Antigen clearance at the peak of the primary immune response induces experimental autoimmune encephalomyelitis.

Autoimmune demyelinating diseases can be induced by an immune response against myelin peptides; however, the exact mechanism underlying the development of such diseases remains unclear. In experimental autoimmune encephalomyelitis, we found that the clearance of exogenous myelin antigen at the peak of the primary immune response is key to the pathogenesis of the disease. The generation of effector T cells requires continuous antigen stimulation, whereas redundant antigen traps and exhausts effector T cells in the periphery, which induces resistance to the disease. Moreover, insufficient antigenic stimulation fails to induce disease efficiently owing to insufficient numbers of effector T cells. When myelin antigen is entirely cleared, the number of effector T cells reaches a peak, which facilitates infiltration of more effector T cells into the central nervous system. The peripheral antigen clearance initiates the first wave of effector T cell entry into the central nervous system and induces chronic inflammation. The inflamed central nervous system recruits the second wave of effector T cells that worsen inflammation, resulting in loss of self-tolerance. These findings provide new insights into the mechanism underlying the development of autoimmune demyelinating diseases, which may potentially impact future treatments.

Toxoplasma gondii tkl1 Deletion Mutant Is a Promising Vaccine against Acute, Chronic, and Congenital Toxoplasmosis in Mice.

In this study, we generated a tkl1 deletion mutant in the Toxoplasma gondii type 1 RH (RHΔtkl1) strain and tested the protective efficacies of vaccination using RHΔtkl1 tachyzoites against acute, chronic, and congenital T. gondii infections in Kunming mice. Mice vaccinated with RHΔtkl1 mounted a strong humoral and cellular response as shown by elevated levels of anti-T. gondii-specific IgG, IL-2, IL-12, IFN-γ, and IL-10. All RHΔtkl1-vaccinated mice survived a lethal challenge with 1 × 103 tachyzoites of type 1 RH or ToxoDB#9 (PYS or TgC7) strain as well as 100 cysts or oocysts of Prugniuad strain. All mock-vaccinated plus infected mice have died. Vaccination also protected against cyst- or oocyst-caused chronic infection, reduced vertical transmission caused by oocysts, increased litter size, and maintained body weight of pups born to dams challenged with 10 oocysts on day 5 of gestation. In contrast, all mock-vaccinated plus oocysts-infected dams had aborted, and no fetus has survived. Vaccinated dams remained healthy postinfection, and their brain cyst burden was significantly reduced compared with mock-vaccinated dams infected with oocysts. In vivo depletion of CD4+ T cells, CD8+ T cells, and B cells revealed that CD8+ T cells are involved in the protection of mice against T. gondii infection. Additionally, adoptive transfer of CD8+ T cells from RHΔtkl1-vaccinated mice significantly enhanced the survival of naive mice infected with the pathogenic strain. Together, these data reaffirm the importance of CD8+ T cell responses in future vaccine design for toxoplasmosis and present T. gondii tkl1 gene as a promising vaccine candidate.

Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice.

Association of Single-Nucleotide Polymorphisms of CD44 Gene with Susceptibility to Breast Cancer in Chinese Women.

BACKGROUND This study aimed to evaluate the association of CD44 gene single-nucleotide polymorphisms with susceptibility to breast cancer. MATERIAL AND METHODS This case-control study included 242 breast cancer patients and 252 normal people without disease. The single-nucleotide polymorphisms of the CD44 gene in the 2 groups were genotyped by PCR-LDR method. The OR and its 95% CI was calculated by chi-square test and logistic regression analysis. The construction of haplotypes and their interaction analysis with relevant factors were carried out by SHEsis and SNPStats online. RESULTS The genotype distribution of CC and CT, CC and CC+CT, and CC+CT and TT in rs13347 showed a significant difference between cases and controls, and the difference in distribution of alleles C and T was statistically significant. The genotype and alleles distribution of rs4756195 and rs8193 showed no statistically significant difference (P>0.05). The haplotypes distribution of CAC, CGT, TAC, and TGT showed a significant difference between the 2 groups (P<0.05). The results of analysis of haplotypes and their interactions with relevant factors showed that breast cancer risk in the PR-negative group was significantly higher than that in the PR-positive group (P=0.016). We found an interaction between haplotypes and PR status. CONCLUSIONS The genotypes CT, CT+TT, TT, and allele T in rs13347 may be risk factors for breast cancer. The haplotype CAC may be a protective factor against breast cancer, and CGT, TAC, and TGT may be risk factors for breast cancer. The PR status interacts with CD44 gene SNP.

MAP7 promotes proliferation and migration of breast cancer cells and reduces the sensitivity of breast cancer cells to paclitaxel.

Breast cancer is a common malignancy that severely threatens women's mental health and lives. The paclitaxel-resistant breast cancer cells were established through a continuous stimulation with paclitaxel in a stepwise escalating concentration manner. The expression of MAP7 was detected by RT-P CR and western blot. The annexin V staining assay was used to measure the cell apoptosis ratio. The expression of cell invasive ability and apoptosis-related proteins was detected by western blot assay. The cellular motility was tested via transwell and wound healing assays. This study indicated that the MAP7 expression was upregulated in breast cancer cells and paclitaxel-resistant breast cancer cells. Moreover, downregulating MAP7 not only suppressed cell viability, motility and invasion, but also enhanced cellular apoptosis in paclitaxel-resistant breast cancer cells. In summary, this study investigated the effect of MAP7 protein on cell critical physiological function, which provided a novel potential target for treating paclitaxel-resistant breast cancer.

[Oridonin ameliorates experimental autoimmune encephalomyelitis by inhibiting NF-κB signaling in T lymphocytes].

Objective To investigate the therapeutic effect and mechanism of oridonin on experimental autoimmune encephalomyelitis (EAE). Methods Female C57BL/6 mice were immunized with MOG/CFA to establish EAE model. The mice were randomly divided into EAE control and oridonin treatment groups. The mice were intraperitoneally injected with oridonin [15 mg/(kg.d)] on day 3, 5 and 7 post immunization, and the control group was injected with the same amount of PBS. EAE scores were recorded and the cell infiltration in the spinal cord was observed by HE staining at the peak of the disease. Flow cytometry analysis was used to detect the proliferation and apoptosis of MOG reactive CD4+ T cells, and the differentiation of pathogenic T helper type 1 (Th1) cells and Th17 cells. The expression of cytokine IFN-γ and IL-17 were detected by ELISA assay. The expression of nuclear factor κBp65 (NF-κBp65), phosphorylated NF-κBp65 (p-NF-κBp65), and phosphorylated IκB (p-IκB) were detected by Western blot analysis. Results Compared with the control group, the incidence and severity of EAE mice in the oridonin-treated groups was reduced, the onset time was delayed, and the immune cell infiltration in the spinal cord was reduced. In vitro and in vivo experiments showed that oridonin inhibited the proliferation of myelin antigen reactive CD4+ T cells and induced their apoptosis. Oridonin inhibited the differentiation of pathogenic Th1 cells and Th17 cells, and the expression of inflammatory cytokines IFN-γ and IL-17. Oridonin inhibited the phosphorylation of IκB and NF-κBp65. Conclusion Oridonin can ameliorate EAE by inhibiting the activation of NF-κB signaling pathway, thereby the proliferation and differentiation of T cells and the secretion of inflammatory factors are inhibited.

RHΔgra17Δnpt1 Strain of Toxoplasma gondii Elicits Protective Immunity Against Acute, Chronic and Congenital Toxoplasmosis in Mice.

: In the present study, a dense granule protein 17 (gra17) and novel putative transporter (npt1) double deletion mutant of Toxoplasma gondii RH strain was engineered. The protective efficacy of vaccination using RHΔgra17Δnpt1 tachyzoites against acute, chronic, and congenital toxoplasmosis was studied in a mouse model. Immunization using RHΔgra17Δnpt1 induced a strong humoral and cellular response, as indicated by the increased levels of anti-T. gondii specific IgG, interleukin 2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-γ). Vaccinated mice were protected against a lethal challenge dose (103 tachyzoites) of wild-type homologous (RH) strain and heterologous (PYS and TgC7) strains, as well as against 100 tissue cysts or oocysts of Pru strain. Vaccination also conferred protection against chronic infection with 10 tissue cysts or oocysts of Pru strain, where the numbers of brain cysts in the vaccinated mice were significantly reduced compared to those detected in the control (unvaccinated + infected) mice. In addition, vaccination protected against congenital infection with 10 T. gondii Pru oocysts (administered orally on day 5 of gestation) as shown by the increased litter size, survival rate and the bodyweight of pups born to vaccinated dams compared to those born to unvaccinated + infected dams. The brain cyst burden of vaccinated dams was significantly lower than that of unvaccinated dams infected with oocysts. Our data show that T. gondii RHΔgra17Δnpt1 mutant strain can protect mice against acute, chronic, and congenital toxoplasmosis by balancing inflammatory response with immunogenicity.

Berberine enhances the radiosensitivity of hepatoma cells by Nrf2 pathway.

Radiation therapy is a major treatment in hepatocellular carcinoma (HCC). Yet, this treatment is ineffective in HCC due to lack of radiosensitivity. For this reason, we examined whether berberine (BBR) might modify the radioresistance of HCC cells. BBR enhances the radiation-induced oxidative stress and apoptosis in Huh7 and HepG2 cells while it protectes HHL-5 cells from radiation damage. To test the importance of Nrf2, a master transcription factor in oxidative damage, the effect of BBR is studied in irradiated Nrf2-deficient cells. BBR fails to induce the radiosensitivity in Nrf2-deficient cells suggesting that Nrf2 is required for the effect of BBR. BBR suppresses the expression of Nrf2 signaling-related proteins (Nrf2, HO-1 and NQO-1) in Huh7 and HepG2 cells, demonstrating that BBR strengthens radiosensitivity via suppressing Nrf2 signaling pathway in HCC cells. Furthermore, experiment using xenografts in nude mice indicated that BBR enhances the growth inhibitory effect of radiation in a Nrf2-dependent manner in vivo. In conclusion, these results suggest that BBR is a promising potential sensitizer for the radiotherapy of HCC.

Characterization of the Role of Amylo-Alpha-1,6-Glucosidase Protein in the Infectivity of Toxoplasma gondii.

In this study, we characterized the role of amylo-alpha-1,6-glucosidase (Aa16GL) in the biology and infectivity of Toxoplasma gondii, using Aa16GL-deficient parasites of type I RH and type II Prugniaud (Pru) strains. The subcellular localization of Aa16GL protein was characterized by tagging a 3 × HA to the 3' end of the Aa16GL gene endogenous locus. Immunostaining of the expressed Aa16GL protein revealed that it is located in several small cytoplasmic puncta. Functional characterization of ΔAa16GL mutants using plaque assay, egress assay and intracellular replication assay showed that parasites lacking Aa16GL exhibit a slight reduction in the growth rate, but remained virulent to mice. Although PruΔAa16GL tachyzoites retained the ability to differentiate into bradyzoites in vitro, they exhibited slight reduction in their ability to form cysts in mice. These findings reveal new properties of Aa16GL and suggest that while it does not have a substantial role in mediating T. gondii infectivity, this protein can influence the formation of parasite cysts in mice.