In vitro and In vivo Effects of Ethanolic Extract of Fumaria parviflora Lam. Embedded in Chitosan Nanoparticles Against Leishmania major.

INTRODUCTION: Fumaria has been traditionally used to treat skin damages due to anti-inflammatory properties. In the present study, we evaluated the effect of the ethanolic extract of Fumaria parviflora Lam. (F. parviflora) against Leishmania major (L. major) using chitosan biopolymer drug delivery system both In vitro and In vivo models. MATERIALS AND METHODS: The ethanolic extract of F. parviflora was analyzed by HPLC to determine its active ingredients content. The extract was then loaded on chitosan nanoparticles (CNPs). The parasite was treated with various concentrations of the ethanolic extract, CNPs and CNPs loaded with F. parviflora extract (CNPs@ F. parviflora). The size of lesions of treated mice were measured on a weekly basis. The parasite burden was evaluated 8 weeks after treatment. RESULTS: The HPLC analysis showed the presence of Fumaric acid at a high concentration. The percentage of the drug released from CNPs@ F. parviflora within 24 and 72 h were 65% and 90% respectively. The results showed that F. parviflora extract and CNPs@ F. parviflora caused 84% and 96% growth inhibition of L. major promastigotes as revealed by Neubauer chamber counting and MTT test respectively. The IC50 values of F. parviflora extract and CNPs@ F. parviflora were 450 and 68.4 µg/ml respectively. In amastigote assay, the best results showed in CNPs@ F. parviflora that only 2% of macrophages were infected with amastigotes. In vivo experiments for mice treated with F. parviflora and CNPs @ F. parviflora in comparison to control group showed a significant reduction (P < 0.05) in the mean diameter of the lesions (2.3 and 1.72 mm and 9.91 mm respectively). CONCLUSION: The ethanolic extract of F. parviflora both as standalone and loaded in CNPs showed promising inhibitory effects against L. major both upon In vitro and In vivo experimentation as well as therapeutic effects for wound healing in infected mice.

Immune system roles in pathogenesis, prognosis, control, and treatment of Toxoplasma gondii infection.

Toxoplasma gondii is the protozoan causative agent of toxoplasmosis in humans and warm-blooded animals. Recent studies have illustrated that the immune system plays a pivotal role in the pathogenesis of toxoplasmosis by triggering immune cytokines like IL-12, TNF-α, and IFN-γ and immune cells like DCs, Th1, and Th17. On the other hand, some immune components can serve as prognosis markers of toxoplasmosis. In healthy people, the disease is often asymptomatic, but immunocompromised people and newborns may suffer severe symptoms and complications. Therefore, the immune prognostic markers may provide tools to measure the disease progress and help patients to avoid further complications. Immunotherapies using monoclonal antibody, cytokines, immune cells, exosomes, novel vaccines, and anti-inflammatory molecules open new horizon for toxoplasmosis treatment. In this review article, we discussed the immunopathogenesis, prognosis, and immunotherapy of Toxoplasma gondii infection.

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model.

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC50) values were 21.66 µg/ml, 43.25 60 µg/ml, 52.58 µg/ml, and 70.38 µg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC50 for positive control (glucantime) was 27.03 µg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 µg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

Molecular detection and characterisation of Theileria in hard ticks of small ruminants in Zarrin Dasht County, Southern Iran.

BACKGROUND: Ticks are important ectoparasites of small ruminants in tropics and subtropics including Iran. They transmit serious zoonotic pathogens such as Babesia and Theileria. These parasites cause major burden on small ruminants jeopardising livelihoods of rural people in Zarrin Dasht County. OBJECTIVES: This study was carried out to investigate the diversity and distribution of hard ticks of small ruminants and their piroplasm infection in a bid to contribute to Theileria and/or Babesia detection and control in Zarrin Dasht County of Fars province, Iran. METHODS: We examined 751 sheep and goats from 10 sites of the County during four seasons for hard tick infestation. The collected hard ticks (994) were taxonomically identified before being separately confined in microtubes coded to indicate their species and host animals as well as site and date of collection. In total 50 pooled samples were analysed by PCR technique for Theileria and Babesia infection. RESULTS: The identified ticks included Hyalomma marginatum 994/362); 36.4%), Rhipicephalus turanicus 994/352); 35.51%), Hyalomma anatolicum 994/264); 26.6%), Hyalomma dromedarii 994/14); 1.41%) and Hyalomma asiaticum 994/2) 0.2%). Molecular analyses showed that 7 out of 50 pooled sample were infected with piroplasm genome in ticks shared by Theileria ovis (6:50) and Theileria lestoquardi (1:50). Babesia was absent in collected hard ticks. CONCLUSIONS: This is the first report on the presence of piroplasm infection in hard ticks of small ruminants in Zarrin Dasht County. Theileria ovis was more prevalent than Theileria lestoquardi but Babesia was absent. Piroplasm infection was detected in Hyalomma marginatum, Hyalomma anatolicum and Rhipicephalus turanicus. Hyalomma marginatum appears to be more competent to vector Theileria spp. This study may contribute to risk assessment and prevention of epizootic theileriosis in the County.

Therapeutic effects of Lucilia sericata larval excretion/secretion products on Leishmania major under in vitro and in vivo conditions.

BACKGROUND: Leishmaniasis is a neglected infectious disease caused by protozoa of the genus Leishmania. The disease generally manifests as characteristic skin lesions which require lengthy treatment with antimonial drugs that are often associated with adverse side effects. Therefore, a number of studies have focused on natural compounds as promising drugs for its treatment. This study aimed to evaluate the effects of larval excretion/secretion products (ES) of Lucilia sericata in crude and fractionated forms on Leishmania major, by using in vitro and in vivo models. METHODS: The in vitro experiments involved evaluation of ES on both promastigotes and macrophage-engulfed amastigotes, whereas the in vivo experiments included comparative treatments of skin lesions in L. major-infected mice with Eucerin-formulated ES and Glucantime. RESULTS: The half maximal inhibitory concentrations of the crude ES, > 10-kDa ES fraction, < 10-kDa ES fraction, and Glucantime were 38.7 µg/ml, 47.6 µg/ml, 63.3 µg/ml, and 29.1 µg/ml, respectively. Significant differences were observed between percentage viabilities of promastigotes treated with the crude ES and its fractions compared with the negative control (P < 0.0001). The crude ES was more effective on amastigotes than the two ES fractions at 300 µg/ml. The macroscopic measurements revealed that the reduction of lesion size in mice treated with the crude ES followed quicker cascades of healing than that of mice treated with Glucantime and the ES fractions. CONCLUSIONS: The present study showed that the larval ES of L. sericata in both crude and fractionated forms are effective for both intracellular and extracellular forms of L. major. Also, the ES exert both topical and systemic effects on mice experimentally infected with L. major.

In vitro and in vivo effects of natural honey on Leishmania major.

Leishmaniosis is an insect-borne disease whose clinical manifestations range from skin ulcer to visceral disease. Antimony compounds are currently known to be the main treatment for leishmaniosis, but there are limitations to their use. This study was performed to determine the in vitro and in vivo efficiency of honey on a standard strain of Leishmania major parasite in comparison with glucantime and amphotericin as the first line treatment. Leishmania major was exposed to different concentrations of honey extract at 400, 200, 100, 50, 25, 12.5, 6.25 µg/ml. The effectiveness of honey concentrations was determined by counting the parasite by Neubauer's chamber. Then, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric method, for promastigotes and macrophages then IC50 was calculated. A flow cytometry test was performed and necrosis and apoptosis diagrams were drawn. Next, the effect of the honey on the amastigotes inside macrophage cells was investigated. Finally, for the in vivo experimentation, the parasite was injected in the base of BALB/c mice tails and the resulting wounds were treated with honey. The results of all tests showed that the honey extract at 400 µg/ml concentration had the best effects on all stages. The honey has lethal effects on Leishmania parasite in vitro as well as therapeutic effects on wounds caused by the parasite. Further experiments are recommended to evaluate the performance of the extract on the parasite in volunteer human models.

Synthesis of Tellurium Oxide (TeO2) Nanorods and Nanoflakes and Evaluation of Its Efficacy Against Leishmania major In Vitro and In Vivo.

PURPOSE: Today, the use of natural products and nanostructures has increased. Given the reports on beneficial effects of various organotellurane compounds on types of visceral leishmaniasis, we decided to investigate the effect of TeO2 NPs on Leishmania major (L. major). Tellurium can cause cell apoptosis in cancer cells without activating the caspase-pathway. METHODS: TeO2 NPs at first synthesized and the structure was checked by XRD, SEM and EDS tests. The cytotoxic effect of TeO2 NPs against L. major promastigotes, amastigotes and macrophages was assessed by MTT test or counting. The possible apoptosis of L. major by TeO2 NPs was evaluated by flow cytometry test. For in vivo assay, the lesions of infected BALB/c mice with L. major promastigotes were treated with TeO2 NPs, then the lesion size and survival rate were evaluated. RESULTS: The synthesis of TeO2 with tetragonal structure was confirmed by XRD. The combination of nanorods and nanoflakes and the presence of Te were proven by SEM and EDS, respectively. According the effects of nanoparticle on promastigotes and amastigotes, the IC50 values of TeO2 after 72 h of incubation were 15.13 and 52.22 µg/ml, respectively. TeO2 NPs induced apoptosis in about 41% of promastigotes. The ulcer greatly healed and survival rate was higher in treated mice compared to those in control group. CONCLUSION: Based on the data, favorable anti-leishmanial properties were observed by using TeO2 NPs. TeO2 NPs have cytotoxic impacts on L. major promastigotes and amastigotes in vitro and in vivo and may be regarded as a therapy option.

A study on the in vitro and in vivo effects of aqueous and alcoholic extracts of Salvia mirzayanii on Leishmania major.

Salvia mirzayanii contains anti-hyperglycemic, antimicrobial, antioxidant, anti-inflammatory, and neuroprotective effects. The purpose of this study was to evaluate the anti-leishmanial efficacy of aqueous and alcoholic extracts of S. mirzayanii (both in vitro and in vivo) against Leishmania major. Aqueous and alcoholic extracts of S. mirzayanii were prepared and tested on L. major promastigotes and amastigotes. MTT test was used to evaluate the cytotoxicity of the plant against L. major. Flow cytometry was performed to assay apoptosis induced by 50 and 100 µg/ml of extracts on the promastigotes and macrophages. For the in vivo assay, the therapeutic effects of aqueous and alcoholic extracts of S. mirzayanii were tested in BALB/c mice. After 72 h, the IC50 value of aqueous and alcoholic extracts of S. mirzayanii against L. major promastigotes was 6.04 and 4.47, respectively. The inhibitory concentration (IC50) of aqueous and alcoholic extracts of S. mirzayanii to amastigotes were determined to be 47.78 µg/ml and 33.58 µg/ml, respectively. Flow cytometry revealed that the apoptosis of promastigotes using 100 µg/ml of aqueous and alcoholic extracts of S. mirzayanii was 5.81% and 5.39%, respectively, while apoptosis induced at 200 µg/ml were 5.09% and 70.71%, respectively. Lesion size was significantly decreased in in vivo experiments, and the survival rate of the treated mice improved in contrast to the control group. Given the efficacy of aqueous and alcoholic extracts of S. mirzayanii on promastigotes both in vitro and in vivo condition, the plant could be considered as a candidate source for the treatment of leishmaniosis.

Toxoplasma gondii in Slaughtered Sheep in High- and Low-Humidity Regions in the South of Iran: Molecular Prevalence and Genotype Identification.

Toxoplasma gondii is one of the most common meat-born zoonoses that infect all warm-blooded animals and humans. Sheep (Ovis aries) is one of the main reservoirs of T. gondii worldwide, and the infections induce various sequels, such as abortion and stillbirth. The present study aimed to identify the effects of humidity on the prevalence of T. gondii in sheep in high- and low-humidity regions. Heart samples from 200 slaughtered sheep (140 samples from a high-humidity region and 60 samples from a low-humidity region) were collected from Hormozgan Province (south of Iran). The samples were tested by nested PCR targeting the RE gene. Genotyping was performed by the PCR-RFLP method using the SAG3 and GRA6 genes. Some isolates were sequenced and recorded in the GenBank. T. gondii DNA was detected in 10.71 percent of the samples from the highly humid region, whereas no positive samples were detected in the low-humidity region. Genotyping revealed that all isolates belonged to the T. gondii type III genotype. Our study indicated that humidity is an important factor for the prevalence of T. gondii in sheep. Additionally, our study also showed the dominance of type III strain of T. gondii in sheep in the south of Iran.

Investigating in Vivo and in Vitro Effects of Ethanolic and Aqueous Extracts of Myrtle (Myrtus communis) on Leishmania major.

BACKGROUND: The extract of myrtle plant contains polyphenolic compounds that show antibacterial, antiviral, and anti-parasitic properties. We aimed to investigate the therapeutic effect of aqueous and ethanolic myrtle extract against leishmaniasis caused by L. major in vivo and in vitro conditions. METHODS: This study was carried out in Tarbiat Modares University, Tehran, Iran in 2018. Aqueous and ethanolic extract of myrtle plant at 6.25 to 400 mg/ml concentrations were tested on Leishmania major promastigotes, non-infected macrophages, and macrophages infected with amastigotes in vitro using counting, MTT and flow cytometry techniques. Then, BALB/c mice were treated with ethanolic, aqueous and a mixture of both extracts of myrtle plant. The treatment was carried out for four weeks. Then, the effectiveness of the herbal medicine was assessed by measuring wounds diameters, mice weights and their mortality rate on weekly basis. RESULTS: The IC50 values of aqueous and ethanolic extracts for promastigotes were 7.86 and 11.66 µg/mL respectively. The IC50 values of the aqueous and ethanolic extracts for amastigotes were 12.5 and 47.2 µg/mL respectively. Flow cytometry indicates 62.88% and 60.16% apoptosis induced by ethanolic and aqueous extract of myrtle plant respectively. The lowest parasitic load was seen in the group treated with ethanolic extract. CONCLUSION: The lesion sizes for treated groups with extracts were similar to those treated with glucantime. Oral administration instead of injection is another advantage of myrtle plant over glucantime, which makes the herb easy and more practical.

In-vitro and in-vivo comparative effects of the spring and autumn-harvested Artemisia aucheri Bioss extracts on Leishmania major.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia aucheri Bioss contains flavonoid, coumarin and santonin with antioxidant, antimicrobial and antileishmanial effects. The current study was aimed to comparatively evaluate the effects of spring and autumn extracts of A. aucheri Bioss on Leishmania major both in-vitro and in-vivo conditions. METHODS: HPLC analysis was used to evaluate the percentages of compounds in spring and autumn extracts of A. aucheri. For in-vitro assay, the effect of different concentrations of spring and autumn extracts of A. aucheri was tested on L. major promastigotes and amastigotes. MTT and flow cytometry methods were used to evaluate the cytotoxicity and probable apoptosis of A. aucheri extracts on L. major promastigotes. On the other hand, for in-vivo assay, the extracts were used as ointments to treat lesions developed on BALB/c mice after 28 days post inoculation of L. major. The diameter of lesions and the survival rates of infected BALB/c mice were measured weekly for a period of two months. RESULTS: The HPLC analysis showed the substance Quercitrin was present in the spring A. aucheri extract but not in the autumn extract. The mean numbers of amastigotes in each treated macrophage with the spring and autumn A. aucheri extracts were 1.2 and 1.8 respectively, which showed statistically significant differences (P < 0.05). Flow cytometry revealed that the spring and autumn A. aucheri extracts caused about 32% and 3.78% apoptosis respectively. The inhibitory concentration (IC50) of spring and autumn A. aucheri extracts to amastigotes were determined to be 90 µg/mL and 183 µg/mL respectiovely. In-vivo, the diameter of lesions treated with the spring A. aucheri extract was significantly less (P < 0.05) compared to those treated with the autumn extract (2.6 and 7.8 mm respectively). Also, mice treated with spring A. aucheri extract had higher survival rates compared to control group. CONCLUSION: Given the above results, it can be concluded that spring A. aucheri extract has a greater fatality effect on L. major promastigotes in-vitro compared to the autum extract. In addition, the spring extract has stronger therapeutic effect on lesions caused by L. major in BALB/c mice than the autum extract.

Determinants of Quality of Life in Children and Adolescents With Hemophilia in Kabul, Afghanistan: First Report.

BACKGROUND: Hemophilia is a rare inherited disorder associated with abnormal repeated bleeding and debilitating joint pain due to deficiency in coagulating factors VIII and IX. This study aimed to provide an updated account on the health-related quality of life (HRQoL) in children with hemophilia in Afghanistan. METHODS: This cross-sectional study included 65 randomly selected hemophiliacs out of 350 children registered with the Afghanistan Hemophilia Patient Association (AHPA). The patients were 8-16 years old and voluntarily entered the study. Data were collected through a demographic questionnaire and a Persian version of Haemo-QoL Questionnaire (short version) for children aged 8-16 years. RESULTS: The patients' age averaged 12.9 ± 3.9 years with a mean QoL score of 75.9 ± 17.4. The patients were suffering from hemophilia A, mostly the severe type (80%). They were born to low income families (95 %) with high illiteracy rates (>50%) and hemophilia family history (90%). Spearman test showed a significant correlation between age and QoL scores (r = 0.8, P = 0.02). One-way ANOVA indicated no significant difference between QoL scores of patients categorized based on hemophilia severity (P = 0.2, F = 1.3), family incomes (P = 0.9, F = 0.01) and parents' levels of education (P = 0.2-0.4, F = 0.82-1.3). The Cronbach alpha for the instrument was 0.82. CONCLUSION: Regardless of hemophilia severity, Family and Sports were the most impaired domains of QoL. Herein, we have presented the first reliable and updated data on hemophiliacs' demographic characteristics and their quality of life in Kabul.

Assessment of DNA vaccine encoding Toxoplasma gondii microneme complete gene and IL-12 as adjuvant in BALB/c mice.

OBJECTIVES: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis in humans and animals. Micronemes (MICs) are effective candidates for DNA vaccine. MATERIALS AND METHODS: In this study, we evaluated the immune response of BALB/c mice against MIC3 gene of Toxoplasma gondii and interleukin 12 (IL-12) as DNA vaccine. The MIC3 gene was cloned into the PTZ57R/T vector before sub-cloning in pcDNA3. Recombinant pc-MIC3 was transformed into Escherichia coli (TOP10 strain). The pc-MIC3 plasmid was then transfected into Chinese Hamster Ovary (CHO) cells, and the expression of the MIC3 gene was evaluated by SDS-PAGE and Western blotting. Sixty female BALB/c mice were divided into 6 groups. Each group received 3 intramuscular immunizations on days 0, 21st and 42nd using one of the following stimulants: phosphate-buffered saline, pcDNA3, pCAGGS-IL12, pc-MIC3 (100 µg), pc-MIC3 (50 µg), or combined pCAGGS-IL12 (50 µg) and pc-MIC3 (50 µg). The enzyme-linked immunosorbent assays was applied to evaluate interferon gamma (IFN-γ) and IL-4 cytokines excretion of lymphocytes stimulated with tachyzoites lysate antigen, as well as the total levels of immunoglobulin G (IgG), IgG2a and IgG1 in immunized mice sera. RESULTS: Our results showed that mice challenged with pc-MIC3 (100 µg) had the highest longevity and quantity of immunoglobulin. Moreover, the highest expression level of IFN-γ was found in mice injected with combined pcMIC3 and pCAGGS-IL12 (P<0.05). CONCLUSION: The MIC3 gene can be an efficient DNA vaccine candidate against toxoplasmosis. While, the single-gene vaccine can confer partial protection to mice against toxoplasmosis, the multigene vaccine can significantly enhance immune responses.

Biodiversity, Leishmania genetic typing and host identification of phlebotomine species in endemic foci of southeastern Iran.

Leishmaniasis is a growing health challenge in many parts of Iran, including Kerman Province. Investigating vector ecology and parasite-harboring capacity is prerequisite to the disease control measures. This study included six provincial sites namely Bam (Bm), Dehbakri (Di), Jiroft (Jt), Mohammad-Abad (Md), Rostam-Abad (Rd) and Darb-e-Behesht (Dt) where sand flies were trapped. The specimens were then identified before being exposed to DNA extraction. PCR-RFLP was used to detect leishmanial infection rates and feeding preference of vectors. Diversity indices indicated that the highest effective numbers of species was in plain sites, whereas, the highest expected numbers of species was in mountainous sites. P. papatasi and P. sergenti showed similar feeding preferences to both human and animal bloods. P. papatasi from indoor catches was found infected with Leishmania major at a 2% rate. The ITS1 gene sequences of isolated parasites were >99% similar to related GenBank haplotypes. Bam and Rostam-Abad remain active foci of both types of cutaneous leishmaniasis (CL). Md and Di are prone to visceral leishmaniasis (VL). Jt is not at risk of anthroponotic cutaneous leishmaniasis (ACL) due to absence of P. sergenti. Sand flies are absent in Dt, probably because of high elevation and cold climate. In conclusion, patterns of climate and ecosystem changes and vector-host-reservoirs interactions must be carefully scrutinized if leishmaniasis is to be controlled in the stricken sites.

Comparative Assessment of Induced Immune Responses Following Intramuscular Immunization with Fusion and Cocktail of LeIF, LACK and TSA Genes Against Cutaneous Leishmaniasis in BALB/c Mice.

In the present study, we evaluated induced immune responses following DNA vaccine containing cocktail or fusion of LeIF, LACK and TSA genes or each gene alone. Mice were injected with 100 µg of each plasmid containing the gene of insert, plasmid DNA alone as the first control group or phosphate buffer saline as the second control group. Then, cellular and humoral responses, lesion size were measured for all groups. All vaccinated mice induced Th1 immune responses against Leishmania characterized by higher IFN-γ and IgG2a levels compared with control groups (p < 0.05). In addition, IFN-γ levels increased in groups immunized with fusion and cocktail vaccines in comparison with LACK (p < 0.001) and LeIF (p < 0.01) groups after challenge. In addition, fusion and cocktail groups produced higher IgG2a values than groups vaccinated with a gene alone (p < 0.05). Lesion progression delayed for all immunized groups compared with control groups from 5th week post-infection (p < 0.05). Mean lesion size decreased in immunized mice with fusion DNA than three groups vaccinated with one gene alone (p < 0.05). While, lesion size decreased significantly in cocktail recipient group than LeIF recipient group (p < 0.05). There was no difference in lesion size between fusion and cocktail groups. Overall, immunized mice with cocktail and fusion vaccines showed stronger Th1 response by production of higher IFN-γ and IgG2a and showed smaller mean lesion size. Therefore, use of multiple antigens can improve induced immune responses by DNA vaccination.

Immunogenicity and efficacy of a bivalent DNA vaccine containing LeIF and TSA genes against murine cutaneous leishmaniasis.

There is no effective vaccine for the prevention and elimination of leishmaniasis. For this reason, we assessed the protective effects of DNA vaccines containing LeIF, TSA genes alone, or LeIF-TSA fusion against cutaneous leishmaniasis pEGFP-N1 plasmid (empty vector) and phosphate buffer saline (PBS) were used as control groups. Therefore, cellular and humoral immune responses were evaluated before and after the challenge with Leishmania major. Lesion diameter was also measured 3-12 weeks after challenge. All immunized mice with plasmid DNA encoding Leishmania antigens induced the partial immunity characterized by increased IFN-γ and IgG2a levels compared with control groups (p < 0.001). Furthermore, the immunized mice showed significant reduction in mean lesion sizes compared with mice in empty vector and PBS groups (p < 0.05). The reduction in lesion diameter was 29.3%, 34.1%, and 46.2% less in groups vaccinated with LeIF, TSA, and LeIF-TSA, respectively, than in PBS group at 12th week post infection. IFN/IL-4 and IgG2a/IgG1 ratios indicated that group receiving LeIF-TSA fusion had the highest IFN-γ and IgG2a levels. In this study, DNA immunization promoted Th1 immune response characterized by higher IFN-γ and IgG2a levels and also reduction in lesion size. These results showed that a bivalent vaccine containing two distinct antigens may induce more potent immune responses against leishmaniasis.

Mechanism of preferential packaging of negative sense genomic RNA by viral nucleoproteins in Crimean-Congo hemorrhagic Fever virus.

The Crimean-Congo Hemorrhagic Fever (CCHF) is an infectious disease of high virulence and mortality caused by a negative sense RNA nairovirus. The genomic RNA of CCHFV is enwrapped by its nucleoprotein. Positively charged residues on CCHFV nucleoprotein provide multiple binding sites to facilitate genomic RNA encapsidation. In the present work, we investigated the mechanism underlying preferential packaging of the negative sense genomic RNA by CCHFV nucleoprotein in the presence of host cell RNAs during viral assembly. The work included genome sequence analyses for different families of negative and positive sense RNA viruses, using serial docking experiments and molecular dynamic simulations. Our results indicated that the main determinant parameter of the nucleoprotein binding affinity for negative sense RNA is the ratio of purine/pyrimidine in the RNA molecule. A negative sense RNA with a purine/pyrimidine ratio (>1) higher than that of a positive sense RNA (<1) exhibits higher affinity for the nucleoprotein. Our calculations revealed that a negative sense RNA expresses about 0.5 kJ/mol higher binding energy per nucleotide compared to a positive sense RNA. This energy difference produces a binding energy high enough to make the negative sense RNA, the preferred substrate for packaging by CCHFV nucleoprotein in the presence of cellular or complementary positive sense RNAs. The outcome of this study may contribute to ongoing researches on other viral diseases caused by negative sense RNA viruses such as Ebola virus which poses a security threat to all humanity.

Causality relationships between coagulation factors in type 2 diabetes mellitus: path analysis approach.

BACKGROUND: Type 2 diabetic mellitus patients are amongst the most susceptible groups to vascular abnormalities, which predominantly lead to myocardial disease. The hypercoagulable state has been widely studied by researchers as being the major suspicious mechanism facilitating the consecutive chain of molecular events leading to these complications. However, there is no consensus on the definition of the hypercoagulable state with respect to coagulation quantities, their interrelations and basic factor(s) initiating this pathogenic event, by which the prognosis of myocardial complications could be determined. METHODS: Path analysis was used to study the interactions between coagulation factors as well as other factors beyond coagulation factors in relation with pathogenic events in both diabetics and healthy subjects. In the present work, coagulation factors of 40 healthy and 40 type 2 diabetics were determined experimentally. The data were then analyzed using SPSS and AMOS software. Multivariate regression analysis was done to draw path diagrams. RESULTS: Our results show that FII, as the main cause for hypercoagulable state, is directly induced by FX and FVIII in normal individuals and by FX, FXI, FV and VWF cofactors in diabetic patients. CONCLUSION: In general, our findings showed complicated relationship between coagulation factors and their effects either separately or combined.

Resistance mechanism of human immunodeficiency virus type-1 protease to inhibitors: A molecular dynamic approach.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors comprise an important class of drugs used in HIV treatments. However, mutations of protease genes accelerated by low fidelity of reverse transcriptase yield drug resistant mutants of reduced affinities for the inhibitors. This problem is considered to be a serious barrier against HIV treatment for the foreseeable future. In this study, molecular dynamic simulation method was used to examine the combinational and additive effects of all known mutations involved in drug resistance against FDA approved inhibitors. Results showed that drug resistant mutations are not randomly distributed along the protease sequence; instead, they are localized on flexible or hot points of the protein chain. Substitution of more hydrophobic residues in flexible points of protease chains tends to increase the folding, lower the flexibility and decrease the active site area of the protease. The reduced affinities of HIV-1 protease for inhibitors seemed to be due to substantial decrease in the size of the active site and flap mobility. A correlation was found between the binding energy of inhibitors and their affinities for each mutant suggesting the distortion of the active site geometry in drug resistance by preventing effective fitting of inhibitors into the enzymes' active site. To overcome the problem of drug resistance of HIV-1 protease, designing inhibitors of variable functional groups and configurations is proposed.

A tri state mechanism for oxygen release in fish hemoglobin: Using Barbus sharpeyi as a model.

Hemoglobin is a porphyrin containing protein with an α2ß2 tetrameric structure and like other porphyrin compounds shows spectral behavior of species specific characteristics. Researchers tend to relate bands in the hemoglobin spectra to certain structural and/or functional features. Given the fact th↔at hemoglobin is the main oxygen carrier in animals functioning through the Oxy↔Deoxy equilibrium, the determination of oxy and deoxy conformations of hemoglobins of different animals may shed light on their oxygen binding properties. Absorption spectra at 280 and 373nm have been widely used to quantitate the formation of hemoglobin deoxy conformation. In the present work, however, we used an optical density ratio of OD373/OD280 as an index for deoxy formation. This ratio was determined for Barbus sharpeyi and human hemoglobins at different SDS concentrations, pH levels and temperatures to compare them from a structure-function point of view. Our data showed that under low concentrations of SDS (<2mM) Barbus sharpeyi hemoglobin folds in a tri-state pattern while human hemoglobin folds through a two-state phenomenon. This finding indicates that in contrast to those of other non aquatic animals, the hemoglobin of Barbus sharpeyi has a loosely folded tetrameric structure with remarkably more oxygen affinity.