Functional insights of three RING-finger peroxins in the life cycle of the insect pathogenic fungus Beauveria bassiana.

Peroxisomes play important roles in fungal physiological processes. The RING-finger complex consists of peroxins Pex2, Pex10, and Pex12 and is essential for recycling of receptors responsible for peroxisomal targeting of matrix proteins. In this study, these three peroxins were functionally characterized in the entomopathogenic fungus Beauveria bassiana (Bb). These three peroxins are associated with peroxisomes, in which BbPex2 interacted with BbPex10 and BbPex12. Ablation of these peroxins did not completely block the peroxisome biogenesis, but abolish peroxisomal targeting of matrix proteins via both PTS1 and PTS2 pathways. Three disruptants displayed different phenotypic defects in growth on nutrients and under stress conditions, but have similar defects in acetyl-CoA biosynthesis, development, and virulence. Strikingly, BbPex10 played a less important role in fungal growth on tested nutrients than other two peroxins; whereas, BbPex2 performed a less important contribution to fungal growth under stresses. This investigation reinforces the peroxisomal roles in the lifecycle of entomopathogenic fungi and highlights the unequal functions of different peroxins in peroxisomal biology.

Functional characterization of two homologs of yeast acetyl-coenzyme A synthetase in the entomopathogenic fungus Beauveria bassiana.

Acetyl-coenzyme A (CoA) synthetase (Acs) links cellular metabolism and physiology by catalyzing acetate and CoA into acetyl-CoA. However, the biological roles of Acs are not well studied in entomopathogenic fungi. In this study, two Acs proteins (BbAcs1 and BbAcs2) was functionally characterized in the filamentous insect pathogenic fungus Beauveria bassiana. BbAcs1 and BbAcs2 localize in cytoplasm and peroxisome, respectively. BbAcs1 contributes to vegetative growth on fatty acids as carbon source, and BbAcs2 did not. Both genes did not contribute to fungal response to stresses. The BbAcs1 loss conferred a slight influence on conidiation, and did not result in the defects in blastospore formation. On the contrary, BbAcs2 significantly contributes to lipid metabolism in germlings, blastospore formation, and virulence. The results indicated that Acs2 played a more predominant role than Acs1 in B. bassiana, which links the acetyl-CoA metabolism with the lifestyle of entomopathogenic fungi.

Succinate Dehydrogenase Subunit C Contributes to Mycelial Growth and Development, Stress Response, and Virulence in the Insect Parasitic Fungus Beauveria bassiana.

Succinate dehydrogenase (SDH), also known as respiratory chain complex II, plays a crucial role in energy production in which SdhC functions as an anchored subunit in the inner membrane of mitochondria. In this study, domain annotation analyses revealed that two SdhC domain-containing proteins were present in the filamentous insect-pathogenic fungus Beauveria bassiana, and they were named BbSdhC1 and BbSdhC2, respectively. Only BbSdhC1 localized to mitochondria; hence, this protein is considered the ortholog of SdhC in B. bassiana. Ablation of BbSdhC1 led to significantly reduced vegetative growth on various nutrients. The ΔBbsdhc1 mutant displayed the significantly reduced ATP synthesis and abnormal differentiation under aerial and submerged conditions. Notably, the BbSdhC1 loss resulted in enhanced intracellular levels of reactive oxygen species (ROS) and impaired growth of mycelia under oxidative stress. Finally, insect bioassays (via cuticle and intrahemocoel injection infection) revealed that disruption of BbSdhC1 significantly attenuated fungal virulence against the insect hosts. These findings indicate that BbSdhC1 contributes to vegetative growth, resistance to oxidative stress, differentiation, and virulence of B. bassiana due to its roles in energy generation and maintaining the homeostasis of the intracellular ROS levels. IMPORTANCE The electron transport chain (ETC) is critical for energy supply by mediating the electron flow along the mitochondrial membrane. Succinate dehydrogenase (SDH) is also known as complex II in the ETC, in which SdhC is a subunit anchored in mitochondrial membrane. However, the physiological roles of SdhC remain enigmatic in filamentous fungi. In filamentous insect-pathogenic fungus B. bassiana, SdhC is required for maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity. These findings improve our understanding of physiological mechanisms of ETC components involved in pathogenicity of the entomopathogenic fungi.

Systematic contributions of CFEM domain-containing proteins to iron acquisition are essential for interspecies interaction of the filamentous pathogenic fungus Beauveria bassiana.

Common in fungal extracellular membrane (CFEM) domain is unique in fungal proteins and some of which contribute to iron acquisition in yeast. However, their roles in iron acquisition remain largely unknown in filamentous fungi. In this study, 12 CFEM-containing proteins were bioinformatically identified in the filamentous entomopathogenic fungus Beauveria bassiana, and the roles of 11 genes were genetically characterized. Transmembrane helices were critical for their association with intracellular membranes, and their number varied among proteins. Eleven CFEM genes significantly contribute to vegetative growth under iron starvation and virulence. Notably, the virulence of most disruptants could be significantly weakened by a decrease in iron availability, in which the virulence of ΔBbcfem7 and 8 strains was partially recovered by exogenous hemin. ΔBbcfem7 and 8 mutants displayed defective competitiveness against the sister entomopathogenic fungus Beauveria brongniartii. All 11 disruptants displayed impaired growth in the antagonistic assay with the saprotrophic fungus Aspergillus niger, which could be repressed by exogenous ferric ions. These findings not only reveal the systematic contributions of CFEM proteins to acquire two forms of iron (i.e. heme and ferric ion) in the entire lifecycle of entomopathogenic fungi but also help to better understand the mechanisms of fungus-host and inter-fungus interactions.

Metagenomic Next-Generation Sequencing Can Clinch Diagnosis of Non-Tuberculous Mycobacterial Infections: A Case Report.

Non-tuberculou Mycobacteria (NTM) is ubiquitous in the environment and is conditional pathogen. Due to NTM and Mycobacterium tuberculosis belong to the genus Mycobacterium, their pathogenic mechanisms and clinical manifestations are similar. Therefore, NTM can cause tuberculosis-like lesions and lead to misdiagnosis. Early diagnosis and treatment greatly improve prognosis. However, traditional pathogenic microorganism detection has limitations, and it is difficult to accurately identify strains in clinical practice. Here, we report a 65-year-old man with NTM who presented with recurrent fever and cough. Computed tomography of the chest revealed a lung infection. The previous improper diagnosis and treatment did not improve his condition. With the aid of metagenomic next-generation sequencing, the pathogen was identified as Mycobacterium avium complex. Subsequently, he received accurate treatment and made significant improvements in clinical and radiology.

Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets.

Liver fibrosis is a reversible wound-healing process aimed at maintaining organ integrity, and presents as the critical pre-stage of liver cirrhosis, which will eventually progress to hepatocellular carcinoma in the absence of liver transplantation. Fibrosis generally results from chronic hepatic injury caused by various factors, mainly viral infection, schistosomiasis, and alcoholism; however, the exact pathological mechanisms are still unknown. Although numerous drugs have been shown to have antifibrotic activity in vitro and in animal models, none of these drugs have been shown to be efficacious in the clinic. Importantly, hepatic stellate cells (HSCs) play a key role in the initiation, progression, and regression of liver fibrosis by secreting fibrogenic factors that encourage portal fibrocytes, fibroblasts, and bone marrow-derived myofibroblasts to produce collagen and thereby propagate fibrosis. These cells are subject to intricate cross-talk with adjacent cells, resulting in scarring and subsequent liver damage. Thus, an understanding of the molecular mechanisms of liver fibrosis and their relationships with HSCs is essential for the discovery of new therapeutic targets. This comprehensive review outlines the role of HSCs in liver fibrosis and details novel strategies to suppress HSC activity, thereby providing new insights into potential treatments for liver fibrosis.

[Effect of Helminthic Infection on the Prevention and Treatment of Inflammatory Bowel Disease and the Mechanisms].

Crohn's disease and ulcerative colitis are two forms of inflammatory bowel diseases. They are autoimmune disorders caused by excessive inflammatory response to antigens in the intestine. In addition to the hygiene hypothesis which suggests the potential application of helminthic infection in the treatment of inflammatory bowel diseases, helminthic infection has shown preventive and treatment effects in animal models of inflammatory bowel disease. Clinical trials have been initiated. For example, Trichuris suis ova infection at a certain dose has a promising efficacy in the treatment of inflammatory bowel diseases. Helminthic infection may also have adverse effects. Therefore, helminth-derived immunomodulatory molecules are needed to overcome these problems. It is traditionally considered that the Th1/Th2 axis is involved in the mechnisms of the efficacy of helminthic infection. More recent research has pointed out the much more participation of the Tregs/Th17 axis. The mechanisms may also involve other palyers such as mucosal barrier, Toll like receptor and macrophages. This paper reviews the effect and mechanism of helminthic infection on the prevention and treatment of inflammatory bowel disease.

Determination of kurarinone in rat plasma by UPLC-MS/MS.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0→301.2 for kurarinone and m/z 168.1→132.1 for IS. The linearity of this method was found to be within the concentration range of 20-2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7-107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats.

Allograft inflammatory factor-1 alleviates liver disease of BALB/c mice infected with Schistosoma japonicum.

Allograft inflammatory factor-1 (AIF-1) plays an important role in various inflammatory conditions. Our previous study demonstrated that AIF-1 was over-expressed in the liver of BALB/c mice infected with Schistosoma japonicum and played significant role in the pathogenesis of schistosomiasis. The aim of this study was to focus on the effect of AIF-1 treatment on liver fibrosis and necrosis of BALB/c mice infected with S. japonicum. Seventy-two BALB/c mice were infected with cercariae of S. japonicum and then divided into three groups: AIF-1-treated group, saline-treated group, and control group. The vital signs, liver function, egg load, and hepatic pathological changes of the mice were assessed, and the levels of AIF-1 and TNF-α in the liver and spleen were measured at 5, 8, and 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum suppressed the expression of TNF-α and increased the effectiveness of AIF-1 in the liver and spleen at 14 weeks postinfection. Histopathological analysis and Masson trichrome staining for the liver tissues showed that the liver fibrosis and necrosis were alleviated previously compared with other infected mice at 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum can alleviate hepatic fibrosis and necrosis which indicate that AIF-1 use may prevent and cure the liver fibrosis.

Effect of cytotoxic T-lymphocyte-associated protein 4 on CD4(+)CD25(+) regulatory T cells in murine schistosomiasis japonica.

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the host's immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.

Effect of Trichinella spiralis infection on the immune response to HBV vaccine in a mouse model.

Vaccination is the most effective and cost-effective way to treat hepatitis B virus (HBV) infection. Collective data suggest that helminth infections affect immune responses to some vaccines. Therefore, it is important to reveal the effects of helminth infections on the efficacy of protective vaccines in countries with highly prevalent helminth infections. In the present work, effects of Trichinella spiralis infection on the protective efficacy of HBV vaccine in a mouse model were investigated. This study demonstrated that the enteric stage of T. spiralis infection could inhibit the proliferative response of spleen lymphocytes to hepatitis B surface antigen (HBsAg) and lead to lower levels of anti-HBsAg antibodies, interferon-γ, and interleukin (IL)-2, along with higher levels of IL-4 and IL-5. However, these immunological differences are absent in the muscle stage of T. spiralis infection. The results suggest that the muscle stage of T. spiralis infection does not affect the immune response to HBV vaccination, while the enteric-stage infection results in a reduced immune response to HBsAg.

Chronic Schistosoma japonicum infection reduces immune response to vaccine against hepatitis B in mice.

BACKGROUND: Hepatitis B and schistosomiasis are most prevalent in Africa and Asia, and co-infections of both are frequent in these areas. The immunomodulation reported to be induced by schistosome infections might restrict immune control of hepatitis B virus (HBV) leading to more severe viral infection. Vaccination is the most effective measure to control and prevent HBV infection, but there is evidence for a reduced immune response to the vaccine in patients with chronic schistosomiasis japonica. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate in a mouse model that a chronic Schistosoma japonicum infection can inhibit the immune response to hepatitis B vaccine (HBV vaccine) and lead to lower production of anti-HBs antibodies, interferon-γ (IFN-γ) and interleukin-2 (IL-2). After deworming with Praziquantel (PZQ), the level of anti-HBs antibodies gradually increased and the Th2-biased profile slowly tapered. At 16 weeks after deworming, the levels of anti-HBs antibodies and Th1/Th2 cytokines returned to the normal levels. CONCLUSIONS/SIGNIFICANCE: The results suggest that the preexisting Th2-dominated immune profile in the host infected with the parasite may down-regulate levels of anti-HBs antibodies and Th1 cytokines. To improve the efficacy of HBV vaccination in schistosome infected humans it may be valuable to treat them with praziquantel (PZQ) some time prior to HBV vaccination.

[Dynamic expressions of IL-17 and IL-23 in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the dynamic expressions of IL-17 and IL-23 in mice with Schistosoma japonicum infection. METHOD: A murine model of the infection of S. japonicum was established. The suspension of spleen cells incubated with ConA was collected at 0, 1, 2, 4, 6, 8 and 10 weeks post-infection. Sandwich ELISA and RT-PCR were used to measure the expressions of IL-17A and IL-23p19 on protein and mRNA level. RESULTS: The dynamic changes of IL-17A and IL-23p19 showed a positive correlation. The level of IL-17A increased and reached the peak at 1 week after the infection, reduced at 4 weeks after the infection, and was even lower at 8 weeks post-infection. The level of IL-17 mRNA increased at 1 week post-infection, and then decreased gradually at 2 weeks post-infection. Being consistent with the dynamic expression of IL-17A, the IL-23p19 expression increased at 1 week post-infection, went up to the peak at 2 weeks post-infection, and gradually reduced into the normal level at 4 weeks. However, the expression of IL-23p19 mRNA fluctuated in the normal range, increased at 4 weeks post-infection, and reached the peak at 6 weeks post-infection. CONCLUSIONS: IL-17 and IL-23 are of co-expression in the mice after schistosome infection. There is a significant increase in the early stage of the infection. IL-17 and IL-23 may play important roles during the immune process in the very early stage of Schistosoma infection.

Application of an immunomagnetic bead ELISA based on IgY for detection of circulating antigen in urine of mice infected with Schistosoma japonicum.

Schistosomiasis is an important zoonosis and some livestock especially bovine and swine play a crucial role on the disease transmission in endemic areas. The gold standard for animal Schistosoma japonicum infection is fecal examination although indirect agglutination assay (IHA) is so far mostly used in field survey and laboratory examination. Lack of sensitivity, poor practicality and high false positivity limit the use of those methods for routine veterinary detection as well as human diagnosis. A novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating schistosomal antigen (CSA) in sera of hosts infected with S. japonicum. To assess the application of this method for diagnosis of domestic animal schistosomiasis with urine sample, the immunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) was employed in the present study to detect CSA in urine of murine schistosomiasis with either light (10 S. japonicum cercariae infection per mouse) or heavy infection (30 S. japonicum cercariae infection per mouse). The results showed that the CSA levels in urine of heavily and lightly infected mice reached a peak in 8 and 10 weeks after infection, respectively, remaining at a constant plateau in both groups by the end of the experiment (14 weeks after infection). The CSA level in urine of heavily infected mice was much higher than that of lightly infected mice from 8 to 14 weeks after infection. The effect of praziquantel treatment on the CSA level in urine of heavily infected mice was also investigated. It was found that the CSA level in urine of heavily infected mice with treatment was much lower than that of untreated mice at 4 weeks post-treatment, although still higher than that of control mice, and then gradually descended to the background level by 8 weeks after treatment. Our findings suggested that the IgY-IMB-ELISA may be an efficient and practical tool in non-invasive diagnosis of schistosome infection based on antigen detection, and evaluation of the efficacy of chemotherapy as well.

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice.

CD4(+) T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 µm vs. 294.4 ± 72.2 µm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.

Delayed tail loss during the invasion of mouse skin by cercariae of Schistosoma japonicum.

A traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm(2) of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.

Evaluation of an IgY-based immunomagnetic enzyme-linked immunosorbent assay system for detection of circulating Schistosoma japonicum antigen in serum samples from patients in China.

We have developed a novel egg yolk antibody (IgY)-coated magnetic beads antigen-capture immunoassay for detection of a circulating antigen of Schistosoma japonicum in serum samples of patients in schistosomiasis-endemic areas of China. This IgY-based immunomagnetic bead enzyme-linked immunosorbent assay (IgY-IMB-ELISA) uses polyclonal IgY-coated magnetic beads as a capture antibody, and a monoclonal IgG as a detection antibody. The sensitivity of the magnetic immunoassay was 100% (40 of 40) in cases of acute infection and 91.5% (107 of 117) in chronic cases of schistosomiasis, and no positive reaction was found in 0 of 49 healthy persons. Cross-reactivity was 3.3% (1 of 33) with clonorchiasis and 0% (0 of 20) with paragonimiasis. There was a significant correlation between ELISA absorbance value and egg count (eggs per gram feces) and a correlation coefficient of 0.88 in a small sample of 14 patients. The results demonstrated that the IgY-IMB-ELISA is a sensitive and specific assay for detection of human schistosomiasis japonica.

[Ageing down-modulates the immune responses to Schistosoma japonicum infection in mice].

OBJECTIVE: To investigate the effect of ageing on the immune responses against Schistosoma japonicum infection in mice. METHODS: Female BALB/c mice were divided into young group (2 months) and old group (18 months), each composed of 8 mice. Each mouse was percutaneously infected with 40 +/- 1 S. japonicum cercariae. At 6 weeks post-infection, the mice were sacrificed, and the spleens were removed and single-cell suspensions of splenocytes were prepared. Worms were perfused from hepatic portal system and counted. The number of eggs in the liver was determined after KOH digestion. Mean single-egg granulomas sizes were determined in stained histological sections. Splenocyte proliferation responses were analyzed by MTT colorimetry. Level of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the splenocyte culture supernatants was determined by ELISA. RESULTS: The worm burden and egg per gram of liver in old mice [19.75 +/- 1.95, (1.59 +/- 1.05) x 10(4)] were significantly lower than that of young mice [26.00 +/- 2.42, (208 +/- 0.87) x 10(4)] (P < 0.05). The mean volume of single-egg granulomas of the livers in old mice [(30.13 +/- 10.97) x 10(3) mm3] was significantly lower than that of the young mice [(47.02 +/- 24.13) x l0(3) mm3] (P < 0.05). RESULTS: of T cell proliferation showed that the splenocytes had poorer immune reactivity to ConA in old mice (SI: 1.08 +/- 0.12) than that in young mice (SI: 131 +/- 0.14) (P < 0.05). Levels of IFN-gamma and IL-4 in the splenocyte culture supernatants [(24.05 +/- 6.24), (4.15 +/- 0.68) pg/ml] from old mice were lower than that of young mice [(34.25 +/- 869), (7125 +/- 0.83) pg/ml](P < 0.05). CONCLUSION: Ageing down-modulates the immune responses and the poorer immune reactivity might decrease pathological alterations in mice infected with Schistosoma japonicum.

Cimetidine enhances the protective effect of GST DNA vaccine against Schistosoma japonicum.

Cimetidine (CIM), a histamine-2-receptor antagonist, has a long history of safe use in gastric acid-mediated gastrointestinal disorders. In this study, we used CIM, as an adjuvant, with pEGFP-Sj26 GST (the recombinant plasmid containing enhanced green fluorescent protein gene and the gene encoding 26 kDa glutathione S-transferase of Schistosoma japonicum) DNA vaccine to immunized mice and attempted to enhance the protective effect against S. japonicum. The results showed that the reduction rate of worm and egg burdens in the pEGFP-Sj26GST plus CIM group were 79.0% and 68.4%, respectively, significantly higher than that in pEGFP-Sj26GST alone group (27.0% and 22.5%, P<0.01). Compared with the pEGFP-Sj26GST alone group, mice immunized with pEGFP-Sj26GST plus CIM showed an elevated level of IFN-γ and IL-12 and a low level of IL-10 in splenocytes, while the levels of IL-4 and IL-5 showed no difference between the two groups. Our data also demonstrated that the percentage of CD4(+)CD25(+) regulatory T cells (Tregs) was significantly decreased in the spleens of mice immunized with pEGFP-Sj26GST plus CIM. All these findings suggest that CIM as a potential schistosome DNA vaccine adjuvant can enhance the protective effect of pEGFP-Sj26GST vaccine.

Effect of CD4+ CD25+ regulatory T cells on the immune evasion of Schistosoma japonicum.

It has been known that parasites developed sophisticated strategies to escape from the host immune assault. More recently, one strategy to induce immune evasion involved CD4(+)CD25(+) regulatory T cells (Tregs). Mice were infected with Schistosoma japonicum cercariae and then injected intraperitoneally with anti-CD25 monoclonal antibody (anti-CD25 mAb). The results showed that the percentages of CD4(+)CD25(+) Tregs in mice were expanded by S. japonicum infection, and it could be partially blocked by anti-CD25 mAb. Worm burden in anti-CD25 mAb group (23.17 ± 6.94) was significantly lower than that in infected group (30.17 ± 5.85). The level of interferon gamma was increased with anti-CD25 mAb administration; meanwhile, lower concentration of interleukin 10 was observed in the same group. These results suggest that CD4(+)CD25(+) Tregs contribute to the escape of S. japonicum from the host immune responses, while anti-CD25 mAb can partially block CD4(+)CD25(+) Tregs and enhance the protective immunity to the parasite by Th1-type immune response.