Adaptive immune responses mediated age-related Plasmodium yoelii 17XL and 17XNL infections in 4 and 8-week-old BALB/c mice.

BACKGROUD: It is important to expound the opposite clinical outcomes between children and adulthood for eradicate malaria. There remains unknown about the correlation between adaptive immune response and age-related in malaria. METHODS: 4 and 8-week-old mice were used to mimic children and adulthood, respectively. Parasitemia and the survival rate were monitored. The proportion and function of Th1 and Th2 cells were detected by FACS. The levels of IFN-γ, IL-4, total IgG, IgG1, IgG2a and Plasmodium yoelii MSP-1-specific IgG were measured by ELISA. RESULTS: The adult group showed greater resistance to P. yoelii 17XL infection, with lower parasitemia. Compared with 4-week-old mice, the percentage of CD4+T-bet+IFN-γ+ Th1 cells as well as IFN-γ production were significantly increased on day 5 p.i. in the 8-week-old mice after P. yoelii 17XNL infection. The percentage of CD4+GATA3+IL-4+ Th2 cells and CD4+CXCR5+ Tfh cells, and IL-4 production in the 8-week-old mice significantly increased on day 5 and day 10 after P. yoelii 17XNL infection. Notably, the levels of total IgG, IgG1, IgG2a and P. yoelii MSP-1-specific IgG were also significantly increased in the 8-week-old mice. PD-1, a marker of exhaustion, was up-regulated on CD4+ or activated CD4+ T cells in the 8-week-old mice as compared to the 4-week-old group. CONCLUSIONS: Thus, we consider that enhanced cellular and humoral adaptive immunity might contribute to rapid clearance of malaria among adults, likely in a PD-1-dependent manner due to induction of CD4+ T cells exhaustion in P. yoelii 17XNL infected 8-week-old mice.

Bacillus Calmette-Guérin-inoculation at different time points influences the outcome of C57BL/6 mice infected with Plasmodium chabaudi chabaudi AS.

Bacillus Calmette-Guérin (BCG) is an attenuated Mycobacterium tuberculosis vaccine. We performed a series of co-infection experiments with BCG-Plasmodium chabaudi chabaudi Landau, 1965 AS using C57BL/6 mice to analyse whether BCG can affect the development of protective immunity to infection with Plasmodium spp. and the mechanism of this protection. We divided mice into four groups: BCG-inoculation 4 weeks prior to P. c. chabaudi AS infection (B-4w-Pc); simultaneous BCG-inoculation and P. c. chabaudi AS infection (Pc+B); BCG-inoculation 3 days post P. c. chabaudi AS (Pc-3-B) infection; and mono-P. c. chabaudi AS infection as control (Pc). The parasitemia level in the B-4w-Pc group was noticeably higher than control group at 6-19 days post infection (dpi). Compared with the control group, the proportion of CD4(+)CD69(+) T cells was significantly reduced 5, 8 and 12 dpi, but the proportion of CD4(+)CD25(+)Foxp3(+) Tregs was significantly increased in the B-4w-Pc group on 5 and 8 dpi. The B-4w-Pc group also demonstrated reduced levels of IFN-γ and TNF-α on 5 and 8 dpi and significantly elevated level of IL-10 on 12 dpi. There were significantly fewer mDCs (CD11c(+)CD11b(+)) and pDCs (CD11c(+)B220(+)) in the B-4w-Pc group than the control group at all the time points post infection and the expression of MHC II was noticeably reduced on day 8 pi. Our findings confirmed that BCG inoculation prior to Plasmodium infection resulted in excessive activation and proliferation of Tregs and upregulation of anti-inflammatory mediators, which inhibited establishment of a Th1-dominant immune response during the early stages of Plasmodium infection by inhibiting dendritive cells response. BCG inoculation prior to P. c. chabaudi AS infection may contribute to overgrowth of parasites as well as mortality in mice.

Parasite densities modulate susceptibility of mice to cerebral malaria during co-infection with Schistosoma japonicum and Plasmodium berghei.

BACKGROUND: Malaria and schistosomiasis are endemic and co-exist in the same geographic areas, even co-infecting the same host. Previous studies have reported that concomitant infection with Schistosoma japonicum could offer protection against experimental cerebral malaria (ECM) in mice. This study was performed to evaluate whether alterations in parasite density could alter this protective effect. METHODS: Mice were inoculated with 100 or 200 S. japonicum cercariae followed by infection with high or low density of Plasmodium berghei ANKA strain eight weeks after the first infection. Then, parasitaemia, survival rate and blood-brain-barrier (BBB) damage were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-4, IL-5, IL-13, IL-10, and TGF-ß levels were determined in splenocyte supernatants using enzyme-linked immunosorbent assay (ELISA). Cell surface/intracellular staining and flow cytometry were used to analyse the level of CD4(+)/CD8(+) T cells, CD4(+)CD25(+)Foxp3(+) Tregs, IL-10-secreting Tregs, and IL-10(+)Foxp3-CD4(+) T cells in the spleen, and CD4(+)/CD8(+) T cells infiltrating the brain. RESULTS: Co-infection with low density P. berghei and increased S. japonicum cercariae significantly increased the levels of IL-4, IL-5, IL-13, TGF-ß and Tregs, but significantly decreased the levels of IFN-γ and the percentage of CD4(+) T cells and CD8(+) T cells in the spleen and CD8(+) T cell infiltration in the brain. Increased worm loads also significantly decreased mortality and BBB impairment during ECM. When challenged with higher numbers of P. berghei and increased cercariae, the observed cytokine changes were not statistically significant. The corresponding ECM mortality and BBB impairment also remained unchanged. CONCLUSIONS: This study demonstrates that protection for ECM depends on the numbers of the parasites, S. japonicum and P. berghei, during co-infection. Alterations in the regulatory response appear to play a key role in this adaptation.

Pre-existing Schistosoma japonicum infection alters the immune response to Plasmodium berghei infection in C57BL/6 mice.

BACKGROUND: Since helminths and malaria parasites are often co-endemic, it is important to clarify the immunoregulatory mechanism that occurs during the process of co-infection. A previous study confirmed that dendritic cells (DCs) are involved in the establishment and regulation of the T-cell-mediated immune response to malaria infection. In the current study, distinct response profiles for splenic DCs and regulatory T cell (Treg) responses were assessed to evaluate the effects of a pre-existing Schistosoma japonicum infection on malaria infection. METHODS: Malaria parasitaemia, survival rate, brain histopathology and clinical experimental cerebral malaria (ECM) were assessed in both Plasmodium berghei ANKA-mono-infected and S. japonicum-P. berghei ANKA-co-infected mice. Cell surface/intracellular staining and flow cytometry were used to analyse the level of splenic DC subpopulations, toll-like receptors (TLRs), DC surface molecules, Tregs (CD4⁺CD25⁺Foxp3⁺), IFN-γ/IL-10-secreting Tregs, and IFN-γ⁺/IL-10⁺-Foxp3⁻CD4⁺ T cells. IFN-γ, IL-4, IL-5, IL-10 and IL-13 levels were determined in splenocyte supernatants using enzyme-linked immunosorbent assay (ELISA). RESULTS: The co-infected mice had significantly higher malaria parasitaemia, compared with the mono-infected mice, on days 2, 3, 7 and 8 after P. berghei ANKA infection. Mono-infected mice had a slightly lower survival rate, while clinical ECM symptoms, and brain pathology, were significantly more severe during the period of susceptibility to ECM. On days 5 and 8 post P. berghei ANKA infection, co-infected mice had significantly lower levels of CD11c⁺CD11b⁺, CD11c⁺CD45R/B220⁺, CD11c⁺TLR4⁺, CD11c⁺TLR9⁺, CD11c⁺MHCII⁺, CD11c⁺CD86⁺, IFN-γ-secreting Tregs, and IFN-γ⁺Foxp3⁻CD4⁺ T cells in single-cell suspensions of splenocytes when compared with P. berghei ANKA-mono-infected mice. Co-infected mice also had significantly lower levels of IFN-γ and higher levels of IL-4, IL-5, and IL-13 in splenocyte supernatants compared to mono-infected mice. There were no differences in the levels of IL-10-secreting Tregs or IL-10⁺Foxp3⁻CD4⁺ T cells between co-infected and mono-infected mice. CONCLUSIONS: A Tregs-associated Th2 response plays an important role in protecting against ECM pathology. Pre-existing S. japonicum infection suppressed TLR ligand-induced DC maturation and had an anti-inflammatory effect during malaria infection not only by virtue of its ability to induce Th2 responses, but also by directly suppressing the ability of DC to produce pro-inflammatory mediators.

Listeria monocytogenes inoculation protects mice against blood-stage Plasmodium yoelii infection.

Listeria monocytogenes (Lm) has been used as the adjuvant or vector for tumor and viral vaccine for its capability of eliciting all aspects of cell-mediated immunity including T cell activation and interferon-gamma (IFN-γ) production. These effector components play critical roles in the protection against Plasmodium infection in both human malaria and mouse models. Therefore, immune response induced by Lm infection may benefit the defense against malaria. To test this hypothesis, we employed blood-stage Plasmodium yoelii (PyL) infected mice and challenged them with Lm. C57BL/6 and BALB/c mice that are sensitive to PyL infection were used in experiments. These two strains are resistant and sensitive, respectively, to Lm infection. The outcomes of double infection with PyL and Lm and the changes of immune response were investigated. We found that live Lm inoculation inhibited PyL multiplication in both C57BL/6 and BALB/c mice. Lm inoculation increased production of IFN-γ, infiltration of CD11b-positive macrophages and generation of nitric oxide in the spleen of C57BL/6 mice at day 5 after parasite infection. Both CD4- and CD8- positive T cells contributed to IFN-γ production induced by Lm inoculation in PyL-infected mice. The protective effect of Lm against PyL infection depended on the viability of the bacteria. Live Lm, rather than heat-killed Lm, stimulated early IFN-γ production which provided essential cytokine environment for the development of Th1 response in PyL-infected mice. Our data show for the first time that Lm inoculation has protective effect against blood-stage murine malaria, which provides a new clue for enhancing anti-Plasmodium immunity.

The expression of malarial invasion-related molecules is affected by two different nitric oxide-based treatments.

The host immune response to parasitic infections plays an important role in controlling multiplication of the parasite and reducing clinical symptoms and life-threatening complications. Nitric oxide (NO), an important innate immune factor and classic Th1 immune effector, may play a role in inhibiting plasmodium infection. In this study, we used two different approaches (L-Arginine [precursor of NO] and NOC5 [short-time NO donor]) to prove the roles of NO in malaria infection. We used 6-8 week-old female BALB/c mice infected with the rodent malaria Plasmodium yoelii Landau, Michel et Adam, 1968 - strain 17XL (P.y17XL) as a model. For L-Arg treatment, mice were administered with an oral dose of 1.5 mg/g L-Arg daily for seven consecutive days prior to infection with Py17XL. L-Arg pretreatment resulted in the decrease of the mRNA level of the apical membrane antigen 1 (AMA1) gene, which encodes a protein involved in host invasion. For NOC5 treatment, NOC5 was injected intraperitoneally into the P.y17XL infected mice on day 5 post-infection or incubated in vitro with purified Py17XL schizonts. Both in vivo and in vitro treatments with NOC5 led to down-regulation of the transcript and protein levels of invasion-related molecules (AMA1, merozoites surface protein 1 and Py235). Our results confirmed the protective role of NO in the asexual blood stage of parasitic infection, which may be partially due to reduced expression of parasite invasion molecules.

Age-related CD4(+)CD25(+)Foxp3(+) regulatory T-cell responses during Plasmodium berghei ANKA infection in mice susceptible or resistant to cerebral malaria.

Different functions have been attributed to CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) during malaria infection. Herein, we describe the disparity in Treg response and pro- and anti-inflammatory cytokines during infection with Plasmodium berghei ANKA between young (3-week-old) and middle-aged (8-month-old) C57BL/6 mice. Young mice were susceptible to cerebral malaria (CM), while the middle-aged mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The levels of pro-inflammatory cytokines, such as TNF-α, in young CM-susceptible mice were markedly higher than in middle-aged CM-resistant mice. An increased absolute number of Tregs 3-5 days post-inoculation, co-occurring with elevated IL-10 levels, was observed in middle-aged CM-resistant mice but not in young CM-susceptible mice. Our findings suggest that Treg proliferation might be associated with the suppression of excessive pro-inflammatory Th1 response during early malaria infection, leading to resistance to CM in the middle-aged mice, possibly in an IL-10-dependent manner.

Age-related susceptibility and resistance to nonlethal Plasmodium yoelii infection in C57BL/6 mice.

In cases of human malaria, children suffer very high rates of morbidity and mortality. To analyse the mechanisms involved in age-dependent protection against malaria, we investigated the characterization of immune responses to Plasmodium yoelii 17XNL (P.y 17XNL) in young (3 weeks) and middle-aged (8 months) C57BL/6 mice. In this study, we found that 100% of young mice succumbed to P.y 17XNL infection with higher parasitemia, while middle-aged mice were able to clear blood parasites and no mortality was observed. These observations suggested that the young C57BL/6 mice were susceptible to P.y 17XNL infection, whereas the middle-aged mice were resistant. Cellular analysis revealed that both the numbers of splenic myeloid dendritic cells (mDCs) as well as the expression of DC maturation markers were higher in middle-aged mice than those in young mice. The numbers of IgG1- or IgG2a-secreting B cells increased markedly in middle-aged mice after infection with P.y 17XNL. The dynamic change of the number of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mice infected with P.y 17XNL was also different between the two groups. In addition, the levels of IFN-gamma and NO increased in both groups during early parasite infection, while there was also an obvious increase in IL-4 production in the infected middle-aged mice. The change in IL-10 levels following infection was consistent with that of the change in the number of Tregs. The survival of middle-aged mice following P.y 17XNL infection was dependent upon the establishment of effective Thl and Th2 responses and a successful switch between Th1 and Th2 responses, as well as appropriate functioning of Tregs.

[L-arginine enhances Th1 immune response against Plasmodium yoelii 17XL infection in DBA/2 mice via activation of dendritic cells].

OBJECTIVE: To investigate the effect of L-arginine (L-Arg) during blood-stage infection by P.y17XL in DBA/2 mice. METHODS: DBA/2 mice were divided into 2 groups, 20 mice in each group. Mice were respectively administered with L-Arg (1.5 g/kg, L-Arg group) and normal saline (control group) 7 days before they were infected intraperitoneally by 1 x 10(6) pRBC. Parasitemia were detected by Giemsa stained thin-smear microscopy and survival rate were monitored daily. Flow cytometry was introduced to detect the subsets of splenic CD4+CD69+ T cells, F4/80+CD36+ macrophages, myeloid dendritic cells (mDCs) (CD11c+CD11b+), plasmacytoid dendritic cells (pDCs) (CD11c+B220+) on day 3, 5 post infection (p.i.). The levels of IFN-gamma and NO in the supernatant of splenocytes culture were detected by ELISA and Griess reaction, respectively. RESULTS: Pre-treatment of mice with L-Arg significantly decreased the parasitemia from 45% to 20% and shortened self-cure time from 22d to 20d after infection. The level of F4/80+CD36+ macrophages [(29.61 +/- 0.47)%], IFN-gamma [(485.84 +/- 39.31) pg/ml], CD4+CD69+ T cells [(7.3 +/- 0.68)%], NO [(42.51 +/- 1.32) micromol/L], mDCs(CD11c+CD11b+) [(5.51 +/- 0.87)%] and pDCs(CD11c+B220+) [(5.60 +/- 0.85)%] in L-Arg group was higher than those in control group [(36.46 +/- 1.33)%, (767.86 +/- 20.56) pg/ml, (11.27 +/- 0.97)%, (78.66 +/- 2.89) micromol/L, (10.02 +/- 0.37)%] and (9.01 +/- 0.53)%, respectively]. CONCLUSION: L-Arg enhances Th1 immune responses during the early stage of P.y17XL infection in DBA/2 mice via the activation of DCs.

Effects of CD4(+)CD25(+)Foxp3(+)regulatory T cells on early Plasmodium yoelii 17XL infection in BALB/c mice.

The outcome of Plasmodium yoelii 17XL-infected BALB/c and DBA/2 mice, ranging from death to spontaneous cure, respectively, depends largely on the establishment of effective pro-inflammatory type 1 responses during the early stages of infection and associates with CD4(+)CD25(+)Foxp3(+)regulatory T cells (Tregs). Here, effects of Tregs were analysed on early P. yoelii 17XL infection in BALB/c and DBA/2 mice. In vivo depletion of Tregs significantly reversed the inhibited establishment of effective pro-inflammatory type 1 responses in BALB/c mice, indicating that this cell population contributed to the suppression of T-cell function in malaria. Moreover, the proportion and absolute numbers of IL-10-secreting Tregs in BALB/c mice were significantly higher than that found in DBA/2 mice by intracytoplasmic staining, and IL-10 production was correlated with the Tregs population. In addition, in vivo Tregs depletion decreased the production of IL-10 and the apoptosis of CD4+ T cells. Consistently, IL-10R blockade also had the same effect as that of Tregs depletion in P. yoelii 17XL-infected BALB/c mice. Our data demonstrate that Tregs perhaps have an important role in regulating pro-inflammatory type 1 responses in an IL-10-dependent manner and induce CD4+ T cell apoptosis during the early stage of P. yoelii 17XL infection.

Plasmodium yoelii: assessment of production and role of nitric oxide during the early stages of infection in susceptible and resistant mice.

There is conflicting evidence regarding the role of nitric oxide (NO) in the process of resistance against blood-stage malaria parasites. In this study, we used two strains of mice infected with Plasmodium yoelii 17XL in order to assess the NO production profile and its possible role during the early stage of malaria infection. We found a greater elevation of NO production associated with a sharp increase in the levels of IFN-gamma in infected DBA/2 mice, compared with infected BALB/c mice. This difference was associated with relatively lower parasitemia, a higher constituent ratio of infected reticulocytes, and greater survival in DBA/2 mice. Endogenous IFN-gamma driving Th1 immunity was responsible for NO production. Moreover, schizonts treated in vitro with NO donors caused a delayed infection to BALB/c mice in a dose and time-dependent manner. These data, thus, suggest that NO may play an inhibitory role in Plasmodium infection.

CD4+CD25+Foxp3+ regulatory T cells prevent the development of Th1 immune response by inhibition of dendritic cell function during the early stage of Plasmodium yoelii infection in susceptible BALB/c mice.

Protective immunity against murine malaria infection depends largely on the establishment of effective Th1 immune response during the early stages of infection. Experimental data suggest that the death of Plasmodium yoelii 17XL (Py 17XL) susceptible BALB/c mice results from the suppression of Th1 immune response mediated by CD4+CD25+Foxp3+ regulatory T cells (Tregs). However, the mechanism by which Tregs regulate Th1 immune response is poorly understood. Since immunity is initiated by dendritic cells (DCs), we analysed DC responses to Py 17XL in control and Treg-depleted BALB/c mice. Myeloid DC proliferation, phenotypic maturation and interleukin-12 (IL-12) production were strongly inhibited in control BALB/c mice. In contrast, plasmacytoid DC proliferation and IL-10 production were strongly enhanced in control BALB/c mice. In-vivo depletion of Tregs resulted in significantly reversed inhibition of DC response, which may contribute to the establishment of Th1 immune response, indicating that Tregs contribute to the suppression of Th1 immune response during malaria. These findings suggest Tregs contribute to prevent Th1 immune response establishment during the early stage of Py 17XL infection by inhibiting DC response.

Modulation of anti-malaria immunity by vitamin A in C57BL/6J mice infected with heterogenic plasmodium.

Vitamin A (VA) is an anti-inflammatory agent that is important in modulating and balancing the immune system. The present study aimed to investigate the immunoregulatory effects of vitamin A supplement (VAS) in C57BL/6J mice infected with Plasmodium yoelii 17XL (P.y17XL) or Plasmodium berghei ANKA (P.bANKA). Following VA treatment, parasitaemia decreased, but survival rate did not significantly change during P.y17XL infection. However, in P.bANKA infected C57BL/6J mice, VA pretreatment decreased parasitaemia, and a lag in cerebral malaria (CM) was observed during the early stages of infection. Furthermore, VA pretreatment was also demonstrated to upregulate MHCII expression in dendritic cells (DCs), downregulate Th1 and Tregs, and downregulate TNF-α and IFN-γ production. The results of the current study indicated that VAS downregulated the inflammation response in CM, but did not exhibit an immunoregulatory effect against P.y17XL infection. VAS protected the onset of CM by reducing inflammation, and was also correlated with the downregulation of Th1 by modifying the function of DCs and Tregs. However, no significant effect was observed during P.y17XL infection.

[In vitro observation on effect of nitric oxide on exflagellation of Plasmodium yoelii].

OBJECTIVE: To observe the effect of nitric oxide (NO) on exflagellation of malaria parasite. METHODS: The level of parasitemia and gametocytaemia in DBA/2 mice infected with Plasmodium yoelii 17XL was measured by scanning Giemsa-stained blood smears, and the NO level in culture supernatant of splenocytes was checked using Griess reaction. The mice were injected with different doses of NO donor (NOC5) on day 4 post-infection, and control mice were injected with NOC5 precursor. On day 6 post-infection, mice were injected with NOS inhibitor (L-NMMA), and control mice were injected with D-NMMA and PBS, respectively. Blood samples were collected from tail vein of mice before injection, 30 and 60 min after being injected with NOC5 and NOC5 precursor, 4 and 8 h after being injected with L-NMMA, D-NMMA, and PBS respectively. Exflagellation number of gametocytes in blood culture was counted under microscope. Results The NO level in culture supernatant of splenocytes from mice on day 4 and 6 post-infection was 16.5 mmol/L and 30.4 mmol/L, and exflagellation number was 11.33 and 0.66, respectively. The number of exflagellation in parasitized erythrocytes, obtained from mice on day 4 post-infection, was 5.33 and 2.66, respectively, 30 and 60 min after injection of 1 mg NO donor (NOC5), significantly lower than that of the control (P<0.01). The number of exflagellation in parasitized erythrocytes derived from mice on day 6 post-infection was 1.83, 8 h after the injection of NOS inhibitor (L-NMMA), which was significantly higher than that of the control (P<0.01). CONCLUSION: NO is a major effector molecule resulting in natural transmission-blocking of malaria parasite by directly inhibiting exflagellation of male gametocytes.

Study on protective immunity against infection of Plasmodium yoelii 17XL in DBA/2 mice.

OBJECTIVE: To investigate the development and dynamic changes of host immune response in DBA/2 mice infected with Plasmodium yoelii 17XL. METHODS: Female DBA/2 mice were infected by intraperitoneal (i. p.) injection of 10(6) P. yoelii 17XL parasitized erythrocytes (PRBC). Levels of IL-12, IFN-gamma, IL-4, IL-10 and P. yoelii 17XL-specific antibody in sera were measured by ELISA. Concentrations of NO in cell supernatants were measured by the Griess reaction. Parasitemia, percentage of mononuclear-macrophages of individual mice were monitored daily, and phagocytosis of mononuclear macrophages was also observed. RESULTS: Primary parasitemia in vein blood was developed on day 3 postinfection, which peaked with a level of 46. 9% on day 9. Most mice cleared the infection and survived by day 20 postinfection. From day 6 to day 16, the phagocytosis of PRBC by rodent macrophages was observed on the blood smear. Infected mice had a continuously increased level of IL-12 in serum from day 1 postinfection. Accordingly, high level of IFN-gamma was also detected in sera from day 1 postinfection, which peaked on day 6. Infected mice produced higher level of IL-4 and IL-10 in serum on day 6 postinfection, which peaked on day 9 and day 15 postinfection respectively. In addition, splenocytes from infected mice produced significantly higher level of NO on day 6 and 20 postinfection. Level of P. yoelii 17XL-specific IgG was determined in the sera of infected mice with a steadily increased trend after infection, which peaked on day 70 postinfection. CONCLUSIONS: Effective polarizing of Th1 cells is significant in inhibition of parasitemia and eventual clearance of the Plasmodium parasites. Activated mononuclear-macrophages play a key role in inhibiting parasitemia in the early phase of infection with P. yoelii 17XL.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei.

OBJECTIVE: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. METHODS: Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. RESULTS: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P < 0.01), and IgG1 and IgG2a all increased (P < 0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P < 0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P < 0.05), and antiserum ookinete numbers (cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. CONCLUSIONS: GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.

An 18-mer Peptide Derived from Prosaposin Ameliorates the Effects of Aß1-42 Neurotoxicity on Hippocampal Neurogenesis and Memory Deficit in Mice.

The pathological hallmarks of Alzheimer's disease (AD) include amyloid-ß (Aß) accumulation, neurofibrillary tangle formation, synaptic dysfunction, and neuronal loss. The present study was performed to investigate the protective effects and mechanism of action of a prosaposin-derived 18-mer peptide (PS18: LSELIINNATEELLIKGL) on mice hippocampal progenitor cell proliferation, neurogenesis, and memory tasks after intracerebroventricular injection of Aß1-42 peptide. Seven days after Aß1-42 injection, significant proliferation of hippocampal progenitor cells and memory impairment were evident. Two weeks after Aß1-42 peptide injection, elevated numbers of surviving 5-bromo-2-deoxyuridine cells and newly formed neurons were detected. Treatment with PS18 attenuated these effects evoked by Aß1-42. Our data indicate that treatment with PS18 partially attenuated the increase in hippocampal neurogenesis caused by Aß1-42-induced neuroinflammation and prevented memory deficits associated with increased numbers of activated glial cells. We observed an increase in ADAM10 and decreases in BACE1, PS1/2, and AßPP protein levels, suggesting that PS18 enhances the nonamyloidogenic AßPP cleavage pathway. Importantly, our results further showed that PS18 activated the PI3K/Akt pathway, phosphorylated GSK-3α/ß, and, as a consequence, exerted a neuroprotective effect. In addition, PS18 showed a protective effect against Aß1-42-induced neurotoxicity via suppression of the caspase pathway; upregulation of Bcl-2; downregulation of BAX, attenuating mitochondrial damage; and inhibition of caspase-3. These findings suggest that PS18 may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative diseases, such as AD.

Blocking of transmission to mosquitoes by antibody to Plasmodium vivax malaria vaccine candidates Pvs25 and Pvs28 despite antigenic polymorphism in field isolates.

We have previously demonstrated that mouse antisera against yeast-produced recombinant forms of the ookinete surface proteins of Plasmodium vivax (Pvs25 and Pvs28) blocks transmission of the homologous P. vivax (Sal I strain). In this study, we developed mouse and rabbit antisera against Pvs25 and Pvs28 and evaluated the efficacy of these vaccine candidates against natural isolates of P. vivax in Thailand. Although both Pvs25 and Pvs28 genes are polymorphic, sera from mice immunized using alum adjuvant completely inhibited oocyst development for most human isolates, whereas sera from rabbits immunized with either alum or Freund's adjuvant were partially inhibitory. All inhibition occurred in an antibody dose dependent fashion. Data from this study clearly demonstrates that antibodies raised against Sal I-based vaccines overcome the genetic polymorphism of Pvs25 and Pvs28 present in natural isolates of P. vivax, suggesting the wide range applicability of Sal I based vaccines.

A rapid genotyping method for the vivax malaria transmission-blocking vaccine candidates, Pvs25 and Pvs28.

The antigenic diversity observed in many vaccine candidates is one of the difficulties to design effective malaria vaccine. Since it is prerequisite to survey genetic polymorphism of the vaccine candidate antigens for the vaccine development, it is necessary to establish efficient screening method to detect the genetic polymorphism from a large number of samples. Here, we have established efficient polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) method to detect nucleotide diversity of the malaria transmission-blocking vaccine candidates Pvs25 and Pvs28. We can distinguish all 4 haplotypes of Pvs25 by this method. By introducing BsmI-digestion step for Pvs28, we can distinguish 15/16 haplotypes by single electrophoresis. Since this method requires neither sequencing nor radioisotope labeling, it will be easy to transfer the method into a field based high throughput screening of genetic polymorphism.

[Transmission-blocking vaccine candidate of Plasmodium vivax Pvs25 is highly conservative among Chinese isolates].

OBJECTIVE: To investigate the genetic diversity of Plasmodium vivax transmission-blocking vaccine candidate antigen (TBV) Pvs25, with P. vivax isolates from Hubei and Zhejiang Provinces, and to compare the genetic polymorphism of Pvs25 with that from Bangladesh. METHODS: The parasite DNA used for the genetic polymorphism assay was obtained from dried filter paper blood spots. The genes were PCR amplified and the products were purified and sequenced directly. RESULTS: 45 complete new sequences were analyzed. Only 3 nucleotide changes were found that would result in amino acid substitutions in Pvs25 in comparison with the sequence from P. vivax Sal-I strain. The measurement of nucleotide diversity (pi) was remarkably similar for the two populations, indicating that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or isolates from the same geographical region. CONCLUSION: The results suggest that Pvs25 has limited antigenic polymorphism, especially compared with candidate antigens expressed by hepatic and erythrocytic stage, which may support the development and application of Pvs25-based transmission-blocking vaccine in China.