RESUMEN
The causative agent of toxoplasmosis, Toxoplasma gondii (T. gondii), is able to influence the health of humans and other vertebrates. Toxoplasma may cause severe illness in the fetus and immunocompromised individuals. The high incidence and intense damages of Toxoplasma infection clearly shows the need to achieve the safe and suitable vaccine. In this study, an immunoinformatics approach was employed to design a multi-epitope DNA vaccine encoding the T. gondii SAG1, SAG3 and SAG5. The bioinformatic outputs supported the immunogenic and non-allergic natures of multi-epitope vaccine. Thereafter, the protective efficacy of the vaccine was evaluated with/without CpG-ODN adjuvant in a laboratory animal model. BALB/c mice were immunized subcutaneously with multi-epitope DNA vaccine. The in vivo findings indicated that the multi-epitope DNA vaccine elicited significant production of IgG antibodies (472.90 ± 2.74 ng/ml) as well as IFN-γ (173.71 ± 26.39 pg/ml) (p < 0.001). Moreover, a significant reduced parasite-burden (17,470 per mg of spleen) and prolonged survival time (9 days) were observed in the immunized groups compared to the controls (p < 0.05). The low values of IL-4 (22.5 ± 0.16 pg/ml) were detected in vaccinated mice compared to the control (PBS) (p > 0.05). In addition, CpG-ODN as an adjuvant increased the immune efficacy of the multi-epitope DNA vaccine. In multi-epitope vaccine+CpG-ODN group, the values of IgG antibodies (535.90 ±7.29 ng/ml) and IFN-γ (358.21 ± 32.70 pg/ml) were significanly higher than the multi-epitope vaccine group. Meanwhile, an increased survival time (10 days) and fewer parasite load (15,485 per mg of spleen) were observed in multi-epitope vaccine+CpG-ODN group. The results revealed that the DNA vaccine containing epitopes of SAG1, SAG3 and SAG5 adjuvanted with CpG-ODN might be a new model for further investigations against acute T. gondii infection.
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Adyuvantes Inmunológicos/uso terapéutico , Antígenos de Protozoos/inmunología , Epítopos/inmunología , Oligodesoxirribonucleótidos/uso terapéutico , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Vacunas de ADN/inmunología , Animales , Antígenos de Protozoos/genética , Femenino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Toxoplasmosis/prevención & controlRESUMEN
Rhoptry proteins (ROPs) are involved in the cell invasion and parasitophorous vacuole (PV) formation and also vital for survival of Toxoplasma gondii (T. gondii) within host cells. ROP8 have a main role during the early phase of infection and can express in tachyzoite and bradyzoite forms. In the present study, we designed a novel multi-epitope DNA vaccine encoding the potential B and T-cell epitopes from ROP8 protein to evaluate the immunity and protective efficacy against acute T. gondii infection in BALB/c mice. For this purpose, several bioinformatics online servers were used. At first, the potential epitopes were selected for T and B cells using immune epitope database (IEDB) and BCPREDS online services. Then, the selected epitopes were fused together by SAPGTP linker. Finally, the physico-chemical features, secondary and tertiary structures, antigenicity, and allergenicity of the peptide were evaluated through different bioinformatics tools. Lastly, the multi-epitope peptide was successfully cloned into pcDNA3.1 expression vector. The DNA vaccine was subcutaneously injected into BALB/c mice and the immune responses of the vaccinated mice and controls were determined. The obtained results revealed that the multi-epitope ROP8 peptide has 183 amino acid residues with average molecular weight (MW) of 18.974 kDa. More than 98 % residues of the peptide were incorporated in favored and allowed regions of the Ramachandran plot. The antigenicity of multi-epitope peptide were estimated 0.8751 and 0.7649 by ANTIGENpro and VaxiJen servers, respectively. BALB/c mice immunized with DNA vaccine showed significantly increased the level of specific anti-T. gondii antibodies (P < 0.05), and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a TH1-type cellular immune response with production of IFN-γ and prolonged survival time, compared with the control groups (P < 0.05). The findings revealed that the multi-epitope ROP8 DNA vaccine induced strong humoral and cellular responses and prolonged the survival time in BALB/c mice, suggesting selection of potential epitopes may be a promising strategy for the design of multi-epitope-based vaccines.
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Linfocitos B/inmunología , Epítopos/inmunología , Vacunas Antiprotozoos/inmunología , Linfocitos T/inmunología , Toxoplasma/inmunología , Toxoplasmosis/prevención & control , Vacunas de ADN/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos B/metabolismo , Modelos Animales de Enfermedad , Mapeo Epitopo , Femenino , Antígenos H-2/inmunología , Inmunización , Ratones , Oligopéptidos/química , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Vacunas Antiprotozoos/administración & dosificación , Linfocitos T/metabolismo , Toxoplasmosis/parasitología , Vacunación , Vacunas de ADN/administración & dosificaciónRESUMEN
BACKGROUND: In this research, the effect of morphine on promastigotes and amastigotes of Leishmania major has been investigated in the presence of nalmefene as a blocking opioid drug and imiquimod as an opioid growth factor receptor. METHODS: This study was conducted at Tarbiat Modares University, Tehran, Iran in 2015-2018. Morphine with different concentration (0.1, 1, 10 and 100 1µg/ml) alone and with imiquimod (0.01, 0.1 and 1µg/ml) and nalmefene (0.1, 1 and 10 µg/ml) on promastigotes and amastigotes in macrophages and also the percentage of infected macrophages was investigated. For evaluation of the apoptosis, we used flow cytometry method. The effect of imiquimod and nalmefene on glucantime and amphotericin B as current drugs for treatment of leishmaniasis was evaluated too. RESULTS: The effect of morphine on promastigotes and amastigotes has a reverse relationship with its concentration. The results of flow cytometry for drug-treated promastigotes revealed that apoptosis and necrosis did not increase markedly relative to the control group. A combination of morphine and imiquimod in concentrations of 0.05, 5 and 5 µg/ml had a pronounced effect on reduction and prevention of macrophage infection with amastigotes. Morphine at a concentration of 0.1 µg/ml plays the role of adjunctive treatment. In amastigote assay we found the better results in group that get glucantime 25 µg/ml+ imiquimod 0.5 µg/ml. CONCLUSION: This effect is strengthened with imiquimod and weakened with nalmefene. Using high dose morphine and nalmefene had reverse effects. They suppress immune system and had no controlling effect in macrophages amastigote infection and reduction of promastigotes.
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In this study we examined enhancement effects of Artemisinin plus Glucantime and shark cartilage extract on promastigotes and amastigotes of L.infantum in in-vitro condition.The toxicity of artemisinin, glucantime, and shark cartilage extract on the L. infantum promastigotes and amastigote-infected macrophages was evaluated using MTT assay. The role of these drugs inducing apoptosis in promastigotes, un- infected, and parasite- infected macrophages was also studied. Using promastigote assay, IC50 values of artemisinin and glucantime as standalone drugs as well as in combination were obtained to be 50, 400, and 100µg/mL respectively. The flow cytometry analysis of apoptotic promastigotes stained with Annexin-V FITC staining showed that artemisinin, glucantime, artemisinin plus glucantime, artemisinin plus shark cartilage extract, and shark cartilage extract alone applied at their IC50 concentrations resulted in 53.5%, 73.92%, 64.46%, 49.9%, and 47.34% apoptosis respectively. The results of MTT assay indicated that cytotoxicity of artemisinin, glucantime, artemisinin plus glucantime, shark cartilage plus artemisinin, and shark cartilage in infected macrophages after 72h was 75%, 84%, 82%, 30%, and 3% respectively. In un- infected macrophages, cytotoxicity of Artemisinin, Glucantime, Artemisinin plus Glucantime and shark cartilage was 15%, 31%, 21%, 2%, and 0% respectively.This study suggests that artemisinin, glucantime, artemisinin plus glucantime, and shark cartilage extract have significant killing effects on promastigotes and amastigotes. Also, it proved that artimisinin alone and in combination with glucantime and shark cartilage extract has little toxic effect on macrophages, but could induce apoptosis in L.infantum promastigotes and amastigote-infected macrophages. Thus, these chemicals can be used as alternative drugs for in-vivo studies.
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Rodents and stray cats are the sources of many parasitic infections including T. gondii, for other animals and human. Toxoplasmosis has a wide range of laboratory factors in its intermediate and definite hosts. Regarding the importance of rodents and stray cats as the hosts that spread the Toxoplasma gondii, it is necessary to obtain comprehensive information about these animals in the life cycle of T. gondii. The objective was to investigate the new prevalence of toxoplasmosis among target animals in Iran, using GRA6 gene in combinacion with ELISA avidity. In this study, 286 rodents and 210 stray cats were collected and their heart tissues extracted to obtain DNA, blood samples and IgG Ab of T.gondii parasite. We detected the positive tissue samples in our study by the nested-PCR method. Then, we examined T. gondii IgG ELISA avidity for assessment of toxoplasmosis among rodents and stray cats. This study, was conducted in January to March 2017, based on the prevalence study. The findings revealed that 246/286 (86.01%) of rodents and 180/210 (85.71%) of stray cats were positive by IgG ELISA avidity methods. moreover, 68 rodents samples and 38 stray cats samples were positive concerning the GRA6 Toxoplasma gene; and these positive samples were at intermediate levels for IgG avidity. We concluded that the new prevalence of toxoplasmosis among rodents and stray cats was at high levels, using the serologic method in Northeast of Iran and the results of quantitative ELISA avidity were as the same as those of the nested-PCR for detecting recent toxoplasmosis in these hosts.
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Toxoplasmosis is a cosmopolitan zoonotic infection, caused by a unicellular protozoan parasite known as Toxoplasma gondii that belongs to the phylum Apicomplexa. It is estimated that over one-third of the world's population has been exposed and are latently infected with the parasite. In humans, toxoplasmosis is predominantly asymptomatic in immunocompetent persons, while among immunocompromised individuals may be cause severe and progressive complications with poor prognosis. Moreover, seronegative pregnant mothers are other risk groups for acquiring the infection. The life cycle of T. gondii is very complex, indicating the presence of a plurality of antigenic epitopes. Despite of great advances, recognize and construct novel vaccines for prevent and control of toxoplasmosis in both humans and animals is still remains a great challenge for researchers to select potential protein sequences as the ideal antigens. Notably, in several past years, constant efforts of researchers have made considerable advances to elucidate the different aspects of the cell and molecular biology of T. gondii mainly on microneme antigens, dense granule antigens, surface antigens, and rhoptry proteins (ROP). These attempts thereby provided great impetus to the present focus on vaccine development, according to the defined subcellular components of the parasite. Although, currently there is no commercial vaccine for use in humans. Among the main identified T. gondii antigens, ROPs appear as a putative vaccine candidate that are vital for invasion procedure as well as survival within host cells. Overall, it is estimated that they occupy about 1%-30% of the total parasite cell volume. In this review, we have summarized the recent progress of ROP-based vaccine development through various strategies from DNA vaccines, epitope or multi epitope-based vaccines, recombinant protein vaccines to vaccines based on live-attenuated vectors and prime-boost strategies in different mouse models.
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Microsporidia are eukaryotic, intracellular obligate parasites that widely involve many organisms including insects, fish, birds, and mammals. One of the genera of Microsporidia is Encephalitozoon, which contains several opportunistic pathogens. Since Encephalitozoon spp. are zoonotic and opportunistic pathogens, it is important to find their reservoir hosts; hence, the current study aimed at isolating and identifying Encephalitozoon spp. in the crows by the light microscopy observations and molecular methods. For this purpose, 36 samples were collected by the dropping method; however, due to the low volume of samples, the total samples were collected in a sterile stool container and the polymerase chain reaction (PCR) was performed to detect Encephalitozoon spp. Accordingly, 300-bp bands, specific to Encephalitozoon spp., were observed and by sequencing E. intestinalis was identified. Crows can be considered as the hosts of E. intestinalis.
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The parasites of genus Leishmania are the causative agents of one of the most widespread and devastating diseases. According to follow-up data, these medications may provoke adverse drug reactions, drug resistance, relapse, as well as financial burden. The mechanism of action of opioid drugs is primarily exerted via transmembrane G-protein coupled receptors. One of the potent synthetic immunomodulator agents is imiquimod with low molecular weight and unknown mechanism of action. Monocyte and macrophage are the primary site of action for imiquimod. Nalmefene is a well-known opioid antagonist agent which simultaneously inhibits these receptors and augments intracellular pathogenicity, hence providing opportunities to investigate their function. The aim of present work was evaluating the effect of morphine, imiquimod and nalmephen on the Leishmania major and investigating cytotoxic effect this drug on the uninfected macrophage and infected macrophage for detected early apoptosis, necrosis and secondry apoptosis by flowcytometry method. In this study we used morphine, imiquimod, nalmefene, and Glucantime. We treated promastigotes, macrophages, and infected macrophages with above drugs, and the apoptosis evaluated by flow cytometry. The results showed that in all concentration of morphine more than 98% of promastigotes remained alive that it is deduced that morphine lacks any lethal effect on L. major after 24 h, whereas in groups treated with Glucantime alone or in combination with Nalmephene and Imiquimod, 84.13%, 88.96% and 86.72% of promastigotes were alive, respectively. The results of macrophage treatment with morphine, imiquimod, and nalmefene demonstrated that most necrosis has occurred in nalmefene group (6.54%).
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Rhoptry proteins (ROPs) are involved in the different stages of Toxoplasma gondii (T. gondii) invasion and are also critical for survival within host cells. ROP8 is expressed in the early stages of infection and have a key role in the parasitophorous vacuole (PV) formation. In this paper, we have combined several bioinformatics online servers for immunogenicity prediction of ROP8 protein. In this study, several bioinformatics approaches were used to analyze the different aspects of ROP8 protein, including the physico-chemical properties, transmembrane domain, subcellular localization, secondary and tertiary structure, B and T-cell potential epitopes, and other important characteristics of this protein. The findings showed that ROP8 protein had 60 potential post-translational modification sites. Also, only one transmembrane domain was recognized for this protein. The secondary structure of ROP8 protein comprises 33.04% alpha-helix, 18.26% extended strand, and 48.70% random coil. Moreover, several potential B and T-cell epitopes were identified for ROP8. In addition, the obtained findings from antigenicity and allergenicity evaluation remarked that this protein is immunogenic and non-allergen. Based on the results of Ramachandran plot, 94.8%, 4.1%, and 1.1% of amino acid residues were incorporated in the favored, allowed, and outlier regions, respectively. This paper provides a foundation for further investigations, and laid a theoretical basis for the development of an appropriate vaccine against toxoplasmosis. More studies are needed experimentally using the ROP8 alone or in combination with other antigens in the future.
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Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/prevención & control , Secuencia de Aminoácidos , Antígenos , Biología Computacional , Epítopos , Humanos , Conformación ProteicaRESUMEN
BACKGROUND: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, GRA7 plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice. METHODS: In this study, 733 bp of GRA7 gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containing GRA7 and ROP2 genes were administered via IM according to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis. RESULTS: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-γ levels were significantly higher than controls (p<0.05), whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group (p<0.01). In contrast, IgG1 antibodies were similar between the two groups (p>0.5). So, survival time in the immune groups was significantly prolonged in comparison to control ones (p<0.01). CONCLUSION: The immunized mice by DNA vaccine produce higher titration of IFNγ, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktail GRA7/ROP2 is a potential vaccine candidate against toxoplasmosis.
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BACKGROUND: In leishmaniasis, some drugs prescribed for treatment have toxic effects and there are reports about drug resistance in some countries. Due to this fact, using herbal drugs such as artemisinin with good efficacy and low toxic effect might be suitable. METHODS: We evaluated the apoptotic effect of artemisinin on Leishmania major in vitro and the antileishmanial activities of artemisinin on leishmaniasis in BALB/c mice and at the end INF-γ and IL-4 cytokines levels were detected by ELISA in spleen cell culture supernatants. During treatment the lesion size and survival rate were measured each four and ten days, respectively. RESULTS: Percentage of early and late apoptosis in promastigotes of control group and promastigotes treated with 10, 25, 50 and 100 µg/ml of artemisinin after 48 h were 0.13, 16.04, 41.23, 49.03 and 81.83, respectively. The IFN-γ in ointment treated group were higher than those of other groups (P<0.05). The in vivo results showed that ointment compounds healed the lesions more effectively rather than intraperitoneal injection method (P<0.05). The survival rate of mice 150 days after challenge in treated group with ointment of artemisinin was 66% while all mice in control groups were died. CONCLUSION: All of in vitro results represented that this drug had antileishmanial effects and these results were confirmed by evaluation effects in vivo condition of leishmaniasis. Interestingly, according to these results it can be concluded that this drug has antileishmanial effects in vitro and in vivo conditions. Artemisinin induces cytotoxic effect on L. major via apoptosis-related mechanism.
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BACKGROUND: Severe and fatal complications of toxoplasmosis urge development of effective vaccines against the disease. The current study was performed to evaluate cocktail DNA vaccine containing plasmids encoding GRA5, SAG1, and ROP2 genes of Toxoplasma gondii in BALB/c mice in Tarbiat Modares University in 2012. METHODS: The plasmids containing complete GRA5, SAG1, and ROP2 genes were mass extracted and then the recombinant plasmids were administered via intramuscular injections according to immunized mice three times with three-week intervals. Then splenocytes were cultured, and proliferation as well as cytokine assays were carried out. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis. RESULTS: The results of cytokine assay for INF-γ were higher in the mice that received the cocktail DNA containing recombinant plasmids. Evaluation of proliferation of splenocytes using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay indicated induction of cellular response. Measurement of total IgG and the isotypes of IgG1 and IgG2a showed that the cocktail DNA stimulated IgG and IgG2a production in comparison with the control groups (P<0.05). Furthermore, the survival rate of mice in the groups that received the cocktail DNA was significantly higher than that in the control groups (P<0.05). CONCLUSION: Administration of the cocktail DNA vaccine led to production of higher levels of IFN-γ, confirmed by secretion of IgG2a, and the immune response was shifted toward Th1. Thus, the cocktail DNA containing the recombinant plasmids can be an appropriate candidate for immunization against toxoplasmosis.
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BACKGROUND: Cutaneous leishmaniasis is a health problem in the world. Lesions should be treated on cosmetically or functionally important sites, such as the face and hands. Cantharidin is a terpenoid compound produced naturally by beetles of Meloidae and Oedemeridae families. OBJECTIVES: The current study aimed to investigate the effect of cantharidin on Cutaneous Leishmaniasis (CL) lesions and IFN-γ and IL-4 patterns in infected BALB/c mice. MATERIALS AND METHODS: INFECTED BALB/C MICE WERE DIVIDED INTO FIVE GROUPS AS: untreated (control group), eucerin-treated and 0.05%, 0.1% and 0.5% cantharidin-treated. Lesions diameter was measured by Vernier caliper every three days for four weeks. Cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA) using U-CyTech kit. RESULTS: The results indicated that treatment with cantharidin exacerbates lesions compared with the controls, except for 0.05% cantharidin dose that restrained lesion growth significantly. Interferon gamma level in cantharidin-treated groups was significantly less than that of the control group. But interlukin-4 level was similar among the groups. CONCLUSIONS: The current study results indicated that high doses of cantharidin exacerbates leishmaniasis lesion, but low dose of cantharidin inhibits lesion growth.
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In the present study, the effect of IL-22 together with the plasmid encoding LACK (Leishmania homolog of receptors for activated C-kinase) gene of Leishmania major on the trend of leishmaniasis in BALB/c mice was evaluated. Evaluation of the cellular and humoral immunity was performed by measurement of IL-4 and IFN-γ, culture of splenocytes and MTT assay, and measurement of total IgG, IgG1, and IgG2a in the control and immunized groups. Clinical evaluations were also carried out by measurement of the lesion size, survival rate, and body weight of mice. Comparison of the mean size of lesions in the LACK and LACK+IL-22 groups demonstrated that the mean size of lesions of the two groups was significantly different from week four (p<0.05). The survival rate at day 170 after challenge for the PBS, pcDNA3 (empty plasmid), pcLACK (pcDNA3 containing LACK gene), and pcLACK+IL-22 groups were 20%, 40%, 60%, and 80%, respectively. According to the results of IFN-γ, IL-4, total IgG, IgG1, and IgG2a measurement and the MTT assay, IL-22 obviously caused an increase in IFN-γ production and a decrease in IL-4 production before and after the challenge (p<0.05). The results showed the effectiveness of IL-22 in DNA vaccine. It showed that IL-22 brought about Th1 cytokine responses and high survival rate of mice.
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Antígenos de Protozoos/genética , Interleucinas/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Peso Corporal , Femenino , Inmunoglobulina G/sangre , Interferón gamma/análisis , Interleucina-4/análisis , Leishmania major/enzimología , Leishmania major/genética , Leishmaniasis Cutánea/mortalidad , Leishmaniasis Cutánea/patología , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/genética , Bazo/citología , Bazo/inmunología , Tasa de Supervivencia , Vacunas de ADN/genética , Interleucina-22RESUMEN
BACKGROUND: It is necessary to develop novel, affordable, and accessible drugs with few side effects as alternatives of the currently available chemical agents for leishmaniasis. OBJECTIVES: The main purpose of this study was to evaluate the effects of these drugs on L. major under in vitro conditions. MATERIALS AND METHODS: In the current study, 5, 10, 25, 50, and 100 µg/mL concentrations of aqueous extract of Artemisia sieberi and chemical artemisinin were tested on promastigotes of Leishmania major (L. major), uninfected macrophages, and infected macrophages with intracellular amastigotes of L. major, by direct counting and 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromid methods. RESULTS: The results obtained for each drug were compared with other drugs and also with the results of the control groups. The results related to promastigote and amastigote assays showed that when the dose of both drugs increased, the parasite number is reduced in comparison with the control groups. Moreover, the parasitic burden in the test cultures decreased significantly. Macrophage assay results showed that the effects of both drugs on uninfected and healthy macrophages were very low. CONCLUSIONS: These results indicate that both drugs have anti-Leishmania effects, which was higher in Artemisia sieberi compared with artemisinin. Thus, carrying out further studies on the effects of Artemisia sieberi in infected animals with L. major is recommended.
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Various methods are available for enhancing the potency of DNA vaccines, including employment of different forms of adjuvant. The current study was carried out to evaluate and compare the effects of genetic and non-genetic adjuvants on the immune response stimulated by DNA vaccine. Thus, two adjuvants, IL-12 (genetic adjuvant) and aluminum hydroxide (alum, non-genetic adjuvant), were used with cocktail DNA vaccine containing plasmids encoding complete rhoptry antigen 2 (ROP-2) and surface major antigen 1 (SAG-1) of Toxoplasma gondii. The efficacy of pcROP2+pcSAG1 in stimulation of the immune response against toxoplasmosis with and without adjuvant was evaluated in female BALB/c mice by measuring the level of total IgG antibody and cytokines. The results obtained indicated that after challenging the mice with the fatal RH strain of T. gondii, the survival rates of mice immunized with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 were significantly greater than that of the control groups (p<0.05). Moreover, measurement of total IgG antibody indicated the significant difference between the control and experimental groups (p<0.05). Finally, the results obtained by measurement of cytokines (IFN-γ and IL-4) showed high levels of IFN-γ and low levels of IL-4 in groups vaccinated with pcROP2+pcSAG1 (DNA cocktail), pcSAG1+pcROP2+alum, and pcSAG1+pcROP2+IL-12 as the experiment groups, in comparison with the controls groups (PBS, pc-DNA3, alum+PBS, and pCAGGS-IL-12+pcDNA3). The results of the study showed that use of adjuvants (IL-12 and alum) coincident with DNA cocktail leads to significant change in the survival rates of the experiment groups in comparison with control groups. Also, there is no significant difference between adjuvants to induce immune responses.