Large-scale Artemisinin-Piperaquine Mass Drug Administration With or Without Primaquine Dramatically Reduces Malaria in a Highly Endemic Region of Africa.

Background: Mass drug administration (MDA), with or without low-dose primaquine (PMQLD), is being considered for malaria elimination programs. The potential of PMQLD to block malaria transmission by mosquitoes must be balanced against liabilities of its use. Methods: Artemisinin-piperaquine (AP), with or without PMQLD, was administered in 3 monthly rounds across Anjouan Island, Union of Comoros. Plasmodium falciparum malaria rates, mortality, parasitemias, adverse events, and PfK13 Kelch-propeller gene polymorphisms were evaluated. Results: Coverage of 85 to 93% of the Anjouan population was achieved with AP plus PMQLD (AP+PMQLD) in 2 districts (population 97164) and with AP alone in 5 districts (224471). Between the months of April-September in both 2012 and 2013, average monthly malaria hospital rates per 100000 people fell from 310.8 to 2.06 in the AP+PMQLD population (ratio 2.06/310.8 = 0.66%; 95% CI: 0.02%, 3.62%; P = .00007) and from 412.1 to 2.60 in the AP population (ratio 0.63%; 95% CI: 0.11%, 1.93%; P < .00001). Effectiveness of AP+PMQLD was 0.9908 (95% CI: 0.9053, 0.9991), while effectiveness of AP alone was 0.9913 (95% CI: 0.9657, 0.9978). Both regimens were well tolerated, without severe adverse events. Analysis of 52 malaria samples after MDA showed no evidence for selection of PfK13 Kelch-propeller mutations. Conclusions: Steep reductions of malaria cases were achieved by 3 monthly rounds of either AP+PMQLD or AP alone, suggesting potential for highly successful MDA without PMQLD in epidemiological settings such as those on Anjouan. A major challenge is to sustain and expand the public health benefits of malaria reductions by MDA.

Temporal changes in genetic diversity of msp-1, msp-2, and msp-3 in Plasmodium falciparum isolates from Grande Comore Island after introduction of ACT.

BACKGROUND: Malaria is still one of the serious public health problems in Grande Comore Island, although the number of annual cases has been greatly reduced in recent years. A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate temporal changes in genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) in Grande Comore 10 years after introduction of ACT. METHODS: A total of 232 P. falciparum clinical isolates were collected from the Grande Comore Island during two sampling periods (118 for 2006‒2007 group, and 114 for 2013‒2016 group). Parasite isolates were characterized for genetic diversity and complexity of infection by genotyping polymorphic regions in merozoite surface protein gene 1 (msp-1), msp-2, and msp-3 using nested PCR and DNA sequencing. RESULTS: Three msp-1 alleles (K1, MAD20, and RO33), two msp-2 alleles (FC27 and 3D7), and two msp-3 alleles (K1 and 3D7) were detected in parasites of both sampling periods. The RO33 allele of msp-1 (84.8%), 3D7 allele of msp-2 (90.8%), and K1 allele of msp-3 (66.7%) were the predominant allelic types in isolates from 2006-2007 group. In contrast, the RO33 allele of msp-1 (63.4%), FC27 allele of msp-2 (91.1%), and 3D7 allele of msp-3 (53.5%) were the most prevalent among isolates from the 2013-2016 group. Compared with the 2006‒2007 group, polyclonal infection rates of msp-1 (from 76.7 to 29.1%, P < 0.01) and msp-2 (from 62.4 to 28.3%, P < 0.01) allelic types were significantly decreased in those from 2013‒2016 group. Similarly, the MOIs for both msp-1 and msp-2 were higher in P. falciparum isolates in the 2006-2007 group than those in 2013-2016 group (MOI = 3.11 vs 1.63 for msp-1; MOI = 2.75 vs 1.35 for msp-2). DNA sequencing analyses also revealed reduced numbers of distinct sequence variants in the three genes from 2006‒2007 to 2013‒2016: msp-1, from 32 to 23 (about 28% decline); msp-2 from 29 to 21 (about 28% decline), and msp-3 from 11 to 3 (about 72% decline). CONCLUSIONS: The present data showed dramatic reduction in genetic diversity and MOI among Grande Comore P. falciparum populations over the course of the study, suggesting a trend of decreasing malaria transmission intensity and genetic diversity in Grande Comore Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Increased IL-27/IL-27R expression in association with the immunopathology of murine ocular toxoplasmosis.

Interleukin 27 (IL-27) is a member of the IL-6/IL-12 family, and IL-27 receptor (IL-27R) consists of WSX-1 (the IL-27Rα subunit) and the signal-transducing subunit gp130. Human and mouse mast cells (MCs) express the IL-27R. To explore the expressions of IL-27/IL-27R subunits (WSX-1 and gp130) during acute ocular toxoplasmosis (OT), we established mouse model by intraocular injection of 500 Toxoplasma gondii RH strain tachyzoites. Histopathological changes were analyzed, MCs were counted by toluidine blue staining, and tryptase+/IL-27+ MCs were examined by immunofluorescence double-staining in the eyes and cervical lymph nodes (CLNs) of T. gondii-infected mice. The mRNA expressions of IL-27p28, WSX-1, gp130, and tachyzoite specific surface antigen 1 (SAG1) in the eyes and CLNs of T. gondii-infected mice, and the expressions of WSX-1 and gp130 in the murine mastocytoma cell line P815 infected with T. gondii tachyzoites in vitro were examined by using quantitative real-time reverse transcription-polymerase chain reaction. Our results showed that, after T. gondii infection, severe histopathological changes, increased numbers of total MCs and degranulated MCs, elevated expressions of IL-27p28, WSX-1, and gp130 were found in the eyes and CLNs, and significant correlations between the levels of IL-27 and SAG1 existed in the eyes and CLNs of T. gondii-infected mice. In addition, increased levels of WSX-1 and gp130 were examined in T. gondii-infected P815 cells. Our data suggested that IL-27/IL-27R expression induced by T. gondii infection may regulate MC-mediated immune response during acute OT in mouse model.

Mast cell activator compound 48/40 is not an effective adjuvant for UV-attenuated Toxoplasma gondii vaccine.

Toxoplasma gondii (T. gondii, Tg) is a globally distributed parasitic protozoan causing different forms of toxoplasmosis in humans. Mast cells (MCs) play a role during T. gondii infection. Several studies suggest that MC activator compound 48/80 (C48/80) may be an effective vaccine adjuvant resulting in a potent and protective antigen-specific immune response against bacteria or virus infections. The present study was performed to determine whether C48/80 had adjuvant activity for ultraviolet (UV)-attenuated T. gondii vaccine to induce protective immune responses against T. gondii in mouse model. Kunming mice were divided into the following groups: naive mice, naive mice administrated with C48/80 intraperitoneal (i.p.) injection, mice infected by i.p. injection of 104 T. gondii RH strain alone (Tg group), mice infected with 104 RH tachyzoites plus C48/80 administration (Tg + C48/80), mice immunized with UV-Tg alone, and mice immunized with UV-Tg plus C48/80 administration (UV-Tg + C48/80). All the vaccinated mice were challenged with 104 tachyzoites of T. gondii RH strain at the same time as the primary infection. The survival rates, liver histopathologies, liver parasite burdens, and mRNA expression levels of Th1 and Th2 cytokines in the livers and spleens detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were compared among the aforementioned groups after primary infection or challenge infection. The results showed that, compared to the Tg group or Tg + C48/80 group, the UV-Tg + Tg group and UV-Tg + C48/80 + Tg group had significantly prolonged survival time, lower liver histopathological scores, decreased liver parasite burdens, and increased levels of Th1 and Th2 cytokines in the livers and spleens. There was no significant difference of survival time between the UV-Tg + Tg group and the UV-Tg + C48/80 + Tg group; however, the UV-Tg + C48/80 + Tg group showed higher parasite burden, more severe liver histopathology, and decreased IL-4 level compared to the UV-Tg + Tg group. These results indicate that C48/80 had no adjuvant activity for the immunization induced by UV-attenuated T. gondii vaccine.

Prevalence of crt and mdr-1 mutations in Plasmodium falciparum isolates from Grande Comore island after withdrawal of chloroquine.

BACKGROUND: In Comoros, the widespread of chloroquine (CQ)-resistant Plasmodium falciparum populations was a major obstacle to malaria control, which led to the official withdrawal of CQ in 2004. Continuous monitoring of CQ-resistant markers of the P. falciparum CQ resistant transporter (pfcrt) and the P. falciparum multiple drug resistance 1 (pfmdr-1) is necessary inder to obtain first-hand information on CQ susceptibility of parasite populations in the field. The objective of this study is to assess the prevalence and evolution of CQ-resistance in the P. falciparum populations on the Comoros' Grande Comore island after withdrawal of CQ. METHODS: A total of 207 P. falciparum clinical isolates were collected from the island, including 118 samples from 2006 to 2007 and 89 samples from 2013 to 2014. Nucleotide substitutions in the pfcrt and pfmdr-1 genes linked to CQ response in parasite isolates were assessed using nested PCR and DNA sequencing. RESULTS: From the pfcrt gene segment sequenced, we detected C72S, M74I, N75E, and K76T substitutions in the parasite isolates collected from both 2006-2007 to 2013-2014 periods. Significant decline of pfcrt resistant alleles at C72S (42.6 to 6.9 %), M74I (39.1 to 14.9 %), N75E (63.5 to 18.3 %), and K76T (72.2 to 19.5 %) from 2006-2007 to 2013-2014 were observed, and the frequency of pfcrt wild type allele was significantly increased from 19.1 % in 2006-2007 to 75.8 % in 2013-2014. Sequence analysis of pfmdr-1 also detected point mutations at codons N86Y, Y184F, and D1246Y, but not S1034C and N1042D, in the isolates collected from both examined periods. An increasing trend in the prevalence of the pfmdr-1 wild type allele (NYD, 4.3 % in 2006-2007; and 28.7 % in 2013-2014), and a decreasing trend for pfmdr-1 N86Y mutation (87.0 % in 2006-2007; and 40.2 % in 2013-2014) were observed in our samples. CONCLUSIONS: The present data indicate that the prevalence and patterns of mutant pfcrt and pfmdr-1 dramatically decreased in the Grande Comore isolates from 2006 to 2014, suggesting that the CQ-sensitive P. falciparum strains have returned after the withdrawal of CQ. The data also suggests that the parasites with wild type pfcrt/pfdmr-1 genes may have growth and/or transmission advantages over the mutant parasites. The information obtained from this study will be useful for developing and updating anti-malarial treatment policy in Grande Comore island.

Upregulated Tim-3/galectin-9 expressions in acute lung injury in a murine malarial model.

Malaria is the most relevant parasitic disease worldwide, and severe malaria is characterized by cerebral edema, acute lung injury (ALI), and multiple organ dysfunctions; however, the mechanisms of lung damage need to be better clarified. In this study, we used Kunming outbred mice infected with Plasmodium berghei ANKA (PbANKA) to elucidate the profiles of T cell immunoglobulin and mucin domain-3 (Tim-3) and its ligand galecin-9 (Gal-9) in the development of ALI. Mice were injected intraperitoneally with 10(6) PbANKA-infected red blood cells. The lungs and mediastinal lymph nodes (MLNs) were harvested at days 5, 10, 15, and 20 post infections (p.i.). The grade of lung injury was histopathologically evaluated. Tim-3- and Gal-9-positive cells in the lungs and MLNs were stained by immunohistochemistry, and the messenger RNA (mRNA) expressions of Tim-3, Gal-9, and related cytokines were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Bronchoalveolar lavage fluid (BALF) analyses were performed from days 18 to 20 p.i. The results showed that the pathological severities in the lungs were increased with times and the total protein level in the BALFs was significantly elevated in PbANKA-infected mice. The numbers of Gal-9(+) and Tim-3(+) cells in the lungs were significantly increased, and the mRNA levels of both Gal-9 and Tim-3 in the lungs and MLNs were over-expressed in PbANKA-infected mice. In conclusion, our data suggested that Tim-3/Gal-9 may play a role in PbANKA-induced ALI.

Increased Gal-9 and Tim-3 expressions during liver damage in a murine malarial model.

Malaria has been one of the most devastating tropical parasite infectious diseases popular around the world. Severe malaria is characterized by multiple organ dysfunctions, especially liver damage. However, the mechanisms of malarial liver injury remain to be better clarified. In this study, Kunming mice inoculated intraperitoneally (i.p.) with 10(6) Plasmodium berghei ANKA (PbANKA)-infected red blood cells (iRBCs) were investigated at days 5, 10, 15, and 20 post-infection (p.i.) to elucidate the profiles of T-cell immunoglobulin and mucin domain-3 (Tim-3) and its ligand galecin-9 (Gal-9) in the development of liver injury. The histopathology of livers and spleens from PbANKA-infected mice were observed, the parasite burdens of the livers and spleens using quantitative real-time PCR (qRT-PCR), Tim-3- and Gal-9-positive cells in the livers and spleens using immunohistochemical staining, and the mRNA levels of Tim-3, Gal-9, and cytokines in both the livers and spleens using qRT-PCR were examined. Our results showed that parasite burdens in the livers and spleens were significantly increased with time after PbANKA infection. Histological scores of both the liver and spleen tissues were significantly increased with time; the numbers of Tim-3- and Gal-9-positive cells were significantly increased in both the livers and spleens using immunohistochemical staining, and the mRNA levels of Tim-3 and Gal-9 in the livers and spleens were also significantly increased after infection. Our data suggests that the increase of Tim-3/Gal-9 expressions may play an important role in the liver damage during P. berghei infection.

Up-regulated TLR2 and TLR4 expressions in liver and spleen during acute murine T. gondii infection.

Toll-like receptors (TLRs) play a central role in the pathogen clearance and pathological processes. The liver is an important innate immune organ, in which Kupffer cells and hepatocytes are important innate immune cells. However, the role of TLR2 and TLR4 in the liver caused by Toxoplasma gondii infection remains less clear. In this study, mice were infected with T. gondii RH strain and the grades of liver and spleen injuries were histopathologically evaluated. TLR2+ and TLR4+ cells in the livers and spleens were detected by immunohistochemistry, and their messenger RNA (mRNA) expressions were detected using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The pathological severities in the livers and spleens were increased with time in T. gondii-infected mice. Compared with uninfected controls, obvious TLR2+ and TLR4+ cells were observed in the livers and spleens infected with T. gondii at 8 days post-infection, accompanied with significantly over-expressed mRNA levels of TLR2 and TLR4 in the livers and spleens after infection. Our data indicated that increased levels of TLR2 and TLR4 in the liver and spleen may play an important role during acute T. gondii infection.

The correlation of Tim-3 and IFN-γ expressions in mice infected with Toxoplasma gondii during gestation.

The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-1 (Tim-1) and Tim-3 participate in the regulation of Th immune response as well as innate immunity. However, there is no report about the expression of Tim genes in Toxoplasma gondii-infected experimental models during pregnancy. In this study, Kunming outbred pregnant mice were infected with RH strain of T. gondii through vagina at days 10 to 16 of gestation, and the mRNA expressions of Tim-1, Tim-3, interleukin (IL)-4, and interferon (IFN)-γ in the placentas, uteri, and draining lumber aortic lymph nodes (LALNs) at day 18 of gestation were analyzed using quantitative real-time PCR (qRT-PCR). Compared with uninfected pregnant controls, significantly increased levels of IFN-γ and Tim-3 were detected in the placentas (P < 0.001), uteri (P = 0.003 and P = 0.017, respectively), and LALNs (P = 0.003 and P = 0.025, respectively) of T. gondii-infected mice; there were positive and significant correlations between Tim-3 and IFN-γ mRNA expression levels in the placentas (R(2) = 0.6331, P = 0.0011), uteri (R(2) = 0.5658, P = 0.003), and LALNs (R(2) = 0.5583, P = 0.0033) of infected mice. Tim-1 (P = 0.002) and IL-4 (P = 0.003) expressions were significantly increased in the placentas, but Tim-1 were significantly decreased in the uteri (P = 0.013) and LALNs (P < 0.001) of infected pregnant mice in comparison of uninfected pregnant controls. Our data suggested that Tim-3 may play a regulatory role in T. gondii-infected pregnant mouse model.

Molecular surveillance of pvdhfr, pvdhps, and pvmdr-1 mutations in Plasmodium vivax isolates from Yunnan and Anhui provinces of China.

BACKGROUND: Plasmodium vivax is the predominant species of human malaria parasites present in China. Although sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been widely used for malaria treatment in China, the resistance profiles of these drugs are not available. Analysis of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps), and multidrug resistance (mdr-1) gene mutations in P. vivax isolates is a valuable molecular approach for mapping resistance to SP and CQ. This study investigates the prevalence of pvdhfr, pvdhps, and pvmdr-1 of P. vivax clinical isolates from China and provides baseline molecular epidemiologic data on SP- and CQ-associated resistance in P. vivax. METHODS: Plasmodium vivax clinical isolates were collected from two malaria-endemic regions of China, subtropical (Xishuangbanna, Yunnan province) and temperate (Bozhou, Anhui province), from 2009 to 2012. All isolates were analysed for single nucleotide polymorphism haplotypes in pvdhfr, pvdhps, and pvmdr-1 using direct DNA sequencing. RESULTS: In pvdhfr, 15% of Xishuangbanna isolates carried wild-type (WT) allele, whereas the majority of isolates carried mutant genes with substitutions at five codons. Eight mutant haplotypes of pvdhfr were detected, while limited polymorphism of pvdhfr was found in Bozhou isolates. A size polymorphism was present in pvdhfr, with the three-repeat type being the most predominate in both Xishuangbanna (79%) and Bozhou (97%) isolates. In pvdhps, mutations at four codons were detected in Xishuangbanna isolates leading to six haplotypes, including WT allele, single-mutation, double-mutation, and triple-mutation alleles. All Bozhou isolates carried WT pvhdps. In pvmdr-1, isolates from Xishuangbanna carried mutations at codons Y976F and F1076L, whereas all isolates from Bozhou had only a single mutation at codon F1076L. CONCLUSIONS: Plasmodium vivax isolates from subtropical and temperate zones of China are shown to have dramatically different frequencies and patterns of mutations in pvdhfr, pvdhps, and pvmdr-1. Whereas P. vivax populations in subtropical China are highly resistant to SP and CQ, those in the temperate zone may still be susceptible to SP and CQ. This information is useful for establishing treatment policy and provides a baseline for molecular surveillance of drug-resistant P. vivax in these areas.

Genetic diversity of Plasmodium vivax population in Anhui province of China.

BACKGROUND: Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp). METHODS: Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing. RESULTS: Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%). CONCLUSIONS: The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.

Dectin-1-CD37 association regulates IL-6 expression during Toxoplasma gondii infection.

Toxoplasma gondii can establish chronic infection and is characterized by the formation of tissue cysts in the brain. Although T. gondii can infect any kind of nucleated cells, macrophages and related mononuclear phagocytes are its preferred targets in vivo. Microglial cells are the resident macrophages in the central nervous system. It has been reported that CD37, a tetraspanin molecule, is expressed exclusively in the immune system; Dectin-1, an important pattern-recognition receptor, is expressed on the surface of murine primary microglia. The Dectin-1-CD37 association can affect Dectin-1-mediated IL-6 secretion. However, there is no report concerning the relationship among the expressions of Dectin-1, IL-6, and CD37 during T. gondii infection. In the present study, Kunming outbred mice were infected with Prugniaud (Pru), a type II strain of T. gondii by oral gavage, and BV-2 murine microglial cells were cocultured with RH tachyzoites of T. gondii. By H&E and immunohistochemical staining, the results showed that marked inflammation and a significantly increased activation of Iba1-positive microglial cells were observed in the brain tissues of mice infected with T. gondii Pru strain at 5 weeks postinfection (p.i.) in comparison of uninfected controls. Using quantitative real-time PCR detection, Dectin-1 messenger RNA (mRNA) expressions were significantly upregulated in both brains at 3 (P < 0.01), 5 (P < 0.01), 7 (P < 0.01), and 9 (P < 0.05) weeks p.i. and spleens at 3, 5, 7, and 9 weeks p.i. (P < 0.01). IL-6 expressions showed similar dynamic tendency as that of Dectin-1 in both the brains and spleens at the same times in comparison of uninfected controls; CD37 expressions were significantly increased in the brain tissues at all the times (P < 0.01) and no significant differences in the spleens at 3 weeks p.i. but significantly downregulated in the spleens at 5, 7, and 9 weeks p.i. (P < 0.01). In vitro study showed that compared with uninfected controls, the mRNA expressions of Dectin-1 at 2, 4, 8, and 10 h (P < 0.01); IL-6 at 8 and 10 h (P < 0.01); and CD37 at 4 (P < 0.05), 8 (P < 0.01), and 10 h (P < 0.01) were significantly upregulated in BV-2 murine microglial cells stimulated with RH tachyzoites of T. gondii. Our data suggested that the expression of Dectin-1 was positively correlated with that of IL-6 in toxoplasmic encephalitis (TE) mouse model; Dectin-1 interaction with tetraspanin CD37 regulated IL-6 expression in both the brain tissues of TE mouse model and in the T. gongdii-infected BV-2 murine microglial cells.

Comparison of dynamic expressions of Tim-3 and PD-1 in the brains between toxoplasmic encephalitis-resistant BALB/c and -susceptible C57BL/6 mice.

T cells and IFN-γ are essential for controlling the reactivation of toxoplasmic encephalitis (TE), regardless of whether mice are susceptible or resistant to TE. It has been demonstrated that CD8(+) T cells exhausted in chronic Toxoplasma gondii infection result in TE reactivation in C57BL/6 mice. However, this phenomenon had not been reported in genetically TE-resistant BALB/c mice. To explore the immune mechanism of TE in different backgrounds of mice, the dynamic expressions of Tim-3, programmed cell death 1 (PD-1), and their ligands (galectin-9, PD-L1, PD-L2) in brain tissues were compared between TE-resistant BALB/c and -susceptible C57BL/6 mice infected with Prugniaud (Pru, a type II strain) of T. gondii in this study. Compared with infected BALB/c mice, there were remarkable pathological changes with significantly higher histological scores in the brains of C57BL/6 mice at 14, 35, 50, and 70 days postinfection (p.i., P < 0.01); significantly increased mRNA expressions of Tim-3 at 35 (P < 0.05) and 70 (P < 0.01) days p.i.; and significantly increased PD-1 at all the times p.i. (P < 0.01) in the brains of infected C57BL/6 mice. Furthermore, there were significantly increased mRNA expressions of PD-L1 in the brain of C57BL/6 mice than that in BALB/c mice at all the times p.i. (P < 0.01). Although the mRNA expressions of galectin-9 (ligand of Tim-3) were increased in the brains of both lineages of mice at all the times p.i., it showed no differences between the two lineages of mice. Our data suggest that the differences of Tim-3 and PD-1/PD-L1 expressions may contribute to the different immune responses between TE-resistant BALB/c and -susceptible C57BL/6 mice infected with Pru strain of T. gondii.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection.

BACKGROUND: Schistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood. METHODS: To investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors. RESULTS: Compared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson's trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-ß, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining. CONCLUSIONS: Our data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.

Coinfection with Clonorchis sinensis modulates murine host response against Trichinella spiralis infection.

Concomitant infections of different species of parasites are common in the field. Infection with one parasite species likely triggers host responses that may influence the subsequent infection of another species and alter disease outcomes. So far, the majority of studies have focused on single species parasite infection, and the mechanisms of protection induced by the first parasite infection against the secondary infection remain poorly defined. In this study, we assess the impact of trematode Clonorchis sinensis infection on the course of another tissue nematode Trichinella spiralis challenge. We observed that mice with preexisting C. sinensis infection had lower worm burden of intestinal T. spiralis than those infected with T. spiralis alone; mice with preexisting C. sinensis also had severe enteric histopathological changes and higher counts of intestinal Paneth cells in responses to T. spiralis challenge. The mRNA levels of interleukin (IL)-4, IL-10, IL-13, and tumor necrosis factor (TNF)-α from the small intestine and spleen of the different groups were analyzed using quantitative real-time polymerase chain reaction. Compared with that in mice infected with T. spiralis alone, the mRNA expression of IL-13 was significantly increased in the small intestine tissues and IL-4, IL-13, and TNF-α were significantly increased in the spleen tissues in the dually infected mice. Our findings suggest that a "preexisting" trematode infection of C. sinensis is a factor which contributes to reducing the establishment of T. spiralis adult worms in the small intestine.

Expression of Tim-1 and Tim-3 in Plasmodium berghei ANKA infection.

Cerebral malaria (CM) is a serious and often fatal complication of Plasmodium falciparum infections; however, the precise mechanisms leading to CM is poorly understood. Mouse malaria models have provided insight into the key events in pathogenesis of CM. T-cell immune response is known to play an important role in malaria infection, and members of the T-cell immunoglobulin- and mucin-domain-containing molecule (Tim) family have roles in T-cell-mediated immune responses. Tim-1 and Tim-3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim family proteins in Plasmodium infection remains unclear. In the present study, the mRNA levels of Tim-1, Tim-3, and some key Th1 and Th2 cytokines in the spleen of Kunming outbred mice infected with Plasmodium berghei ANKA (PbANKA) were determined using real-time polymerase chain reaction (qRT-PCR). Compared with uninfected controls, Tim-1 expression was significantly decreased in infected mice with CM at day 10 postinfection (p.i.) but significantly increased in infected mice with non-CM at day 22 p.i.; in contrast, Tim-3 expression was significantly increased in infected mice both with CM at day 10 p.i. and with non-CM at day 22 p.i. The expressions of IFN-γ, TNF-α, IL-10, and IL-12 were significantly increased but IL-4 was significantly decreased in infected mice with CM at days 10 p.i., whereas the expressions of IFN-γ, TNF-α, IL-4, IL-10, and TGF-ß were significantly increased but IL-12 was significantly decreased in infected mice with non-CM at days 22 p.i. Furthermore, the expression of Tim-1 and Tim-3 could reflect Th2 and Th1 immune response in the spleen of PbANKA-infected mice, respectively. Our data suggest that PbANKA infection could inhibit the differentiation of T lymphocytes toward Th2 cells, promote the Th1 cell differentiation, and induce Th1-biased immune response in the early infective stage, whereas the infection could promote Th2 cell differentiation and induce Th2-biased immune response in the late infective stage. Our data indicate that both Tim-1 and Tim-3 may play a role in the process of PbANKA infection, which may represent a potential therapeutic target.

Evaluation of the adjuvant effect of pidotimod on the immune protection induced by UV-attenuated Toxoplasma gondii in mouse models.

The current anti-Toxoplasma gondii drugs have many shortcomings and effective vaccines against T. gondii may contribute to the control of this pathogen. Pidotimod is a synthetic substance capable of stimulating both cellular and humoral immunity. To investigate the possible adjuvant effect of pidotimod on the immune response to T. gondii in Kunming mice induced by ultraviolet-attenuated T. gondii (UV-T.g), in this study, mice were immunized intraperitoneal (i.p.) with UV-T.g or UV-T.g co-administered with pidotimod (UV-T.g + PT). After infection or challenge by i.p. injection of 10(2) RH tachyzoites, the animal survival rate, parasite burden in peritoneal lavage fluids, liver histopathology, the level of serum anti-toxoplasma IgG antibody, and the mRNA expressions of IL-2, IFN-γ, and TNF-α from spleen analyzed using real-time PCR, were compared among different groups. The results showed that, compared with infected controls, infected mice treated with pidotimod had significantly increased survival rate and extended survival time, decreased parasite burden, improved liver histopathology, increased level of anti-toxoplasma IgG antibody, and increased mRNA expressions of Th1-type cytokine (IL-2, IFN-γ, and TNF-α) (P < 0.01), while mice vaccinated with UV-T.g and then challenged had even significantly increased survival rate and extended survival time, decreased parasite burden, improved liver histopathology, and increased mRNA expressions of Th1-type cytokines (IL-2, IFN-γ, and TNF-α) (P < 0.01); furthermore, vaccinated mice co-administered with pidotimod had even more lower parasite burden, milder liver histopathology, and higher levels of Th1-type cytokine and anti-toxoplasma IgG antibody (P < 0.01). Our data demonstrated that pidotimod in vivo could promote strong and specific humoral and cellular immune response to T. gondii challenge infection when co-administered with UV-attenuated T. gondii. It suggests that pidotimod may have the potential to be used as an effective vaccine adjuvant.

Upregulated expression of Tim-3 involved in the process of toxoplasmic encephalitis in mouse model.

Toxoplasma gondii can establish chronic infection and is characterized by the formation of tissue cysts in the brain. The cysts may remain throughout the life of the host but can reactivate and cause life-threatening toxoplasmic encephalitis (TE) in immunocompromised patients. T cell-mediated immune responses are essential for preventing the reactivation of chronic infection of T. gondii in the brain. The immunoinhibitory receptor T cell immunoglobulin and mucin domain (Tim)-1 and Tim-3 are expressed on terminally differentiated T helper (Th) 2 and Th1 cells, respectively, participating in the regulation of Th immune response. However, there is no report concerning the role of Tim genes in TE. In this study, Kunming outbred mice were infected with Prugniaud (Pru), a type II strain of T. gondii by oral gavage. Compared with the uninfected controls, there were mild brain inflammations at 3 weeks postinfection (p.i.), moderate brain inflammations at 5 weeks p.i., and aggravated brain inflammations and necrosis at 7 and 9 weeks p.i. The expressions of tachyzoite stage-specific genes in brains were consistent with the severity of brain histopathology of TE at 5 and 7 weeks p.i., while the expressions of bradyzoite stage-specific genes in brains were significantly increased at 7 and 9 weeks p.i. Using quantitative real-time PCR detection and immunohistochemistry staining, our results showed that the expressions of Tim-3 were significantly upregulated in both brains and spleens at 5 weeks p.i. and in spleens at 9 weeks p.i., which showed the similar dynamic tendency as that of interferon-γ expressions in both brains and spleens at the same times. In contrast, the Th2-specific marker Tim-1 expressions were significantly downregulated in both brains and spleens at 3 weeks p.i. and upregulated in both brains and spleens at 7 and 9 weeks p.i., which showed the similar dynamic tendency as that of interleukin-4 expressions in both brains and spleens at the same time. Our data indicate that Tim-3 may involve in the process of TE in mice infected with T. gondii Pru strain.

In vitro effects of aqueous extracts of Astragalus membranaceus and Scutellaria baicalensis GEORGI on Toxoplasma gondii.

Toxoplasma gondii is a parasite that infects animals and humans worldwide. The standard treatment for toxoplasmosis is limiting due to toxic adverse effects, thus there is a need to identify new drugs that are less toxic. Both Astragalus membranaceus and Scutellaria baicalensis GEORGI are popular traditional Chinese herbs widely used for the treatment of various inflammatory diseases in Asia, and we have previously demonstrated that water extracts of A. membranaceus (AmE) and S. baicalensis GEORGI (SbE) have good efficacy in controlling T. gondii replication in mouse models. This study was designed to further evaluate their effects against developing tachyzoites of the RH strain of T. gondii in HeLa cell cultures. AmE, SbE, and TMP-SMX (trimethoprim-sulfamethoxazole) were added into the wells containing both HeLa cells and replicating T. gondii of green fluorescent protein (GFP)-expressing RH tachyzoites. The proliferation and morphous of the tachyzoites were observed, the fluorescence intensity expressed as the fluorescence gray scale value was measured, and the living tachyzoites were counted at different culture times after treatment. The results showed that, compared to untreated controls, parasites treated with either AmE or SbE had significantly decreased intracellular replication at 72, 96, and 120 h after treatment (P < 0.01); while compared to either AmE- or SbE-treated groups, SMX-treated groups had even significantly decreased replication (only a few living parasites were detected) at the above times (P < 0.01). Our data demonstrated that both AmE and SbE had remarkable in vitro activities against T. gondii.

Cryopreservation of Toxoplasma gondii in infected murine tissues.

Laboratory maintenance of the RH strain of Toxoplasma gondii is generally done by passage in mice, in vitro propagation in fibroblasts, or cryopreservation of peritoneal exudates from mice infected with T. gondii. To explore alternative techniques for preserving laboratory T. gondii tachyzoites, we propose a new method of freezing tissues from infected mice. The effect of storage of T. gondii tissue tachyzoites in two different cryoprotectant combinations and at two different temperatures was studied. The liver and spleen tissues, and peritoneal exudates from mice infected with RH-GFP strain of T. gondii, suspended in RPMI 1640 medium supplemented with 12 % glycerol plus 20 % calf serum, or 12 % dimethyl sulfoxide (DMSO) plus 20 % calf serum, were stored for 3 months at -20 °C in an ordinary refrigerator or at -80 °C in a deep freezer, respectively. The viability of tissue T. gondii tachyzoites was determined by animal inoculation method, which was assessed by monitoring survival and tissue parasitemia in recipient mice. Our data showed that toxoplasma tachyzoites in the above tissues remained viable after cryopreservation in 12 % DMSO plus 20 % calf serum at -80 °C, the infectivity of tachyzoites from the tissues and peritoneal fluids was demonstrated in inoculated murine tissues. Our data indicate that freezing infected murine tissues at -80 °C provides a simple and appropriate method for preservation of T. gondii tachyzoites in laboratory without the need for costly liquid nitrogen preservation procedures.