Construction of multi-epitope gene of periodic Brugia malayi and its expression in eukaryotic cells / 中华地方病学杂志

Objective:To construct a eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi cysteine protease inhibitor/glyceraldehyde 3-phosphate dehydrogenase (CPI502/GAPDH720) multi-epitope gene and observe its protein expression in Hela cells. Methods:Primers were designed according to the predicted gene sequences of T cell epitopes. The target gene fragment was amplified by reverse transcription (RT)-PCR using plasmid pGEM-T-CPI621 as template. The fragment was cloned into prokaryotic expression plasmid pET-28a(+) to construct prokaryotic expression plasmid pET28a(+)-BmCPI502. The BmCPI502 gene and BmGAPDH720 gene were amplified by RT-PCR, respectively. The gene fragments of pcDNA3.1(+), BmCPI502 and BmGAPDH720 were digested by double enzyme digestion and ligated with the target gene. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was constructed. The recombinant plasmid was transfected into Hela cells and verified by RT-PCR to obtain the desired target bands. The expression product was detected by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).Results:The recombinant plasmid pET28a(+)-BmCPI502 was obtained, and the 502 bp specific fragment was identified by enzyme digestion, which was in line with the expected value; the eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 of the multi-epitope gene was successfully constructed, and the fragment size was in line with the expected value. The eukaryotic expression plasmid pcDNA3.1(+)-BmCPI502/BmGAPDH720 was transfected into Hela cells and the recombinant protein was stable expressed. SDS-PAGE analysis showed that the relative molecular weight ( Mr × 10 3) of the recombinant protein was about 50. Conclusions:The eukaryotic expression plasmid of pcDNA3.1(+)-BmCPI502/BmGAPDH720 of periodic Brugia malayi multi-epitope gene is successfully constructed, and the corresponding recombinant protein is obtained in eukaryotic cells. This study has laid a foundation for further study of the purification and biological activity of the recombinant protein.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of ～65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN-γ, TNF-α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

Effect of short-And long-Term immunization of recombinant disorganized muscle protein-1 (rDIM-1) against human filarial parasite Brugia malayi in rodents

Objective: To evaluate the effect of short-Term and long-Term immunization of recombinant disorganized muscle protein-1 (rDIM-1) in rodents against human filarial parasite Brugia malayi. Methods: Recombinant Brugia malayi DIM-1 (rDIM-1bm) protein was cloned, expressed and purified using a Ni-NTA affinity column. Mastomys coucha were immunized with rDIM-1bm in three immunization schedules: short-Term (3-dose of rDIM-1bm), and long-Term (booster doses till 3-And 6-week) and subsequently challenged with infective third-stage larvae of filarial parasite Brugia malayi (L3). Microfilaraemia was monitored in L3 exposed groups on day 90 post larval inoculation (p.l.i.) and continued till day 205 p.l.i. On day 205 p.l.i. all the infected animals were killed and total worm burden was estimated. Cellular proliferative response, macrophage activity, nitric oxide (NO) release, specific IgG and its subtypes, IgE, IgA and Th1 (IFN-γ, TNF-α and IL-2) and Th2 (IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release were determined. Results: Of the 3 different immunization schedules, short-Term immunization (3-dose schedule) showed better reduction in microfilarial burden (36%-63%) in the peripheral circulation, adult worm load (52%), whereas long-Term immunization (3-And 6-week schedule) exerted less effect on peripheral microfilariae count (9%-58%), and adult worm burden (9%-12.5%). Short-Term immunization resulted in upregulation of cellular proliferation, macrophages activity, NO release, specific IgG, IgG1, IgG2a, IgG2b, IgE and IgA levels and both Th1 (IFN-γ, TNF-α and IL-2) and Th2 (IL-4, IL-5, IL-6, IL-10 and IL-13) cytokine release whereas long-Term immunization (3-And 6-week schedule) exerted less effect on parasite burden and showed mixed immunological responses. None of the rDIM-1bm administration schedules induced any pathology in lymphoid tissues, or alteration in mast cell number and granularity. Conclusions: The short-Term immunization with rDIM-1bm (3-dose schedule) induces robust immune responses and protects the host from filarial parasite infection.

Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

Objective: To evaluate the immunostimulatory potential of cross-reactive molecule heat shock protein 60 (HSP60) of filarial parasite Brugia malayi and Leishmania donovani. Methods: HSP60 of Brugia malayi (BmHSP60) was amplified using gene-specific primer, cloned in pTriEx4 vector, expressed in BL21-DE3 cells, and recombinant HSP60 (rHSP60) of 65 kDa was purified by affinity chromatography using Ni-NTA column. The recombinant protein was desalted by the dialysis membrane, and the presence of endotoxin level was determined by Limulus amebocyte lysate assay. The recombinant protein was tested for cell proliferation, nitric oxide release, expression of Th1 and Th2 cytokines, and transcription factors (STATs) in vitro using murine macrophage cell line (J774A.1). Results: Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential. rBmHSP60 exposure upregulated the expression of iNOS, STAT1, STAT4, Th1 cytokines (IFN-γ, TNF-α, IL-12), and nitric oxide release. In addition, no remarkable change was observed in the expression of IL-6, IL-10, and STAT3 in macrophage cell line J774A.1. The ELISA analysis showed the levels of IFN-γ, TNF-α, and IL-12 were upregulated while IL-10 level was downregulated, revealing that BmHSP60 triggered a Th1 immune response. Conclusions: Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses, and can be used as an immunoprophylactic agent against leishmaniasis. Furthermore, in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective:To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR).Methods:A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected.Results:Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences.Conclusions:This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

High resolution melting real-time PCR detect and identify filarial parasites in domestic cats

Objective: To detect and identify filarial parasites in dried blood spots (DBS) collected from domestic cats using high resolution melting real-time PCR (HRM RT-PCR). Methods: A total of 208 DBS were collected from domestic cats in a brugian filariasis endemic areas in Surat Thani Province, southern Thailand. Microfilariae were found in 9 blood slides using Giemsa-stained thick blood film. The extracted DNA from blood spot volumes of 10 and 20 μL DBS with positive filarial parasites in cats were performed using HRM RT-PCR method. The primers were designed based on the partial mitochondrial 12S rRNA gene for identifying Brugia malayi, Brugia pahangi, Dirofilaria immitis. All purified samples were then detected. Results: Using different volumes of 10 μL and 20 μL DBS could easily distinguish filarial parasites and showed similar results. PCR amplicons of Brugia malayi, Brugia pahangi and Dirofilaria immitis were determined at melting peak (temperature) of 75.70 77.46 and 73.56 respectively. All 9 positive DBS samples showed positive Brugia pahangi and similar nucleotide sequences. Conclusions: This HRM RT-PCR method is able to diagnose, identify and discriminate filarial parasites collected from DBS, which is simple and inexpensive compared with other probe-based genotyping methods. Furthermore, this method is useful to survey, prevent and control filariasis.

Follow-up Study of Patients Previously Diagnosed with Lymphatic Filariasis in Korea

OBJECTIVES: Korea was an endemic area for lymphatic filariasis (LF), caused by the nematode parasite Brugia malayi, until the 1970s. The World Health Organization recognized Korea as LF-free in June 2008. However, it is necessary to confirm that patients that have had LF in the past still test negative, to prevent the re-emergence of LF in Korea. METHODS: We followed up a total of 83 patients who had been diagnosed with LF between 2002 and 2010 in endemic LF areas. RESULTS: Fifty-two of the 83 subjects were negative for LF, whereas 31 subjects had re-located to a different city or province, were dead, or were unaccounted for. Most subjects with negative test results still exhibited edema in the legs or the arms, and some complained of redness and swelling in the legs or ankle joints. However, we found that these symptoms were due to diseases other than LF. CONCLUSION: In this follow-up study, we did not find any evidence indicating the potential re-emergence of LF in Korea.

Antifilarial actions of green tea extract and a synthetic heterocyclic thiazolidine derivative, Im8 compound in experimental mouse model.

In spite of the advances in drug development and research against human lymphatic filariasis following the WHO mandate to address the disease-associated socioeconomic burden, diethylcarbamazine (DEC, N, N-diethyl-4-methyl-1-piperazine carboxamide) is the only available antifilarial drug to date. The major obstacle for further development of antifilarial drugs is the lack of validation of candidate drugs in the experimental animal models. Both, green tea extract and a synthetic heterocyclic thiazolidine derivative (Im8; 2-chloro-N-(4-phenylthiazol-2-yl), showed efficacy of antifilarial action in our earlier in vitro study and hence, they were screened in the present study for their antifilarial potential in the BALB/c mouse filariasis model. Mice were treated with 25 mg/kg dose of either Im8 or green tea extract or DEC or only with their respective vehicles. The untreated mice served as controls. Following insertion of the micropore chamber laden with microfilariae (Mf) of Brugia malayi, the drug or vehicle was administered s.c. in mice at 12 h intervals as 4 doses. After 12 h of administration of the last dose, the micropore chambers were removed to determine the action of the treatments as the loss of Mf motility. The green tea extract showed a significant antifilarial action and Im8 showed relatively less but significant antifilarial action as compared to the respective vehicle controls. Both the green tea extract and Im8 showed higher activity than that was exerted by DEC. These results revealed a greater efficacy of green tea and thiazolidine derivative, Im8 as the novelantifilarial agents in the experimental mouse model of filariasis.

A recombinant plasmid of composite cysteine proteinase inhibitor/ glyceraldehyde‑3‑phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host.

Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine‑elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription‑polymerase chain reaction in muscle tissues. The stimulation index (SI) of T‑lymphocyte proliferation and the levels of interferon‑gamma (INF‑γ) and interleukin‑4 (IL‑4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme‑linked immunosorbent assays. Results: The pcDNA3.1(+)‑BmCPI/ BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF‑γ and IL‑4 of pcDNA3.1(+)‑BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF‑γ of pcDNA3.1(+)‑BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)‑BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)‑BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.

Targeting folate metabolism for therapeutic option: A bioinformatics approach.

Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.

Prevalence of filarial parasites in domestic and stray cats in Selangor State, Malaysia

Objective: To determine the prevalence of the filarial parasites,. ie.,. Brugia malayi, Brugia, Brugia pahangi(B. pahangi), Dirofilaria immitisand. Dirofilaria repens (D. repens) in domestic and stray cats. Methods: A total of 170 blood sample were collected from domestic and stray cats and examined for filarial worm parasites in two localities, Pulau Carey and Bukit Gasing, Selangor State, Malaysia. Results: The overall prevalence of infection was 23.5% (40/170; 95% CI = 17.4-30.6). Of this, 35% (14/40; 95% CI = 22.1-50.5) and 50% (20/40; 95% CI = 35.2-64.8) were positive for single B. pahangi nd D. repens, respectively. The remaining of 15% (6/40; 95% CI = 7.1-29.1) were positive for mixed B. pahangi and D. repens. In addition, 75% of the infected cats were domestic, and 25% were strays. No Brugia malayi and Dirofilaria immitis was detected. Eighty-four cats were captured at Pulau Carey, of which 35.7% (30/84) were infected. Among the cats determined to be infected, 93% (28/30; 95% CI = 78.7-98.2) were domestic, and only 6.7% (2/30; 95% CI = 19.0-21.3) were strays. Conversely, the number of infected cats was three times lower in Bukit Gasing than in Pulau Carey, and most of the cats were stray. Conclusions: B. pahangi and D. repens could be the major parasites underlying filariasis in the study area. Adequate prophylactic plans should be administrated in the cat population in study area.

Prevalence of filarial parasites in domestic and stray cats in Selangor State, Malaysia

OBJECTIVE@#To determine the prevalence of the filarial parasites,ie.,Brugia malayi, Brugia, Brugia pahangi(B. pahangi), Dirofilaria immitisandDirofilaria repens (D. repens) in domestic and stray cats.@*METHODS@#A total of 170 blood sample were collected from domestic and stray cats and examined for filarial worm parasites in two localities, Pulau Carey and Bukit Gasing, Selangor State, Malaysia.@*RESULTS@#The overall prevalence of infection was 23.5% (40/170; 95% CI = 17.4-30.6). Of this, 35% (14/40; 95% CI = 22.1-50.5) and 50% (20/40; 95% CI = 35.2-64.8) were positive for single B. pahangi nd D. repens, respectively. The remaining of 15% (6/40; 95% CI = 7.1-29.1) were positive for mixed B. pahangi and D. repens. In addition, 75% of the infected cats were domestic, and 25% were strays. No Brugia malayi and Dirofilaria immitis was detected. Eighty-four cats were captured at Pulau Carey, of which 35.7% (30/84) were infected. Among the cats determined to be infected, 93% (28/30; 95% CI = 78.7-98.2) were domestic, and only 6.7% (2/30; 95% CI = 19.0-21.3) were strays. Conversely, the number of infected cats was three times lower in Bukit Gasing than in Pulau Carey, and most of the cats were stray.@*CONCLUSIONS@#B. pahangi and D. repens could be the major parasites underlying filariasis in the study area. Adequate prophylactic plans should be administrated in the cat population in study area.

Successful Treatment of Brugia pahangi in Naturally Infected Cats with Ivermectin

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 microg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

IgG subclass responses to proinflammatory fraction of Brugia malayi in human filariasis.

Background & objectives: Earlier we demonstrated that immunization with F6, a proinflammatory molecular fraction isolated from the human filarial parasite Brugia malayi, protected the host and eliminated the infection in Mastomys coucha by a Th1/Th2 response including IgG2a antibody response. Whether F6 molecules become accessible to human host during natural course of infection and elicit similar response is not known. The present study was undertaken to determine the profile of IgG subclasses specifically reactive to F6 in different categories of bancroftian filariasis cases to infer any relationship between the levels of a particular F6-specific IgG subclass and the infection or disease status. Methods: Serum samples of normal individuals from filariasis non-endemic regions of India like Jammu & Kashmir, Uttarakhand, and Chandigarh [(NEN-W; n=10), healthy subjects from USA (NEN-U; n=10) and three categories of bancroftian filariasis cases from endemic areas: endemic normals (EN; n=10) with no symptoms and no microfilariae, asymptomatic microfilaremics (ASM; n=10) and chronic symptomatic amicrofilaremics (CL; n=10) were assayed for F6-specific IgG1, IgG2, IgG3 and IgG4 by ELISA using SDS-PAGE-isolated F6 fraction of B. malayi adult worms. Results: Significantly high levels of F6-specific IgG1, IgG2 and IgG3 were found in CL (P<0.001) and EN (P<0.01-0.001) bancroftian filariasis cases compared to NEN-U. Significant levels of F6-specific IgG1 (P<0.01) and IgG2 (P<0.01) but not IgG3 were found in ASM cases compared to NEN-U. The most abundant was IgG2 which when compared to NEN-U, was significantly high in CL (P<0.001) and EN cases (P<0.001), followed by ASM (P<0.01). F6-specific IgG4 response in EN, ASM and CL subjects was not significantly different from the levels of NEN-U. Among the non-endemic normals, the NEN-W subjects showed significant reactivity with IgG2 (P<0.001) but not with IgG1, IgG3 and IgG4 as compared to NEN-U subjects. IgG subclass levels were different in different categories. Interpretation & conclusions: The high levels of F6 reactive IgG1, IgG2 and IgG3 in endemic normals and chronic symptomatic bancroftian patients, and IgG1 and IgG2 in asymptomatic microfilaraemics, suggest that F6 molecules of parasite are accessible in these subjects for IgG subclass-specific immune response and IgG2 may be related to pathogenesis. Studies using individual F6 molecules will be done to identify the molecule(s) involved in infection and protective immunity.

Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity.

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Construction of periodic Brugia malayi cysteine protease inhibitor and glyceraldehydes-3-phosphate dehydrogenase eukaryotic recombinant plasmid and its immunogenicity / 中华传染病杂志

ObjectiveTo construct the eukaryotic expression plasmids containing cysteine protease inhibitor (CPI) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene from periodic Brugia malayi (Bm),and to observe its cellular immune response in mouse.Methods pcDNA3.1 (+)-BmCPI/BmGAPDH was constructed.The recombinant plasmids were screened and identified by digestion with restriction enzyme.BALB/c mice were injected intramuscularly with a dosage of 100 μg purified recombinant plasmid DNA with GpG oligodeoxynucleotide (CpG ODN) and two same doses were administrated at 2-week intervals.pcDNA3.1 (+) and phosphate buffered solution (PBS) were used as controls.The tissue of muscles at 4 weeks after the third injection was collected and the target gene was detected by reverse transcription-polymerase chain reaction (RTPCR).Two weeks after the third immunization,the stimulation index (SI) of spleen lymphocytes of immunized mice was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method and the serum levels of interleukin (IL)-4 and interferon (IFN)-γ were detected by enzyme-linked immunosorbent assay (ELISA).The data were analyzed by t test.ResultsBmCPI/BmGAPDH gene in the injected muscle of the immunized mice was detected by RT-PCR. At 6 weeks after immunization,the SIot spleen T lymphocytes in pcDNA3.1 (+)-BmCPI/CpG group and pcDNA3.1 (+)-BmCPI/BmGAPDH/CpG group were 1.466 ± 0.635 and 1.610 ± 0.112,respectively,which were both higher than PBS group and pcDNA3.1( +)-CpG group (1.004 ± 0.019 and 1.078 ± 0.129,respectively) (t=64.438,45.318,42.749 and 34.314,respectively; all P＜0.05).At 4 weeks after immunization,the serum levels of IL-4 and IFN-γ of mice in pcDNA3.1 ( + )-BmCPI/BmGAPDH/ CpG group were significantly higher than those in pcDNA3.1 (+)-CpG group (t=288.053 and 76.453,respectively; both P＜0.05),while the serum level of IFN-γ was also higher than that in pcDNA3.1 (+)-BmCPI/CpG group (t=129.642,P＜0.05). ConclusionThe recombinant eukaryotic plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH could be expressed in mice,and could elicit specific cellular immune responses in immunized mice.

Pathology of lymphatic filariasis

Developing and adult worms of the human lymphatic filarial parasites (Wuchereria bancrofti, Brugia malayi, and Brugia timori) are located mainly in the lymphatic system and occasionally in aberrant sites like subcutaneous and conjunctival cysts. Lymphatic pathology ranging from dilatation of lymphatic channels and lymphangiectasia are detected on ultrasonography in apparently healthy, amicrofilaraemic, but filarial antigen positive individuals in endemic areas. Microfilariae are distributed in various organs and may be associated with immune mediated pathology at these sites; tropical pulmonary eosinophilia is characterized by intense immune mediated destruction of microfilariae in the lung parenchyma. In the spleen and other sites, nodular granulomatous lesions can occur where microfilariae are trapped and destroyed. The finding of Wolbachia endosymbionts in all stages of lymphatic filarial parasites has provided new insight on the adverse reactions associated with anti-filarial chemotherapy. Inflammatory molecules mainly lipopolysaccharide (LPS)-like molecules released from endosymbionts on death of the parasites are largely responsible for the adverse reactions encountered during anti-filarial chemotherapy. Prenatal tolerance or sensitization to parasite derived molecules can immune-modulate and contribute to both pathology and susceptibility/resistance to infection. Pathological responses thus depend not only on exposure to filarial antigens/infection, but also on host-parasiteendosymbiont factors and to intervention with antifilarial treatment. Treatment induced or host mediated death of parasites are associated with various grades of inflammatory response, in which eosinophils and LPS from endosymbionts play prominent roles, leading to death of the parasite, granulomatous formation, organization and fibrosis. The non-human primate (Presbytis spp.) model of Brugia malayi developed for the tertiary screening of anti-filarial compounds has provided unique opportunities for the longitudinal study of the pathology associated with lymphatic filariasis. The pathology in this non-human primate model closely follows that seen in human lymphatic filarial infections and correlates with clinical evidence of lymphatic pathology as detected with ultrasonography. These studies also show that successful treatment as detected by loss of motility and calcification of worms on ultrasonography is associated with reversal of early dilatations of lymphatic channels.

Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells / 中国地方病学杂志

Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and cysteine protease inhibitor ( CPI ) gene from periodic Brugia malayi (Bm) and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 (+). pcDNA3.1 (+)/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE). Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass (Mr) of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell.