, the cytoplasmic appearance jumbled. The OMPs of the virulent strain agglutinated only in RA serotype 2 antisera, whereas the OMPs of the avirulent strain agglutinated in antisera of RA 1, 2, 10 and 11. SDS-PAGE Analysis showed the OMPs profiles of both strains were similar but the immunoblotting profiles were different. The protective immunity against Riemerella anatipestifer infection was investigated by immunizations with OMPs in ducks. ELISA results showed that the OMPs induced the production of antibodies in immunized ducks, but the OMPs of virulence strain induced higher antibody titers than the attenuated strain (P < 0.05). RA2 group showed significantly higher survival rates (100%) than RA200 group (0%) after challenged with the homologous virulent strain. The ompA gene of both stains were also amplified by PCR, nucleotide homology was 99.9%. In conclusion, OMPs of virulent RA strain are suitable candidates for vaccine development. Biological properties of OMPs undergoes significant changes during serial passage and suggest that vigilance should be used when extrapolating data obtained from the study of high-passage strains.