Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Expression of IL-23 and IL-17 in immune injury of the liver of mice sensitized by trichloroethylene and aggravated by Poly I:C / 中国职业医学

OBJECTIVE: To observe the expression of interleukin(IL)-23 and IL-17 in the liver of mice sensitized by trichloroethylene(TCE), and to explore the role of IL-23 and IL-17 in polyinosinic: polycytidylic acid(Poly I:C) exacerbated TCE-sensitized mice with immune injury of the liver. METHODS: Female specific pathogens free BALB/c mice were randomly divided into control group(n=5), solvent control group(n=5), TCE group(n=20), and TCE+Poly I:C group(n=20). A TCE-sensitized mouse model was established in TCE group and TCE+Poly I:C group. Three hours before the last challenge, mice in TCE+Poly I:C group was intraperitoneally injected with a mass concentration of 0.5 g/L poly I:C, 100 μL per mouse. The two groups of mice were divided into sensitized and non-sensitized subgroups according to the results of skin sensitization. After 48 hours of the final challenge, serum alanine aminotransferase(ALT) was detected by colorimetric method. The histopathological changes of mouse liver were observed, and the expression of IL-23 and IL-17 in liver tissues was detected by immunohistochemistry and Western blotting method. RESULTS: The sensitization rates of TCE and TCE+Poly I:C groups were 35.0%(7/20) and 40.0%(8/20) respectively, with no significantly statistical difference(P>0.05). Pathological examination showed that there was cell edema in some areas of the liver tissues of mice in the TCE-sensitized subgroup, while the TCE+Poly I:C sensitized subgroup showed cell vacuolar degeneration and loose cytoplasm. Serum ALT activity and the expression of IL-23 and IL-17 in liver tissues in the TCE-sensitized subgroup were higher than that in the blank control group, the solvent control group and the TCE non-sensitized subgroup(P<0.05). Serum ALT activity and IL-23 and IL-17 expression in the TCE+Poly I:C sensitized subgroup were higher than that in the TCE-sensitized subgroup(P<0.05). The relative expression of IL-23 and IL-17 protein in liver tissues in TCE-sensitized subgroup was higher than that of the blank control group and the solvent control group(P<0.05), while that in TCE+Poly I:C sensitized subgroup was higher than that of TCE-sensitized subgroup(P<0.05). CONCLUSION: IL-23/IL-17 axis may play an important role in the development of immune injury of liver in the TCE-sensitized mice and Poly I:C exacerbated TCE-sensitized mice.

The modelling of the autism spectrum disorder of Sprague-Dawley rat exposed to Poly-IC in the pregnancy and the preliminary study of neuropathologic changes / 中华实用儿科临床杂志

Objective Through modelling autism spectrum disorder(ASD) in condition of Poly-IC,to analyze the development,nervous pathological changes and behavior of rats in experiments,and to define whether the Sprague-Dawley(SD) rats are suitable for the study of ASD.Methods Ten SD rats were randomly divided in 2 groups with 5 rats in each group.The experimental group rats were injected intraperitoneally with 5 mg/kg Poly-IC at gestational day 12,while the rats in control group were injected intraperitoneally with 9 g/L normal saline at the gestational day 12.The body weight,eye-opening time,swimming performance,and conduct parallel water maze test,social communication ability test of each offspring were recorded,and the developmental state of rats and the existence of autism-like social behavior were evaluated.The alterations in hippocampus morphology of offspring rats were indirectly observed with immunofluorescence double standard dyeing.Results Compared with the control group,the weights of the experimental rats were lighter,opening-eye time was delayed,swimming score was lower,in each measurement point.The differences above all were significant(all P ＜0.05).The near-escape scores of experiment rats in social communication ability test were lower than those of rats in control group.The rats in the experimental group needed more time to get the Morris water maze platform,and the times of rightly run through the Morris water maze decreased significantly,and the differences were significant (all P ＜ 0.05).Pathological results revealed that the mineralocorticoid receptor (MR) average optical density value (0.061 3 ± 0.028 8) and the glucocorticoid receptor (GR) mean optical density value (0.041 9 ± 0.040 3) in hippocampal CA1 area of experiment rats were lower than those of the control group (MR:0.081 3 ±0.053 9;GR:0.061 2 ±0.043 6) (t =10.319,10.241;all P ＜0.05).There was no significant difference between MR/GR ratio(the ratio of MR and GR optical density value in the same vision area) in experimental group and in the control group.Conclusions The Poly-IC during early pregnancy can cause lag behind in motor development,harm the social communication ability,lose memory ability to learn,and develop ASD symptoms of SD rat,and these symptoms may be associated with abnormal expression of MR and GR on the hippo-camp cells surface.AS a consequence,early pregnant SD rats exposed to Poly-IC can be used for the establishment of the model of ASD which can provide a platform for the research of ASD.

Expression of intrahepatic chemokine CXCL13 in a mouse model of primary biliary cholangitis / 医学研究生学报

Objective The expression of chemokine C-X-C motif ligand 13 (CXCL13) within liver in primary biliary cholangitis (PBC) patients is significantly increased, but its origin and mechanism is not clear yet.The study aimed to investigate the expression of CXCL13 in the liver of mice through establishing a mouse model of PBC.Methods C57BL/6 mice were randomly divided into experiment group (n=20) control group(n=10).The mice in the experimental group were intraperitoneally injected with polyriboinosinic polyribocytidylic acid (Poly I:C) while the mice in control group were injected with PBS of the same volume.The level of serum AMA was quantified by ELISA and intrahepatic inflammatory cells were assessed by HE staining.Kupffer cells, liver sinusoidal endothelial cells, and infiltrating lymphocytes in the liver of mice were collected by in situ perfusion enzyme digestion and magnetic bead separation methods.The transcriptional level of intrahepatic CXCL13 in liver tissues and cell subpopulations were detected by qPCR.Results The serum AMA titers of the mice in experiment group increased gradually with the prolonging of modeling time and the positive rates at the 4th, 8th, and 12th week after the first injection of Poly I:C were 5.9%, 52.9% and 76.5% respectively.While the serum AMA titers of the mice in control group were at a lower level through the modeling process, with only 2 mice presenting a little higher level above positive cutoff value at the 12th week.The results of HE staining in liver tissues of both groups showed that there were a great amount of intensely infiltrating inflammatory cells in the mice of experimental group while no inflammatory cell infiltration were found in the mice of control group.The separation purity of Kupffer cells and liver sinusoidal endothelial cells in the mice of experiment group tested by flow cytometry were 76%-80%, 68%-72% respectively.Compared with the CXCL13 mRNA level in Kupffer cells [2.34(0.22-8.64)], the expression levels in liver sinusoidal endothelial cells and infiltrating lymphocytes declined[0.27(0.03-1.64), 0.05(0-0.22), P<0.05].Conclusion The chemokine CXCL13 is predominantly produced by Kupffer cells in the liver of PBC mice established by Poly I:C injection.

Observation of adequate stimulus methods for key initiative factor expression in human keratinocytes of anaphylactic disease / 中国药理学通报

Aim To explore the expressed level of ini-tiative key factors TSLP and IL-33 in a human kerati-nocyte cell line,HaCaT cells were chosen to be stimu-lated by different stimulants,and develop a stable and effective in vitro model to observe allergic sensitization. Methods HaCaT cells were cultured in K-SFM with different stimulants to screen out the stimulants which could significantly improve the expressed level of TSLP and IL-33.Expressed level of TSLP and IL-33 was an-alyzed by ELISA kits and immunofluorescence.Re-sults (1 )The dose-response relationship of single stimulant indicated that both Poly(I:C)and TNF-αcould significantly improve expressed level of TSLP and IL-33 in HaCaT cells,but the rest of stimulants was not observed significant stimulation in concentration range of this experiment.(2)Dose-effect relationship of combined stimulants indicated that poly(I ∶C)1 00 mg·L -1 combined with TNF-α20 μg·L -1 was the most efficient.(3)Time-effect relationship of the a-bove-mentioned combined stimulants showed that 1 2 h was the optimal time of stimulation.Conclusions Different stimulants and different time result in various expressed levels of TSLP and IL-33 in HaCaT cells.1 2 h stimulus duration of Poly(I:C)1 00 mg·L -1 com-bined with TNF-α20 μg · L -1 is the most efficient stimulating way.This result provides an effective in vitro model to study the pathomechanism and drug effi-cacy of allergic sensitization.

Differential microglial and astrocytic response to bacterial and viral infection in the developing hippocampus of neonatal rats.

Polyinosinic:polycytidylic acid (Poly I:C; 5 mg/kg body weight, ip) and lipopolysaccharide (LPS; 0.3 mg/kg body weight, ip) induced microglial and astrocytic activation in Sprague Dawley rats. Higher microglial and astrocytic activities were noticed in Poly I:C infused rats throughout the hippocampus till postnatal day 21 with a comparatively weaker response in LPS group. However, LPS induced inflammation persisted even after postnatal day 21, indicating thereby, that the Poly I:C (viral mimic) produces an acute inflammation, while LPS (bacterial endotoxin) produces chronic inflammation when exposed during early neonatal life.

Induction of Potent Antigen-specific Cytotoxic T Cell Response by PLGA-nanoparticles Containing Antigen and TLR Agonist

Previously we showed that biodegradable nanoparticles containing poly-IC or CpG oligodeoxynucleotide (ODN) together with ovalbumin (OVA) were efficient at inducing MHC-restricted presentation of OVA peptides in dendritic cells. The CTL-inducing activities of the nanoparticles were examined in the present study. Nanoparticles containing poly-IC or CpG ODN together with OVA were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid), and then were opsonized with mouse IgG. The nanoparticles were injected into the tail vein of mice, and 7 days later the OVA-specific CTL activities were measured using an in vivo CTL assay. Immunization of mice with the nanoparticles containing poly-IC or CpG ODN together with OVA elicited potent OVA-specific CTL activity compared to those containing OVA only. In accordance with these results, nanoparticles containing poly-IC or CpG ODN together with OVA exerted potent antitumor activity in mice that were subcutaneously implanted with EG7.OVA tumor cells. These results show that encapsulation of poly-IC or CpG ODN together with antigen in biodegradable nanoparticles is an effective approach for the induction of potent antigen-specific CTL responses in vivo.

Polyinosinic-polycytidylic acid and tumor immunity / 国际肿瘤学杂志

Polyinosinic-polycytidylic acid is a synthetic analog of double-stranded RNA,which plays a vital role in the regulation of immune system.Toll-like receptor 3 ( TLR3 ),melanoma differentiation-associated gene 5 (MAD-5) and retinoic acid inducible gene - Ⅰ ( RIG- Ⅰ ) are the main three intracellular receptors binding to polyinosinic-polycytidylic acid which activates human anti-tumor immune responses including the activation of innate immunity and acquired immunity through TIR-domain-containing adapter-inducing inter feron-β (TRIF) and interferon-β.Thus,polyinosinic-polycytidylic acid plays an important role in regulating anti-tumor immune responses.The immunoregulation mechanisms ofpolyinosinic-polycytidylic acid in melanoma,breast cancer,malignant glioma and lung cancer have been clarified,which will provide new strategies for tumor immunotherapy.

Curative effect observation on the treatment of chronic type B hepatitis with Gankang Granule / 国际中医中药杂志

Objective To observe the clinical effects of treating chronic type B hepatitis with Gankang Granule combined with poly I-C and immune ribonucleic acid. Methods A total of 100 cases of chronic type B hepatitis were randomly recruited into a control group and a treatment group, 50 cases in each group. The control group was treated with poly I-C and immune ribonucleic acid, while the treatment group was treated with Gankang Granule on the basis of the control group. Results Symptoms in both groups were improved. 23 eases in the treatment group showed negative value of HBeAg,contrasting to 13 cases in the control group. 38 cases in the treatment group turned to be negative value of HBV- DNA, higher than the number of 23 cases in the control group. The treatment group demonstrated a significantly better therapeutic results than the control group (χ2 value was 6.267 and 6.345 respectively, both P＜0.05). Conclusion Gankang Granule combined with poly I-C and immune ribonucleic acid can obviously improve hepatic functions and symptoms of chronic type B hepatitis,shorten the course of disease, and increase the rate of negative value of HBeAg and HBV- DNA.

Expression of Toll-like receptor 3 on the peripheral blood dendritic cells after stimulated by poly I: C in patients with chronic hepatitis B / 中华传染病杂志

Objective To elucidate the expression of Toll-like receptor 3 (TLR3) on dendritic cells(DCs) in patients with chronic hepatitis B (CHB), and to explore the correlation between hepatitis B virus (HBV) persistent infection and TLR3 expression. Methods Sixty CHB patients (CHB group) and 20 healthy controls (control group) were enrolled. The peripheral blood mononuclear cells (PBMCs) were isolated and CD14~+ monocytes were sorted by immunomagnetic beads. Immature DCs (imDC) were induced and proliferated in vitro and mature DCs (mDC) were obtained after the poly I:C stimulation. The expression of intracellular TLR3 mRNA was detected by real-time polymerase chain reaction (PCR), and surface markers [CD80 and human leucocyte antigen (HLA)-DR] were determined by flow cytometry after 48 h of stimulation. The comparison of quantitative data was done using t test. The qualitative data were compared using chi-square test.Results The mean fluorescence intensities (MFI) of intracellular TLR3 of imDC before poly I:C stimulation in CHB group and control group were 1212.05 ± 250.80 and 1192.95 ± 301.40,respectively, which were not significantly different (t = 0. 280, P>0. 05). While after stimulation,those were 1352.98± 313.67 and 1593. 00± 349. 65, respectively, the latter was significantly higher than the former (t = 2. 880, P<0. 05). The levels of TLR3 mRNA inside mDCs in both groups were increased after poly I:C stimulation, which were 0. 1204 ±0.0267 and 0. 1780 ± 0.0664, respectively in CHB group and control group, and that in control group was significantly higher (t = 3. 909, P<0.05). Furtherly, patients in CHB group were divided into HBeAg(+ ) and HBeAg( -) subgroups.After stimulation, the MFI and mRNA of TLR3 inside mDC were greatly elevated in both subgroups,but there were no difference between these two subgroups (t = 0. 366, P>0. 05). Conclusions The intracellular expressions of TLR3 in mDC in CHB group and control group are obviously increased after the poly I:C stimulation, but the increased level in CHB group is lower than that in control group. The results suggest that the insufficiency of TLR3 synthesis may be related to the HBVpersistent infection.

Defective activation induced cell death of CD4+ T cells in primary biliary cirrhosis murine model / 中华微生物学和免疫学杂志

Objective To study the activation induced cell death (AICD) of CD4+ T cells in primary biliary cirrhosis(PBC)murine model induced by poly I∶C. Methods Thirty female C57BL/6 mice were divided into model and control group randomly, and the former were injected with 5 mg/kg of poly I∶C, the later with PBS. PBC mice were detected 16 weeks after injection. CD4+ T cells isolated from spleen were stimulated in vitro by Con A and anti-CD3, and the apoptosis were determined by Annexin-V and PI staining. The expression of Fas, FasL and TRAIL were assayed by relative quantitative real-time PCR. Bcl-2 was detected by Western blot. Results Compared with control group, the portal areas of mice in model group were infiltrated with mononuclear cells obviously. The positive rate of serum antimitochondrial antibody(AMA) and the level of alkali phosphatase (ALP) were higher than that in control group (P<0.001). AICD of splenic CD4+ T cells in model group was lower than that of control group (P<0.001). The mRNA of FasL and TRAIL in model mice was down-regulated. Simultaneously, the anti-apoptosis protein Bcl-2 was up-regulated in model group. Conclusion These observations suggest that a defect in AICD of auto-reactive TH1 cells may contribute to the pathogenesis of PBC model. Furthermore, this defect in AICD may results from the change of Fas/FasL, TRAIL pathway and the up-regulation of Bcl-2.

The Expression of TLR3 and Cytokines Induced by Poly I:C in Human Retinal Pigment Epithelial Cells

In this study, we examined the expression of Toll-like receptor3 (TLR3) by human retinal pigment epithelial cells (RPE) and determined whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly I:C) would induced the expression of cytokines in these cells. RT-PCR revealed that TLR3 was constitutively expressed in human RPE, and its expression was increased by treatment with poly I:C. After treatment with poly I:C, we determined the expression levels of pro-inflammatory cytokines in human RPE using RT-PCR and ELISA. We demonstrated that poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human RPE. Upon exposure to poly I:C, human RPE initiated antiviral response resulting in the induction of IFN-beta mRNA expression and 2',5'-oligoadenylate synthetase mRNA expression. These results suggest that human RPE may participate in ocular defense mechanism against viral infection through TLR3.

Prophylactic efficacy of BCG and polyinosinic : Polycytidylic acid on implanted murine bladder tumor / 대한비뇨기과학회지

A study was done to evaluate whether intraperitoneal Polyinosinic: Polycytidylic acid (Poly I:C) administration in addition to BCG could enhance the anticancer effect of BCG on subcutaneously implanted syngeneic murine bladder tumor. The appropriate administration schedules of Poly I:C and BCG were defined by evaluating the change of natural killer cell activity and cell mediated cytotoxic effect against syngeneic bladder tumor cells or splenic and peritoneal lymphocyte at regular time interval. The prophylactic anticancer effect of Poly I:C alone and Poly I:C plus BCG were evaluated by observing the tumor take rate and growth change of the taken tumor compared to the control group. Bladder tumor take rate and growth of the taken tumor were suppressed after administration of Poly I:C alone. Natural killer cell activity of the splenic and peritoneal lymphocytes were enhanced and in vitro cytotoxicity against MBT-2 was increased after Poly I:C administration. When Poly I:C was administrated in addition to BCG, tumor take rate and tumor growth were more suppressed and the time for appearance of the visible tumor was prolonged compared to BCG therapy alone. In conclusion, Poly I:C showed prophylactic anticancer effect against subcutaneously implanted murine bladder tumor when used alone or in combination with BCG, and the cell mediated cytotoxicity including enhanced NK cell activity seemed to play an important role in their anticancer effect. These results suggest a possibility of Poly I:C, alone or in combination with BCG, would become a new prophylactic treatment modality against bladder tumors.

THE ANTITUMOR ACTION OF ADRIAMYCIN-POLY I:C COMPLEX IN VIVO AND IN VITRO / 中国药理学通报

The binding of adriamycin to poly I:C, a double-stranded thetic homoribopolymor,and antitumor activity of adriamycin-Poly I:C complex were studied. Results showed that the affinity for adriamycin to poly I:C is no linear relationship. Adriamycin binding rate( % ) = 20.6 + 0.2268?Poly I:C(W)/adriamycin(W)? The increased therapeu-tic effect of the complex was comparable to that of the free and the Poly I:C alone on bearing Sl80 mice. In vitro cytotoxic effect of the complex is lower than free adriamycin following lh exposure. The complex is recommended for clinical therapeutic studies.

COMPARATIVE OBSERVATION OF TWO METHODS TREATING THE VERRUCA ACUMINATE / 重庆医科大学学报

This paper reports the treatment of 65 cases with Verruca Acuminate, 35 cases with the fulguration while 30 cases with the combined fulguration with injection poly i:c and both were compared. The results showed that the cure rate of the fulguration group was 68.6%, and that of the combined therapy group was 96,5%. The cure rate of the latter group revealed higher (P