مقالات خارجی
  • Summary:

     

    An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

    Authors: دکتر فریدون مهبودی، دکتر دوامی، احمد عادلی، برخورداری، آل بویه
    Keywords: TGE-SGE Expression, Disposable Bioreactors, PAI-1-Resistant t-PA, CHO DG44 Cells
  • Summary:

    The midgut microbiota associated with Anopheles stephensi and Anopheles maculipennis (Diptera: Culicidae) was investigated for development of a paratransgenesis-based approach to control malaria transmission in Eastern Mediterranean Region (EMR). Here, we present the results of a polymerase chain reaction (PCR) and biochemical-based approaches to identify the female adult and larvae mosquitoe microbiota of these two major malaria vectors, originated from South Eastern and North of Iran. Plating the mosquito midgut contents from lab-reared and field-collected Anopheles spp. was used for microbiota isolation. The Gram-negative and Gram-positive bacterial colonies were identified by Gram staining and specific mediums. Selected colonies were identified by differential biochemical tests and 16S rRNA gene sequence analysis. A number of 10 An. stephensi and 32 An. maculipennis adult mosquitoes and 15 An. stephensi and 7 An. maculipennis larvae were analyzed and 13 sequences of 16S rRNA gene bacterial species were retrieved, that were categorized in 3 classes and 8 families. The majority of the identified bacteria were belonged to the γ-proteobacteria class, including Pseudomonas sp. and Aeromonas sp. and the others were some closely related to those found in other vector mosquitoes, including Pantoea, Acinetobacter, Brevundimonas, Bacillus, Sphingomonas, Lysinibacillus and Rahnella. The 16S rRNA sequences in the current study aligned with the reference strains available in GenBank were used for construction of the phylogenetic tree that revealed the relatedness among the bacteria identified. The presented data strongly encourage further investigations, to verify the potential role of the detected bacteria for the malaria control in Iran and neighboring countries.

    Authors: Dinparast Djadid N, Jazayeri H, Raz A, Favia G, Ricci I, Zakeri S.
    Keywords: Midgut Microbiota, An. stephensi, An. maculipennis, Paratransgenic Tool , Malaria
  • Summary:

    The increasing use of sulfadoxine/pyrimethamine (SP) for treatment of chloroquine-resistant Plasmodium falciparum has resulted in increased exposure of Plasmodium vivax parasites in areas where both species co-exist. In this study, the extent of mutations/haplotypes in pvdhfr and pvdhps was examined using PCR-RFLP methods in 427 P. vivax isolates in Iran after 4 years of introducing SP as the first-line anti-malarial drug in Iran. Mutations were detected in three codons of pvdhfr (F57L, S58R and S117N) and in one of pvdhps (A383G) and the majority of isolates had double mutations (58R/117N, 45.4%). In addition, the frequency of 57L mutation was detected in 8.2% of P. vivax isolates. This frequency was significantly increased when compared with a similar study on P. vivax isolates in 2005 (X(2) test, P<0.0001). Moreover, there was an increase in the frequency of single nucleotide polymorphisms at position 383G in pvdhps (0-2.6%) was found. Furthermore, the number of haplotypes increased from 6 to 12 in the study areas during 2006-2010. Interestingly, when combining the two loci, the frequency of parasites carrying pvdhfr/pvdhps pure mutations (L(57)R(58)/G(383), R(58)N(117)/G(383)) increased from 0% in 2006 to 2.1% in 2010. In conclusion, the present results suggest that SP could be effective in treatment against the erythrocytic stages of vivax malaria in Iran; however, the increased frequency of mutant haplotypes in Iran since 2006 is worrying and indicates the emergence of drug-tolerant/resistant P. vivax isolates in Iran in near future.

    Authors: Afsharpad M, Zakeri S, Pirahmadi S, Djadid ND.
    Keywords: Molecular assessment, dhfr/dhps mutations, Plasmodium vivax, sulfadoxine/pyrimethamine, artesunate
  • Summary:

    The main objective of this investigation was whether the combination therapy of sulfadoxine pyrimethamine (SP) plus artesunate (AS) protects against the spread of resistance to SP in malaria-endemic south-eastern Iran. Infected blood samples of Plasmodium falciparum (n=170) were collected during 2008-2010 after the adoption of SP-AS as the first line treatment in Iran. Four different genes of P. falciparum [dihydropteroate synthetase (pfdhps), dihydrofolate reductase (pfdhfr), chloroquine (CQ) resistance transporter (pfcrt K76T) and multidrug resistance1 (pfmdr1 N86Y)], associated with SP and CQ resistance were analyzed using PCR-RFLP methods. The result showed 4.1, 95.9 and 100% prevalence of pfdhfr 51I, 59R and 108N, respectively and the majority of patients (95.9%) were found to carry both 59R and 108N. The prevalence of single mutation at pfdhps 437G gene was 26.9% before the adoption of SP-AS, but as SP was used as the first line treatment; this mutation started to increase and reached a high level of 55.5% in 2008 (χ(2) test, P<0.05). However, three years after the introduction of SP-AS, this prevalence was reduced from 55.5% (in 2008) to 39.1% (in 2009) and then 40.5% (in 2010). The frequency of parasites carrying pfdhfr/pfdhps mutations (N(51)R(59)N(108)/G(437)) decreased from 53.3% in 2008 to 39.1% in 2009 and 38% in 2010. In addition, no significant reduction was seen in the frequency of mutant alleles of pfcrt 76T and pfmdr1 86Y after CQ was discontinued from study areas as a treatment for P. falciparum. This is explained by the fixation of pfcrt 76T in the falciparum populations that need more time to recover from CQ sensitivity in the absence of drug pressure in this region. In conclusion, the present findings suggest that in Iran, SP is still effective for the treatment of uncomplicated falciparum malaria as a partner drug of Artemisinin Combination Therapy (ACT) in this region.

    Authors: Afsharpad M, Zakeri S, Pirahmadi S, Djadid ND.
    Keywords: Molecular monitoring, Plasmodium falciparum, antimalarial drugs, sulfadoxine-pyrimethamine, artesunate
  • Summary:

    Carboxy-terminus of merozoite surface protein-1 (MSP-1(19) ) is the major protein on the surface of the plasmodial merozoite that acts as one of the most important blood-stage vaccine candidates. The present investigation was designed to evaluate the immune responses when either two recombinant antigens (rPvMSP-1(19) + rPfMSP-1(19)) or two plasmid constructs (pcDNA3.1 hygro-PvMSP-1(19) + pcDNA3.1 hygro-PfMSP-1(19)) were administered in combination at a single site in mice by using different immunization strategies (protein/protein, DNA/DNA and DNA/protein) at weeks 0, 5 and 8. All mice were monitored for the level of MSP-1(19) -specific antibody for up to 40 weeks. The inclusion of both recombinant antigens in a vaccine mixture could not inhibit induction of antibodies to the other antigen when the two recombinant antigens were combined in immunization formulation. Interestingly, antisera from immunized mice with either recombinant antigen failed to cross-react with heterologous antigen. Moreover, the results of this study showed that co-immunization with both antigens at a single site generated a substantial PvMSP-1(19) - and PfMSP-1(19) -specific antibody responses and also IFN-γ cytokine production (Th1 response) in DNA/protein prime-boost immunization strategies. The increased humoral response to PvMSP-1(19) and PfMSP-1(19) lasted nearly a year after immunization. Therefore, the results of this study are encouraging for the development of multi-species malaria vaccine based on MSP-1(19) antigen.

    Authors: Mehrizi AA, Zakeri S, Rafati S, Salmanian AH, Djadid ND.
    Keywords: Immune responses, co-immunization , Plasmodium vivax, P. falciparum MSP-1, prime-boost immunization strategies
  • Summary:

     

    Envenomation caused by Hemiscorpius (H.) lepturus from Liochlidae family presents clinical features that have not been previously described for the Buthidae family scorpions. The most significant manifestations of H. lepturus envenomation are hemolysis and dermonecrosis which could lead in severe cases to renal, cardio-respiratory failure, and death. In this study, we aimed to identify and characterize the protein(s) causing these effects. We have purified a 33 kDa protein from the venom of H. lepturus and named it Heminecrolysin. Tryptic digestion and MS/MS analysis of obtained peptides showed homology with previously described brown spider sphingomyelinases D. Functional characterization of Heminecrolysin indicated a sphingomyelinase D, a complement-dependent hemolysis properties and a dermonecrosis activity. Heminecrolysin displayed higher hemolytic activity to human erythrocytes (ED50 of 0.025 μg/ml), a stronger inflammatory and dermonecrotic effects when injected intra-dermally to rabbit skins, while its efficiency to hydrolyze sphingomyelin seems weaker than other known spider dermonecrotic SMasesD (149 ± 32.5 nmol/mg). Step of sensitization of human erythrocytes by Heminecrolysin was shown to be Mg²⁺and Ca²⁺-independent while hemolysis step in the presence of complement required both bivalent ions. Heminecrolysin is the first hemolytic dermonecrotic toxin identified in venom other than spiders. Except in spider Loxosceles genus and some pathogenic strains of Corynebacteria, sphingomyelinase D activity is unknown in the animal kingdom.

    Authors: Borchani L, Sassi A, Shahbazzadeh D, Strub JM, Tounsi-Guetteti H, Boubaker MS, Akbari A, Van Dorsselaer A, El Ayeb M.
    Keywords: Heminecrolysin, hemolytic dermonecrotic toxin, scorpion venom
  • Summary:

     

    Epidemiological and clinical survey of scorpion envenomation was carried out by statistical method of stratified cluster random sampling in Khuzestan, the southern province of Iran, cross-sectionally. We analyzed 12,150 cases recorded in Emergency Unit of the hospitals of six cities in Khuzestan province during the year 2003. The prevalence rate of human scorpion stings in the province is 3.1/1000 inhabitants. The percentage of prevalence in selected cities was as follows: Masjed-Soleiman (27.1%), Ramhormoz (26.6%), Izeh (15.3%), Shush (12%), Baghmalek (11.7%), and Behbahan (7.3%). The scorpions, responsible for the majority of stings in Khuzestan province of Iran were identified as 53.3% yellow (Mesobuthus eupeus, Hottentotta saulcyi, Odonthobuthus doriae and Hemiscorpius lepturus), and 17.4% black (Androctonus crassicauda and Hottentotta schach), and 29.3% unknown colors. Most stings occurred throughout the year, however, the highest and lowest frequency occurs in June (16.0%) and February (0.6%), respectively. Nocturnal envenomations (60.9%) were more common than diurnal (39.1%), and 39.3% of stings were on the hands and 37.3% on the feet Most envenomings were mild (74.5%) that all evolved to cure, except for three deaths. Envenomation was characterized by local pain (63.3%), erythema (10.1%), vomiting (1.3%), restlessness (0.6%), hyperthermia (0.5%), sweating (0.4%), and spasmic (0.3%). With respect to the outcoming results, scorpionism in Khuzestan province of Iran is a public health problem, which needs to be monitored carefully by the government.

    Authors: Shahbazzadeh D, Amirkhani A, Djadid ND, Bigdeli S, Akbari A, Ahari H, Amini H, Dehghani R.
    Keywords: scorpionism
  • Summary:

     

    In Saccharomyces cerevisiae, Nce102 encodes a 173 amino acid transmembrane protein, which acts as a key player in eisosome assembly and plasma membrane organization. Here we describe the characterization of Nce102 homologue in the human pathogen, Aspergillus fumigatus. Our results demonstrated that AfuNce102 is continuously expressed during fungal growth. In addition, microscopic examination of an AfuNce102-GFP expressing transformant confirmed the localization of the fusion protein to the endoplasmic reticulum with higher density fluorescence at the tip of the mycelium. During conidiogenesis, the protein was localized to the conidiophores and the conidia. Abnormal conidiation of AfuNce102 deletion mutant suggests a potential role for AfuNce102 in sporulation process.

    Authors: Khalaj V, Azizi M, Enayati S, Khorasanizadeh D, Mirabzadeh Ardakani E.
    Keywords: NCE102 homologue, Aspergillus fumigatus, sporulation,
  • Summary:

     

    Annexin C4 has been identified as a new member of fungal annexin family. In search of function, we have generated an annexin C4 disruptant strain of human pathogen, Aspergillus fumigatus. Detailed phenotypic analysis confirmed a non essential role of annexin C4 in the growth and sporulation of this pathogen. We applied a comparative proteomics strategy to understand the possible role of this protein in the fungus. The modification of respiratory chain proteins and stress response proteins suggests the occurrence of a mild oxidative stress in anxC4 disruptant strain. This may indicate a possible anti stress function of annexin C4 in A. fumigatus.

     

    Authors: Khalaj V, Azarian B, Enayati S, Vaziri B.
    Keywords: Annexin C4, A. fumigatus, proteomics approach
  • Summary:

     

    Human tissue plasminogen activator (t-PA) plays a pivotal role in the treatment of acute myocardial infarction, ischemic stroke, and deep vein thrombosis. It has the benefit of generating no adverse effects such as fibrinogen depletion, systemic hemorrhage, and immunologic reactions. Human t-PA is a serine-protease enzyme containing 527 amino acid residues in five structural domains. The correct folding of t-PA requires the correct pairing of 17 disulfide bridges in the molecule. A gene encoding full-length human t-PA was cloned into pPICZαA expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae and flush with the kex2 cleavage site to express the protein with a native N terminus. The methylotrophic yeast, Pichia pastoris GS115 strain, was transformed with this cassette, and methanol utilizing (mut+) transformants were selected for production and secretion of human t-PA into culture media. SDS-PAGE and Western blot analysis showed the expressed bands of t-PA protein. Zymography test indicated suitable folding and proper function of the expressed recombinant human t-PA in conversion of plasminogen to plasmin and gelatin lysis. Amidolytic activity test showed the amidolytic activity of 1,650 IU/ml. The results of this study concluded that P. pastoris methylotrophic yeast can be a suitable alternative for mammalian and prokaryotic expression systems to produce t-PA.

     

    Authors: Majidzadeh-A K, Khalaj V, Fatemeh D, Mahdi H, Farzaneh B, Ahmad A, Mahboudi F.
    Keywords: Cloning and expression, human tissue plasminogen, activator, Pichia pastoris
  • Summary:

    An episomal RNAi silencing construct containing the inducible cbhB promoter and a hairpin structure has been made to downregulate the alb1 gene in the human pathogen Aspergillus fumigatus. Transformation of fungal protoplasts resulted in a high number of transformants with an inducible silenced phenotype (white spores). Efficient downregulation of the alb1 gene using this system suggests that this approach may overcome the variable downregulation observed with integrative constructs.

    Authors: Khalaj V, Eslami H, Azizi M, Rovira-Graells N, Bromley M.
    Keywords: downregulation, alb1 gene, AMA1-based episomal expression, RNAi construct, Aspergillus fumigatus
  • Summary:

     

    This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

    Authors: Alibolandi M, Mirzahoseini H.
    Keywords: Purification and Refolding, Human Basic Fibroblast, Growth Factor, Escherichia coli
  • Summary:

     

    Escherichia coli is one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins in E. coli. In vitro refolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

    Authors: Alibolandi M, Mirzahoseini H.
    Keywords: Chemical assistance, refolding, bacterial inclusion bodies
  • Summary:

    The -α(3.7) rightward deletion is the most frequent α-globin mutation worldwide, while frequencies of the ααα(anti 3.7) triplication are only sporadically known. Carriers of the ααα(anti 3.7) triplication show no clinical symptoms or significant hematological changes, but co-inheritance with β-thalassemia (β-thal) has been reported to worsen the clinical and hematological features of the patient as well as the trait. We have screened the α-globin gene rearrangements of 280 individuals with normal hematological indices and 117 persons with borderline hematological parameters. We used multiplex polymerase chain reaction (m-PCR) and multiplex ligation-dependent probe amplification (MLPA) technology to detect triplications and quadruplications. Only the ααα(anti 3.7) triplication was observed. The carrier frequency in the first group was 2.14% and in the second group 1.7%. No phenotype aggravation was noticed in two carriers of β-thal and the ααα(anti 3.7) triplication, while a mild β-thalassemia intermedia (β-TI) was observed in a β-thal carrier with six α-globin genes. Due to the high consanguinity in the country, homozygosity for the ααα(anti 3.7) triplication and for other rearrangements can be expected. Therefore, an accurate determination of the frequencies and a routine control for these mutations is essential for a correct genotype-phenotype prediction during genetic counseling for β-thal.

    Authors: Moosavi SF, Amirian A, Zarbakhsh B, Kordafshari A, Mirzahoseini H, Zeinali S, Karimipoor M.
    Keywords: α-globin gene, triplication, Iranian population, hematological parameters
  • Summary:

    OBJECTIVE:

    To compare the gene dosage results achieved by a novel multiplex quantitative assay with cytogenetic and quantitative fluorescent polymerase chain reaction (QF-PCR) analysis for prenatal screening of trisomy 21 syndrome on corresponding fetal samples.

    DESIGN AND METHODS:

    Fetal samples (n=134) were collected from pregnant women considered high risk for having trisomy 21 affected fetus. Cytogenetic analysis and QF-PCR were performed. Then, the relative gene dosage of DSCAM and DYRK1A2 genes was determined on corresponding samples using comparative delta cycle of threshold (ΔC(T)) method.

    RESULTS:

    The mean gene dosage ratio was 1.55±0.11 (95% CI:1.51-1.58) in trisomy 21 cases and 1.01±0.12 (95% CI:0.98-1.03) in normal samples (p value<0.001). The results were in agreement to the results of cytogenetic and QF-PCR analysis with the overall specificity of 0.96 (95% CI:0.91-0.98) and the sensitivity of 0.80 (95% CI:0.49-0.94).

    CONCLUSIONS:

    This gene dosage assay is appropriate for the screening of high risk pregnant women and is readily amenable to automation.

     

    Authors: Kamyab AR, Shahrokhi F, Shamsian E, Hayat Nosaied M, Dibajnia P, Hashemi M, Mahdian R.
    Keywords: sensitivity, specificity, gene dosage assay, prenatal screening, trisomy 21 syndrome
  • Summary:

    Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.

    Authors: Hemayatkar M, Mahboudi F, Majidzadeh-A K, Davami F, Vaziri B, Barkhordari F, Adeli A, Mahdian R, Davoudi N.
    Keywords: Increased expression, recombinant, human tissue plasminogen activator, Leishmania tarentolae
  • Summary:

    OBJECTIVES:

    To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene.

    DESIGN AND METHODS:

    We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA.

    RESULTS:

    The results showed the gene dosage ratio of 0.99+/-0.14 and 1.09+/-0.19 for normal individuals and 0.48+/-0.06 and 0.50+/-0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA.

    CONCLUSION:

    Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.

     

    Authors: Hayat Nosaeid M, Mahdian R, Jamali S, Maryami F, Babashah S, Maryami F, Karimipoor M, Zeinali S.
    Keywords: Validation and comparison, quantitative, real-time PCR assay, DMD/BMD carriers
  • Summary:

    beta-Thalassemia is mainly caused by mutations involving single base substitution and small deletions. However, a considerable number of carriers are suspected to have large deletions in beta-globin gene cluster. Common strategy for identifying deletions with definite breakpoints is based on Gap PCR. There are, however, some cases with indefinite breakpoints which usually cannot be detected by this method. We developed and optimized a quantitative real-time PCR assay for copy number analysis of beta-globin gene cluster. The copy number of target fragments (i.e. beta, delta or (G)gamma-globin genes) was determined using comparative threshold cycle method. In addition, gene dosage was analyzed using multiplex ligation-dependent probe amplification (MLPA) method in all suspected carriers. Using these relative quantitative assays, normal or carrier statuses of all 26 unknown samples were successfully determined according to the ranges obtained from the ratios of normal and definite carrier samples. Interestingly, large deletions involving the entire beta-globin gene cluster were observed in six carrier individuals. This study showed that the MLPA as a preliminary screening test can be followed by SYBR Green real-time PCR for analysis of copy number variations in beta-globin gene cluster. Combination of these relative quantitative PCR methods could be an appropriate approach for accurate diagnosis of unknown beta-thalassemia deletions in routine diagnosis of beta-thalassemia mutations.

    Authors: Babashah S, Jamali S, Mahdian R, Nosaeid MH, Karimipoor M, Alimohammadi R, Raeisi M, Maryami F, Masoudifar M, Zeinali S.
    Keywords: unknown deletions, beta-globin gene, quantitative PCR methods
  • Summary:

    Vascular endothelial growth factor receptor-2 (VEGFR2) is an important tumor-associated receptor and blockade of the VEGF receptor signaling can lead to the inhibition of neovascularization and tumor metastasis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids. Here, we describe the identification of a VEGFR2-specific Nanobody, named 3VGR19, from dromedaries immunized with a cell line expressing high levels of VEGFR2. We demonstrate by FACS, that 3VGR19 Nanobody specifically binds VEGFR2 on the surface of 293KDR and HUVECs cells. Furthermore, the 3VGR19 Nanobody potently inhibits formation of capillary-like structures. These data show the potential of Nanobodies for the blockade of VEGFR2 signaling and provide a basis for the development of novel cancer therapeutics.

    Authors: Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K, Khabiri A, Schoonooghe S, Habibi Anbouhi M, Hassanzadeh-Ghassabeh G, Muyldermans S.
    Keywords: Generation, characterization, functional Nanobody, vascular endothelial, growth factor, receptor-2, angiogenesis cell receptor
  • Summary:

    BACKGROUND:

    In the previous study, we have shown that the presence of A allele at position -588 in Agamma-globin gene was highly frequent and closely associated with fetal hemoglobin elevation among beta-thalassemia intermedia patients. Therefore, we decided to investigate whether this allele (A allele at -588) could result in an increase in Agamma-globin gene expression to ameliorate the severity of the disease in thalassemia patients.

    METHODS:

    Three constructs containing mu locus control region, Agamma-globin and beta-globin genes were designed and employed in the transient expression assay. The difference among constructs was in the promoter region of Agamma-globin gene (A and G alleles at -588). A construct with T to C base substitution at -175 of Agamma-globin, created by site-directed mutagenesis, was selected as positive control. The K562 cell line was transfected with the above constructs. Subsequently, the expression of Agamma-globin gene was determined by quantitative real-time reverse transcription-PCR.

    RESULTS:

    There was not a significant increase in the expression of Agamma-globin gene in the construct containing A allele comparing the one with G allele at -588.

    CONCLUSIONS:

    -588 (A>G) mutation does not play a major role in regulation of Agamma-globin gene, suggesting that other factors may be involved.

    Authors: Hamid M, Mahjoubi F, Akbari MT, Khanahmad H, Jamshidi F, Zeinali S, Karimipoor M
    Keywords: expression assay, Agamma-588 (A/G), mutation, K562 cell line
  • Summary:

    Here we report the result of three cases referred to our lab that had a combination of β-thalassemia and hemoglobin D (Hb D) traits. These individuals had no symptoms of profound anemia and hematological indices were similar to that of a β-thalassemia heterozygote. In all three cases, the Hb D level was elevated and no HbA was detected electrophoretically. The electrophoresis pattern suggested that all cases were homozygotes for Hb D. PCR followed by digestion with EcoRI and sequencing of the β-globin gene confirmed the presence of Cd 121 GAA>CAA in the heterozygous form with another β-globin mutation. In all cases, the mutations in the β-globin gene were detected by ARMS PCR technique and they were either IVSII-I or IVSI-5. Hematological studies of the family members showed that thalassemia which caused the mutations and Hb D were in the trans position.

    Authors: Taghavi Basmanj M, Karimipoor M, Amirian A, Jafarinejad M, Katouzian L, Valaei A, Bayat F, Kordafshari A, Zeinali S
    Keywords: Co-inheritance, hemoglobin D, β-thalassemia, Iranian families
  • Summary:

    BACKGROUND:

    Co-inheritance of β- and δ-globin mutations in Iran is not uncommon. This situation may interfere with correct diagnosis and genetic counseling of α- and β-thalassemia in screening programs. Here we report the co-inheritance of β- and δ-globin gene mutations in an individual with microcytosis, hypochromia and a normal hemoglobin A₂ (HbA₂) level.

    METHODS:

    Genomic DNA extraction, amplification refractory mutation system (ARMS) polymerase chain reaction and direct DNA sequencing of δ- and β-globin genes were exploited for detection of the mutations in these two genes in an individual with low hematological indices and normal HbA₂.

    RESULTS:

    ARMS-PCR technique revealed the β(+) IVSI-5 (G to C) mutation and direct DNA sequencing of the δ-globin gene detected a previously reported delta codon 12 (AAT-->AAA) HbA2-NYU. This study reports HbA2-NYU in association with the β IVSI-5 (G to C) mutation in Iran.

    DISCUSSION:

    This report emphasizes that normal HbA₂ expression in a β-goblin carrier is due to mutation in the δ-globin gene and may cause misdiagnosis of thalassemia.

    Authors: Amirian A, Karimipoor M, Jafarinejad M, Taghavi M, Kordafshari A, Fathi Azar S, Mohammadi MS, Zeinali S
    Keywords: co-inheritance, beta-globin IVS-I-5 (G-->C) thalassemia, delta globin CD12, {Asn-->Lys (AAT-->AAA)}HbA₂-NYU
  • Summary:

    δ-Thalassemia (δ-thal) has no clinical symptoms, but its coinheritance with β-thal may cause misdiagnosis, especially in countries with a high prevalence of β-thal where prevention programs have been implemented. The molecular basis of most β-thal syndromes have been defined, while the spectrum of mutations causing δ-thal have not been well characterized. A couple was referred to us for thalassemia molecular screening. Since she had rather low values of Hb A₂ and normal Hb F, her δ-globin gene was amplified and directly sequenced. We found two different mutations on her δ-globin genes: HBD: c.92+5G>T/HBD:c.428C>A. The c.92+5G>T mutation has not been previously reported. Two different mutations in trans may explain the reduced Hb A₂ level.

    Authors: Amirian A, Jafarinejad M, Kordafshari AR, Mosayyebzadeh M, Karimipoor M, Zeinali S
    Keywords: novel, δ-globin gene, mutation, Iranian family
  • Summary:

    Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process.

    Authors: Teimoori-Toolabi L, Azadmanesh K, Amanzadeh A, Zeinali S.
    Keywords: Selective suicide, gene therapy, colon cancer, urokinase plasminogen, activator, receptor, promoter
  • Summary:

    Fibroblast growth factor 18 (FGF18) is one of the genes downstream of Wnt, one of the most important signaling pathways activated in colon cancer. An FGF18 promoter containing a single T-cell factor/lymphocyte enhancing factor 1 (TCF/LEF1) binding site was inserted upstream of a thymidine kinase (TK) suicide gene module, while a bacterial beta-Gal (LacZ) element served as the reporter gene. Following transient transfection with pUCFGF18LacZ, beta-Gal staining showed that 5% of SW480, 10% of HCT116, 0% of human umbilical vein endothelial cells (HUVECs) and 0% of normal colon cells (NCCs) had expressed LacZ. beta-Gal enzyme-linked immunosorbent assay revealed that the ratio of pUCFGF18LacZ activity to that of positive control was 0.09 and 0.25 in SW480 and HCT116, respectively (significantly higher than mock plasmid), while there were no significant changes in the beta-Gal expression in HUVEC and NCC cells transfected with pUCFGF18LacZ or mock plasmid. Following transfection with pUCFGF18TK and pUCCMVTK (positive control), cytotoxicity analysis of transfected cells showed that treatment with ganciclovir (GCV) significantly decreased SW480 and HCT116 cell survival at GCV concentrations above 20 microg/mL. An inverse correlation between GCV concentration and cell viability was evident in both colon cancer cell lines following transfection with these suicide plasmids. pUCFGF18TK and pUCCMVTK induced apoptosis after the administration of GCV in HCT116, but not in SW480, as demonstrated by M30 cytodeath antibody. This discrepancy may stem from differences in the mechanisms of TK/GCV-induced apoptosis in p53-proficient (HCT116) and -deficient (SW480) cells. The specific activity of the FGF18 promoter in HCT116 and SW480 may reflect the advantage of this promoter over artificial promoters containing artificial TCF/LEF binding sites.

    Authors: Teimoori-Toolabi L, Azadmanesh K, Zeinali S.
    Keywords: Selective suicide gene therapy of colon cancer cell lines exploiting fibroblast growth factor 18 promoter.
  • Summary:

    BACKGROUND:

    Multipotent mesenchymal stromal cells (MSC) are promising candidates in the field of regenerative medicine and in several studies have been genetically modified to bring a new property to or enhance an existing one in these cells. Furthermore, MSC have been used as gene delivery vehicles. The success of these experiments depends on selecting an appropriate method for gene delivery to the cells.

    METHODS:

    MSC were isolated from rat bone marrow; their authenticity was checked by differentiation experiments as well as staining for cell-surface markers. A systematic approach was used to optimize five cationic polymer-based gene delivery methods (Lipofectamin2000, Effecten, Superfect, Polyfect and FuGENE HD). The transfection yield and cell viability of each method was measured after 48 h in three to six separate experiments with nine to 12 different ratios and amounts of DNA/transfection reagent.

    RESULTS:

    The isolated MSC were successfully differentiated to osteoblasts, adipocytes and chondroblasts. They were positive for rat CD90 and CD73 and negative for CD31, CD45, CD11b and VEGFR2 markers. The average transfection rates with optimum conditions were 5.18+/-2.72 (FuGENE HD), 8.72+/-4.52 (Effecten), 9.59+/-3.12 (Superfect), 16.29+/-7.44 (Polyfect) and 19.60+/-3.12 (Lipofectamine 2000). The toxicity was below 20% for all reagents.

    DISCUSSION:

    Moderate levels of transfection and acceptable cell viability could be achieved using Lipofectamine 2000 and Polyfect in optimized conditions. The results could be improved by gating and sorting live cells using a simple FSC-SSC gating.

    Authors: Gheisari Y, Soleimani M, Azadmanesh K, Zeinali S.
    Keywords: mesenchymal, stromal cells, optimization, comparison, cationic polymer-based gene delivery method
  • Summary:

    Hemophilia B, a recessive X-linked coagulopathy, is rare in females, and only a few cases have been reported so far. In this report, we describe a 9-year-old female, offspring of a consanguineous marriage, with a clinically severe course of hemophilia B and a normal 46,XX karyotype. Polymerase chain reaction and conformation sensitive gel electrophoresis techniques have been applied to the important regions of the factor IX gene,and an abnormal conformation sensitive gel electrophoresis profile was identified in exon 5 of the gene. After sequencing, the mutation was found to be C17761T (R116X) in homozygous form. Then, polymerase chain reaction-restriction fragment length polymorphism using the EcoRV restriction enzyme was applied for confirmation of the homozygous mutation in the proband and for carrier testing in the relatives. In addition, haplotype analysis was informative at the HhaI polymorphic site for the female patient.

    Authors: Karimipoor M, Kokabee L, Kamali E, Karizi SZ, Zeinali S.
    Keywords: Molecular analysis, factor IX gene, Iranian female, hemophilia B
  • Summary:

    An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

    Authors: Davami F, Barkhordari F, Alebouyeh M, Adeli A, Mahboudi F.
    Keywords: Combined, TGE-SGE expression, novel PAI-1-.resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors
  • Summary:

    Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase®. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase®), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.

    Authors: Davami F, Sardari S, Majidzadeh-A K, Hemayatkar M, Barkhordari F, Enayati S, Adeli A, Mahboudi F.
    Keywords: variant, t-PA resistant, plasminogen activator inhibitor-1, expression, CHO cells, in silico experiments
  • Summary:

    Tissue plasminogen activator (t-PA) is one of the fibrin-specific serine proteases that play a crucial role in the fibrinolytic system. The rapid clearance of the drug from the circulation, caused by its active uptake in the liver, has lead to complicated clinical applications. Different forms of plasminogen activators have been developed to treat thrombotic disease. Deletion of the first three domains of t-PA by gene manipulation techniques has shown a significant increase in its plasma half life. In order to compensate the disadvantage of higher bleeding risk, a novel chimeric truncated form of t-PA with 394 amino acids and more fibrin affinity compared to the truncated form was designed to be expressed in Chinese Hamster Ovarian (CHO) cells. The recombinant chimeric plasminogen activator consists of kringle 2 and serine protease (K2S) domains of t-PA, namely GHRP-SYQ-K2S. The level of expression was found to be 752 IU/ml with 566,917 IU/mg specific activity, based on amidolytic activity. The fibrin binding of this novel chimeric truncated t-PA was 86% of the full length t-PA at a fibrinogen concentration of 0.2 mg/ml. This could be a promising approach with more desirable pharmacodynamic properties compared to existing commercial forms.

    Authors: Davami F, Sardari S, Majidzadeh-A K, Hemayatkar M, Barkhrdari F, Omidi M, Azami M, Adeli A, Davoudi N, Mahboudi F
    Keywords: Expression, chimeric t-PA, truncated, CHO cells, in silico experiments
  • Summary:

    PURPOSE:

    To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits.

    METHODS:

    A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples.

    RESULTS:

    The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value.

    CONCLUSIONS:

    Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.

    Authors: Mohammadi M, Talebkhan Y, Khalili G, Mahboudi F, Massarrat S, Zamaninia L, Oghalaei A
    Keywords: ELISA kit, Helicobacter pylori infection, imported kits
  • Summary:

    A variety of recombinant protein expression systems have been developed for heterologous genes in both prokaryotic and eukaryotic systems such as bacteria, yeast, mammals, insects, transgenic animals and transgenic plants. Also, it has been reported that Leishmania tarentolae, a trypanosomatid protozoan parasite of the white-spotted wall gecko (Tarentola annularis), has the capability of expressing heterologous genes. Trypanosomatidae are rich in glycoproteins, which can account for more than 10% of total protein. The oligosaccharide structures of their glycoproteins are similar to those of mammals with N-linked galactose, and sialic acid residues. For a variety of reasons, including the glycosylation patterns and the secondary structures of some of these proteins, synthesis in eukaryotic system is highly preferable. In addition, formation of native disulfide bonds in complex eukaryotic proteins is tremendously important. In the present study, we tried to express the tPA (tissue plasminogen activator) gene in L. tarentolae. This protein is a thrombolytic agent with 527 amino acid residues. tPA possesses serine-protease activity, with 35 cysteine residues that participate in the formation of 17 disulfide bonds. We have used an expression cassette, including the alpha intergenic regions of Leishmania major and two sites at the 3'- and 5'-ends, for homologous recombination in L. tarentolae, in addition to antibiotic-resistant genes. Southern-blot analysis showed that the human tPA gene had been inserted into the genome of the parasite. The expression of the tPA at the mRNA and protein levels was confirmed. It was shown that the expressed tPA in this system was 70 i.u. (international units)/ml of culture media, which is much higher than levels reported previously in other systems.

    Authors: Soleimani M, Mahboudi F, Davoudi N, Amanzadeh A, Azizi M, Adeli A, Rastegar H, Barkhordari F, Mohajer-Maghari B
    Keywords: Expression, human tissue plasminogen, activator, trypanosomatid protozoan, Leishmania tarentolae
  • Summary:

    The rate of human immunodeficiency virus type 1 (HIV-1) infection in Iran has increased dramatically in the last few years. While the earliest cases were found in hemophiliacs, intravenous drug users are now fueling the outbreak. In this study, both the 122 clones of HIV-1 gag p17 and the 131 clones of env V1-V5 region were obtained from 61 HIV-1 seropositives belonging to these two groups in Iran. HIV-1 subtyping and phylogenetic analysis was done by heteroduplex mobility assays (HMA) and multiple clone sequencing. The result indicated all hemophiliacs are infected with HIV-1 subtype B and all intravenous drug users are infected with HIV-1 subtype A. Since intravenous drug abuse is the major transmission route in Iran, HIV-1 subtype A is likely to be the dominant viral subtype circulating in the country. The analysis of genetic distances showed subtype B viruses in Iran to be twice as heterogeneous as the subtype A viruses. In conclusion, this first molecular study of HIV-1 genotypes in Iran suggests two parallel outbreaks in distinct high-risk populations and may offer clues to the origin and spread of infection in Iran.

    Authors: Sarrami-Forooshani R, Das SR, Sabahi F, Adeli A, Esmaeili R, Wahren B, Mohraz M, Haji-Abdolbaghi M, Rasoolinejad M, Jameel S, Mahboudi F
    Keywords: Molecular analysis, phylogenetic characterization, HIV, Iran
  • Summary:

    The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.

    Authors: Mahboudi F, Irina NA, Chevalier A, Ghadiri A, Adeli A, Amini-Bavil-Olyaee S, Barkhordari F, Farzamfar B, Alinejad M
    Keywords: serological screening assay, immunodeficiency, virus type 1 antibodies, recombinant protein p24-gp41, fusion protein, Escherichia coli
  • Summary:

    In spite of hepatitis B virus (HBV) vaccination, HBV infection remains an important public health problem worldwide. Although the HBV genotype distribution has been determined in some parts of South Central Asia, no survey has been conducted to determine the HBV genotype in Afghanistan. Twelve Afghan patients infected with HBV living in Afghanistan were enrolled in this study. Partial HBsAg and basic core promoter, precore, and core (BCP/preC/C) regions were amplified and subjected for direct sequencing. In parallel, precore G1896A mutation was also determined by an amplification-created restriction site method. Results revealed HBV genotype D (95% bootstrap value), sub-genotype D1 (98% bootstrap value), and subtype ayw2 in all Afghan isolates. Afghan isolates clustered in a separate branch in the D1 sub-genotype called D1', while supported by 82% bootstrap value. The percentage of intra-genotypic distance among Afghan isolates was 1.05% and inter-genotypic distance with the other genotype D was 2.87% and with other genotypes was 7.50%-11.1%. The wild-type, mixed infection, and precore mutant were found in six, two, and four HBV isolates, respectively. The A1762T/G1764A BCP dual mutation was found in one isolate. Three isolates presented single mutation in the BCP dual mutation region, whereas two showed a novel G1764T mutation. In conclusion, this preliminary study revealed HBV genotype D, sub-genotype D1, and subtype ayw2 of HBV among hepatitis B infected patients from Afghanistan. Further investigation should be carried out.

    Authors: Amini-Bavil-Olyaee S, Alavian SM, Adeli A, Sarrami-Forooshani R, Sabahi F, Sabouri E, Tavangar HR, Azizi M, Mahboudi F
    Keywords: Hepatitis B virus, genotyping, core promoter, precore/core mutations, Afghan patients, hepatitis B
  • Summary:

    Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.

    Authors: Amini-Bavil-Olyaee S, Sarrami-Forooshani R, Adeli A, Sabahi F, Abachi M, Azizi M, Mahboudi F
    Keywords: genomic sequence, phylogenetic relatedness, hepatitis B virus, isolates from Iran
  • Summary:

    The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.

    Authors: Amini-Bavil-Olyaee S, Sarrami-Forooshani R, Adeli A, Mahboudi F, Sabahi F, Nafisi H, Zali MR, Azizi M
    Keywords: accurate amplification, restriction site method, wild type and the precore mutant hepatitis B virus variants
  • Summary:

    To provide a safer live challenge strain for use in clinical vaccine trials, a double drug sensitive strain of Leishmania major was derived using advances in gene targeting technology by stably introducing into the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV), and a Saccharomyces cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC). In vitro studies showed that the homozygous L. major (tk-cd+/+) promastigotes were killed by either drug alone, and together the drugs acted synergistically. In vivo infection studies showed that progressively growing lesions in BALB/c mice, caused by L. major (tk-cd+/+), were completely cured by 2 weeks of treatment with either drug alone or in combination. Treated animals showed no signs of reoccurrence of infection for at least 4 months when the experiments were terminated.

    Authors: Davoudi N, Tate CA, Warburton C, Murray A, Mahboudi F, McMaster WR
    Keywords: recombinant Leishmania, sensitive, ganciclovir 5-fluorocytosine, live vaccine challenge, clinical trials
  • Summary:

    Hepatitis B virus (HBV) is one of the major causative agents of acute and chronic liver disease worldwide and is believed to be responsible for a million deaths annually. Eight genotypes of HBV, A to H, have been described on the basis of similarity of the complete genomes sequence. Although, it is reported that the predominant HBV genotype in the Mediterranean area and the middle east is genotype D, there are no reports on HBV genotypes prevalent in Iran. In this study, the C and S regions of HBV from 26 chronic hepatitis B Iranian patients were amplified and sequenced. Phylogenetic analysis revealed that all Iranian HBV isolates sequences were classified into genotype D with bootstrap values of 100%, 73%, and 100% (1,000 replicates each) for S, C, and preS2 regions, respectively. The mean percent intra-distance of S and C regions were 0.8% and 2.3%, respectively. The mean percent inter-distance of S and C regions between Iranians and genotype D isolates were 1.7% and 3.0%, respectively, and the range of mean percent nucleotide distance of S and C regions between Iranians and the other reference isolates were 7.9%-17.5% and 4.8%-14.7%, respectively. Thirteen out of 23 HBV C region sequences showed nucleotide "A" at position 1896 (precore mutant) in C region. Nucleotide 1858 showed presence of "T" in all isolates. No insertion or deletion was found in both regions. SimPlot and BootScanning analyses did not show any recombination between Iranian isolates and other genotypes in both regions.

    Authors: Amini-Bavil-Olyaee S, Sarrami-Forooshani R, Mahboudi F, Sabahi F, Adeli A, Noorinayer B, Azizi M, Reza Zali M.
    Keywords: Genotype characterization, phylogenetic analysis, hepatitis B virus, isolates, Iranian patients
  • Summary:

    The variable and conserved sequence boxes of kinetoplast DNA (kDNA) of 11 standard strains of 6 complexes of New and Old World Leishmania were amplified using PCR. Four strains from 2 complexes of Old World Leishmania - L. major (MRHO/IR/64/Nadim-1), with 2 bands at 850 and 620 bp, L. major (MHOM/SU/73/5-ASKH), with a band at 620 bp, L. donovani, with a band at 800 bp and L. infantum, with a band at 650 bp - could be differentiated from each other and from the New World strains, with the exception of L. infantum. Seven Leishmania strains from 4 complexes of New World Leishmania - L. mexicana and L. pifanoi, with a band at 730 bp, L. guyanensis, with 2 bands at 730 and 650 bp, L. peruviana, with a band at 710 bp and L. amazonensis, L. garnhami and L. braziliensis, each with a band at 650 bp - were identified. Of these strains, L. guyanensis and L. peruviana could be differentiated from each other and from the Old World strains. These results show that using PCR amplification of kDNA we could differentiate between New and Old World Leishmania at both complex and strain levels. The amplified kDNA PCR products, together with other techniques, could be useful as a diagnostic tool for the identification of Leishmania species.

    Authors: Mahboudi F, Abolhassani M, Tehrani SR, Azimi M, Asmar M
    Keywords: Differentiation , world leishmania species, complex and species levels, PCR
  • Summary:

    We describe the specific identification of Leishmania species in Iran using PCR DNA amplification of kDNA. For this purpose, we designed a pair of primers--upstream 5' TCGCAGAACGCCCCTACC 3' and downstream 5'-AGGGGTTGGTGTAAAATAGGC 3'--specific for conserved sequences of kDNA of Leishmania. Using this primer, we identified 3 different amplified fragments from the kDNA of the WHO reference Leishmania species. Two bands at 620 and 850 bp were identified for L. major (MRHO/IR/64/Nadim-1 strain) and only 1 band at 620 bp was identified for L. major (P strain). Therefore, we could differentiate 2 Leishmania species. Also, 1 band at 830 bp was identified for L. tropica (MHOM/Sudan/58/OD strain). We determined the sequence analysis of 2 DNA bands (620 and 850 bp) obtained from kDNA of L. major (MRHO/IR/64/Nadim-1). A total of 157 bp from the 5' site and 234 bp from the 3' site were sequenced and showed about 28% homology between 620 and 850 bp fragments. This technique could amplify as little as 1 fg of DNA and was used to differentiate kDNA samples isolated from Iranian patients with cutaneous leishmaniasis. These data indicate that the primer used for PCR amplification of kDNA is specific and can be used for diagnostic and epidemiological purposes.

    Authors: Mahboudi F, Abolhassan M, Yaran M, Mobtaker H, Azizi M
    Keywords: Identification, differentiation, Iranian Leishmania species, PCR amplification, kDNA
  • Summary:

    Helicobacter pylori (Hp) is a major risk factor for gastrointestinal disorders including gastric cancer. We evaluated host serum antibody responses toward outer membrane protein18 in comparison with Urease A and B subunits. omp18 and ureA-ureB gene fragments were PCR amplified, cloned, and expressed in E. coli expression system. The expressed proteins were visualized on SDS-PAGE and confirmed by immuno-blotting. Purified proteins were applied in western blotting assays in comparison with local and foreign ELISA kits. ROC curve analysis identified the optimum cut-off points for each protein. rOmp18 represented the highest rates of sensitivity (94%), specificity (89%), PPV (97.4%), NPV (77.4%), and accuracy (93.2%) in comparison with urease A and B subunits. These immunologic indices were in "substantial" agreement (Κ = 0.7) with the gold standard tests for Hp detection. This study recommends Hp conserved Omp18 as a reliable serologic marker for accurate detection of Hp infection particularly for application in population screening approaches.

    Authors: Talebkhan Y, Ebrahimzadeh F, Esmaeili M, Zamaninia L, Nahvijoo A, Khedmat H, Fereidooni F, Mohagheghi MA, Mohammadi M
    Keywords: Helicobacter pylori Omp18, serologic screening of infection
  • Summary:

    Helicobacter pylori bacterial ghosts, HPBG, were generated by PhiX174 mechanism and loaded with recombinant Omp18, which were then applied in therapeutic immunization of Hp-infected C57BL/6 mice. Recombinant Omp18 loaded HPBG plus cholera toxin stimulated serum anti-Hp and Omp18-specific antibodies which resulted in significant reduction of gastric Hp colonization (P<0.05).

    Authors: Talebkhan Y, Bababeik M, Esmaeili M, Oghalaei A, Saberi S, Karimi Z, Afkhami N, Mohammadi M
    Keywords: Helicobacter pylori, bacterial ghost, recombinant Omp18, putative vaccine
  • Summary:

    AIM:

    Considering the fact that histology and its grading systems are accepted gold standards in diagnosis of diverse clinical disorders, assessing the accuracy and reliability of this method of diagnosis is of utmost importance. Thus, this study was performed to measure the agreement values between four independent histopathology readings for identical indices under one scoring guideline using three different approaches.

    METHODS:

    Four independent pathologists participated in this study and were blinded to the clinical diagnosis of patients. Various histological features were examined on gastric tissue specimens according to the updated Sydney system.

    RESULTS:

    Statistical analysis revealed that our null hypothesis about the existing agreement between different histopathological observations is rejected for chronic gastritis, the presence of inflammatory activity, atrophy and Helicobacter pylori, whereas there were significant inter-observation agreements in regard to the presence of lymphoid follicles, intestinal metaplasia and dysplasia. Pairwise analysis showed that different grading scales resulted in different kappa values ranging from poor to excellent agreements. The best kappa values were observed for the evaluation of dysplasia between two independent pathologists.

    CONCLUSIONS:

    This assessment has demonstrated that standardisation of less quantitative grading scales resulting in consistent readings is essential for affirmative clinical diagnosis and devising effective treatment strategies.

    Authors: Talebkhan Y, Mohammadi M, Rakhshani N, Abdirad A, Fayaz Moughadam K, Fereidooni F
    Keywords: Interobserver variations, histopathological assessment, gastric pathology
  • Summary:

    BACKGROUND/AIM:

    The identification of the vacA intermediate region has provided new insights into the role of vacA heterogeneity in relation to gastro-duodenal pathogenesis. The aim of this study was to assess vacA polymorphism in Iranian Helicobacter pylori strains and its association with cagA as a major virulence determinant, gastric histopathology and disease.

    METHODS:

    vacA polymorphism and serum antibody responses were studied in 207 H. pylori-infected (139 NUD, 34 PUD, and 34 GC) patients and correlated with gastric histopathology.

    RESULTS:

    Multivariate logistic regression analysis found intermediate region typing superior to signal or mid region typing for screening high risk patients. vacA i1 allele was identified as an independent predictor of dysplasia (OR = 9.044; 95% CI: 1.11-73.33). Possession of s1/i1/cagA(+) strains was also identified as a predictor of intestinal metaplasia (OR = 3; 95% CI: 1.13-7.95), dysplasia (OR = 9.9; 95% CI: 1.23-80.86) and risk of GC (OR = 6.9; 95% CI: 2.5-18.66) as well as induction of anti-VacA sero-positivity (OR = 5.04; 95% CI: 1.8-13.6). Anti-VacA serology correctly detected 83.8% of s1/i1/cagA(+) strains carried by high-risk patients.

    CONCLUSIONS:

    The current study emphasizes the implication of vacA polymorphic structure, especially the s1/i1/cagA(+) genotype, in increasing the risk of GC by revealing their association with gastric pre-neoplastic changes and their reflection in VacA sero-positivity which encourages the application of noninvasive procedures in population screening.

    Authors: Douraghi M, Talebkhan Y, Zeraati H, Ebrahimzadeh F, Nahvijoo A, Morakabati A, Ghafarpour M, Esmaili M, Bababeik M, Oghalaie A, Rakhshani N, Hosseini ME, Mohagheghi MA, Mohammadi M
    Keywords: gene status, Helicobacter pylori strains,gastric cancer
  • Summary:

    Plasmodium vivax remains an important cause of morbidity outside Africa, and no effective vaccine is available against this parasite. The P. vivax Duffy binding protein (PvDBP) is essential during merozoite invasion into erythrocytes, and it is a target for protective immunity against malaria. This investigation was designed to evaluate naturally acquired antibodies to two variant forms of PvDBP-II antigen (DBP-I and -VI) in malaria individuals (N = 85; median = 22 years) who were living in hypoendemic areas in Iran. The two PvDBP-II variants were expressed in Escherichia coli, and immunoglobulin G (IgG) isotype composition and avidity of naturally acquired antibodies to these antigens were measured using enzyme-linked immunosorbent assay (ELISA). Results showed that almost 32% of the studied individuals had positive antibody responses to the two PvDBP-II variants, and the prevalence of responders did not differ significantly (P > 0.05; χ(2) test). The IgG-positive samples exhibited 37.03% and 40.8% high-avidity antibodies for PvDBP-I and PvDBP-VI variants, respectively. Furthermore, high-avidity IgG1 antibody was found in 39.1% of positive sera for each examined variant antigen. The avidity of antibodies for both PvDBP variant antigens and the prevalence of responders with high- and intermediate-avidity IgG, IgG1, and IgG3 antibodies were similar in patients (P > 0.05; χ(2) test). Moreover, the prevalence of IgG antibody responses to the two variants significantly increased with exposure and host age. To sum up, the results provided additional data in our understanding of blood-stage immunity to PvDBP, supporting the rational development of an effective blood-stage vaccine based on this antigen.

    Authors: Zakeri S, Babaeekhou L, Mehrizi AA, Abbasi M, Djadid ND
    Keywords: Antibody responses, avidity, anti-Plasmodium vivax Duffy binding protein (PvDBP, antibodies,unstable malaria transmission
  • Summary:

    Plasmodium falciparum remains globally an important cause of mortality and morbidity and despite decades of research, no effective vaccine is available against this deadly parasite. The 19-kDa C-terminal fragment of P. falciparum merozoite surface protein 1 (PfMSP-1(19)) is a target for protective immunity against malaria and the major concern in development of vaccine based on this antigen is the presence of polymorphisms. This investigation was designed to evaluate naturally acquired antibodies and antigen-binding avidity of IgG antibodies to three variant forms of PfMSP-1(19) antigen (E/TSG/L, E/KNG/F and Q/KNG/L) in malaria individuals who are living in hypoendemic areas in Iran (n=92, 4-75 years old). The three variant forms of PfMSP-1(19) were expressed in Escherichia coli and IgG isotype composition and avidity of naturally acquired antibodies to the 19-kDa antigen were measured by ELISA assay. Results showed that almost 72% of the studied individuals had positive antibody responses to three PfMSP-1(19) variants and the prevalence of responders did not differ significantly (P>0.05). High-avidity IgG (62.7%, 65.7% and 47.76%) and IgG1 (64.2%, 50.75%, and 50.75%) were found in positive sera for E/TSG/L, E/KNG/F and Q/KNG/L variants, respectively. Moreover, the prevalence and titers of IgG1 antibody responses to the three variants increased with age (P<0.05). In summary, individuals in low transmission areas in Iran can develop and maintain equal immune responses with high avidity to the PfMSP-1(19) variants (E/TSG/L, E/KNG/F and Q/KNG/L); however, the precise role of the total IgG and its isotypes in protection requires further investigation. These results could support the design of a universal PfMSP-1(19)-based vaccine.

    Authors: Mehrizi AA, Asgharpour S, Salmanian AH, Djadid ND, Zakeri S
    Keywords: IgG subclass, antibodies, Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)), unstable malaria transmission, Iran
  • Summary:

    BACKGROUND:

    Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and their activation leads to the induction of effector genes involving inflammatory cytokines that may have contribute to controlling parasite growth and disease pathogenesis. The current immunogenetic study was designed to analyse the key components of innate immunity, TLRs and TIRAP (Toll-interleukin-1 receptor domain-containing adaptor protein), also known as MAL (MYD88 adaptor-like), in Iranian patients with mild malaria.

    METHODS:

    The tlr-4 (D299G and T399I), tlr-9 (T-1486C and T-1237C) and tirap (S180L) genes were assessed in 640 Baluchi individuals (320 Plasmodium falciparum-infected and 320 non-infected, median age of 28 years) from malaria-endemic regions using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods.

    RESULTS:

    Common tlr-4 SNPs and promoter SNPs of tlr-9 were distributed among P. falciparum-infected and non-infected groups (P > 0.05) that showed no association of these variants with mild clinical manifestation. The comparison of the tirap S180L genotypes between patients with mild malaria and those healthy individuals showed that the frequency of heterozygosity was significantly higher in infected than non-infected individuals (33.8 vs. 25.6; OR, 1.479; 95% CI, 1.051-2.081; P = 0.024). The result also revealed a significant association of tirap S180L (P < 0.05) with development of mild malaria, which is common in Baluchi populations, who are living in malaria hypoendemic region of Iran but not in African populations (0%-6%).

    CONCLUSION:

    These data point towards the need for addressing the exact role of TLRs in contributing to human genetic factors in malaria susceptibility/resistance/severity within different malaria settings in the world.

    Authors: Zakeri S, Pirahmadi S, Mehrizi AA, Djadid ND
    Keywords: Genetic variation, TLR-4, TLR-9, TIRAP genes Iranian malaria patients
  • Summary:

    BACKGROUND:

    The C-terminal region of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)) is a leading malaria vaccine candidate antigen. However, the existence of different variants of this antigen can limit efficacy of the vaccine development based on this protein. Therefore, in this study, the main objective was to define the frequency of PfMSP-1(19) haplotypes in malaria hypoendemic region of Iran and also to analyse cross-reactive and/or variant-specific antibody responses to four PfMSP-1(19) variant forms.

    METHODS:

    The PfMSP-1(19) was genotyped in 50 infected subjects with P. falciparum collected during 2006-2008. Four GST-PfMSP-1(19) variants (E/TSR/L, E/TSG/L, E/KNG/F and Q/KNG/L) were produced in Escherichia coli and naturally occurring IgG antibody to these proteins was evaluated in malaria patients' sera (n = 50) using ELISA. To determine the cross-reactivity of antibodies against each PfMSP-1(19) variant in P. falciparum-infected human sera, an antibody depletion assay was performed in eleven corresponding patients' sera.

    RESULTS:

    Sequence data of the PfMSP-1(19) revealed five variant forms in which the haplotypes Q/KNG/L and Q/KNG/F were predominant types and the second most frequent haplotype was E/KNG/F. In addition, the prevalence of IgG antibodies to all four PfMSP-1(19) variant forms was equal and high (84%) among the studied patients' sera. Immunodepletion results showed that in Iranian malaria patients, Q/KNG/L variant could induce not only cross-reactive antibody responses to other PfMSP-1(19) variants, but also could induce some specific antibodies that are not able to recognize the E/TSG/L or E/TSR/L variant forms.

    CONCLUSION:

    The present findings demonstrated the presence of non-variant specific antibodies to PfMSP-1(19) in Iranian falciparum malaria patients. This data suggests that polymorphism in PfMSP-1(19) is less important and one variant of this antigen, particularly Q/KNG/L, may be sufficient to be included in PfMSP-1(19)-based vaccine.

    Authors: Zakeri S, Mehrizi AA, Zoghi S, Djadid ND.
    Keywords: Non-variant, antibody responses, C-terminal region, merozoite surface protein-1, Plasmodium falciparum (PfMSP-1(19)), Iranians unstable malaria transmission
  • Summary:

    BACKGROUND &#38; OBJECTIVES:

    Species identification and information on transmission pattern of malaria parasite in any malaria endemic area is key to success for a malaria control programme. In this investigation, malaria diagnosis using molecular method was used to assess the transmission pattern of malaria parasite in three malaria endemic regions: Afghanistan, Iran and Pakistan.

    METHODS:

    Blood samples were collected from the patients presenting with vivax malaria from Afghanistan (n=108), Iran (n=200) and Pakistan (n=199). Malaria parasite detection was made by the gold standard (microscopy) and also nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene.

    RESULTS:

    Based on microscopy method, the level of mixed infection was zero to 2.5 per cent; however, nested-PCR assay detected 6.5, 22 and 23.5 per cent mixed infections in samples collected from Afghanistan, Iran and Pakistan, respectively. The present results showed that the co-infection of P. vivax with P. falciparum was frequent in malaria endemic regions of Iran and Pakistan.

    INTERPRETATION &#38; CONCLUSION:

    The present data suggest the need for improving microscopy diagnosis method and the clinician should also have careful clinical observation, along with the reports on Giemsa- stained thick blood films, particularly in summer time when P. vivax is predominant. Also sharing information on transmission pattern of mixed infection among these countries may help in designing better control strategies for malaria.

    Authors: Zakeri S, Kakar Q, Ghasemi F, Raeisi A, Butt W, Safi N, Afsharpad M, Memon MS, Gholizadeh S, Salehi M, Atta H, Zamani G, Djadid ND
    Keywords: mixed Plasmodium falciparum, P. vivax infections, nested-PCR
  • Summary:

    The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR-RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I(13)P(33)F(57)S(58)T(61)S(117)I(173)/A(383)A(553) (43.9%), I(13)P(33)F(57)S(58)T(61)N(117)I(173)/A(383)A(553) (33.6%) and I(13)P(33)F(57)R(58)T(61)N(117)I(173)/A(383)A(553) (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.

    Authors: Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Kakar Q, Atta H, Djadid ND
    Keywords: Plasmodium vivax, mutations, sulfadoxine-pyrimethamine, resistance, Plasmodium vivax, clinical isolates, Pakistan
  • Summary:

    The extract from Artemisia annua, containing artemisinin, has been proven active against multidrug resistant Plasmodium falciparum in previous studies. The purpose of this paper was to study five Artemisia species from Iran for their in vitro and in vivo antimalarial property and detection of artemisinin in the active species by chromatographic and spectroscopic methods including nuclear magnetic resonance (NMR) spectroscopy. Dried plants were extracted by 80% ethanol, and total extracts were investigated for antiplasmodial property and artemisinin content by TLC, HPLC, and (1)H-NMR techniques. Two plants (A. annua L. and Artemisia absinthium L.) showed good antiplasmodial activity against multidrug resistant and sensitive strain of P. falciparum. A. absinthium and A. annua at concentrations of 200 mg/kg for 4 days reduced parasitemia in BALB/C mice infected with Plasmodium bergei by 94.28% and 83.28%, respectively, but we could not detect artemisinin in all plants studied in this research. The antiplasmodial property of these two herbs is possibly related to essential oils that present in high amounts in their extracts.

    Authors: Ramazani A, Sardari S, Zakeri S, Vaziri B
    Keywords: In vitro, antiplasmodial and phytochemical study, Artemisia species, Iran, in vivo activity
  • Summary:

    BACKGROUND:

    There is an urgent need to identify new anti-malarial drug targets for both prophylaxis and chemotherapy, due to the increasing problem of drug resistance to malaria parasites. In the present study, the aim was to discover novel, effective plant-based extracts for the activity against malaria.

    METHODS:

    Ten plants found in Iran were selected by ethnobotanical survey of medicinal plants. The crude ethanolic extracts were tested for in vitro anti-plasmodial activity against two strains of Plasmodium falciparum: K1 (chloroquine-resistant strain) and CY27 (chloroquine-sensitive strain), using the parasite lactate dehydrogenase (pLDH) assay. The anti-plasmodial activity of the extracts was also assessed in the 4-day suppressive anti-malarial assay in mice inoculated with Plasmodium berghei (ANKA strain). Crude ethanolic extracts showed good anti-plasmodial activity were further fractionated by partitioning in water and dichloromethane.

    RESULTS:

    Of 10 plant species assayed, three species: Boerhavia elegans (Choisy), Solanum surattense (Burm.f.) and Prosopis juliflora (Sw.) showed promising anti-plasmodial activity in vitro (IC50 < or = 50 microg/ml) and in vivo with no toxicity. The dichloromethane fraction of three extracts revealed stronger anti-plasmodial activity than the total extracts.

    CONCLUSION:

    Anti-plasmodial activities of extracts of B. elegans and S. surattense are reported for the first time.

    Authors: Ramazani A, Zakeri S, Sardari S, Khodakarim N, Djadidt ND
    Keywords: In vitro and in vivo anti-malarial activity, Boerhavia elegans, Solanum surattense
  • Summary:

    This study was conducted to investigate the prevalence of Leptospira species in Mazandaran Province of Iran by using nested polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) methods and sequencing analysis. Blood samples (n = 119) were collected from humans suspected of having leptospirosis from different parts of the province in 2007. By using an indirect immunofluorescent antibody test (IFAT), we determined that 35 (29.4%) of 119 suspected cases had leptospiral antibody titers >/= 1:80, which confirmed the diagnosis of leptospirosis. Nested PCR assay also determined that 60 (50.4%) of 119 samples showed Leptospira infection. Furthermore, 44 (73.3%) of 60 confirmed leptospirosis amplified products were subjected to sequencing analysis. Sequence alignment identified L. interrogans, L. kirschneri, and L. wolffii species. All positive cases diagnosed by IFAT or PCR were in patients who reported contact with animals, high-risk occupational activities, and exposure to contaminated water. Therefore, it is important to increase attention about this disease among physicians and to strengthen laboratory capacity for its diagnosis in infected patients in Iran.

    Authors: Zakeri S, Sepahian N, Afsharpad M, Esfandiari B, Ziapour P, Djadid ND
    Keywords: Molecular epidemiology, leptospirosis, northern Iran, nested polymerase chain, reaction/restriction, fragment length, polymorphism, sequencing methods
  • Summary:

    OBJECTIVE:

    The objective of this study was to determine the frequency of dhfr and dhps resistance-associated haplotypes in Plasmodium falciparum isolates, three years after the introduction of sulfadoxine-pyrimethamine (SP) as the first-line antimalarial treatment in Iran.

    METHODS:

    Blood samples (N=182) were collected from patients presenting with falciparum malaria from southeastern Iran, and analyzed by nested-PCR/restriction fragment length polymorphism, followed by sequencing analysis.

    RESULTS:

    In pfdhfr, double mutation at positions 59R and 108N was a predominant allele with a prevalence of 95.7%. The pure double mutations of pfdhfr (I(51)N(108)) were detected, and showed an increase from 0.7% to 4.3% after the introduction of SP as first-line drug. Furthermore, a significant decrease in double mutations/wild-type of pfdhfr/pfdhps (R(59)N(108)/A(437)) was observed from 2004 (83.5%) to 2008 (44%) after changes in treatment policy. With regards to pfdhps, the results showed a rapid increase in frequency of the single pure form of pfdhps at position 437G (54.4%) and that of triple pfdhfr/pfdhps (R(59)N(108)/G(437)) mutant haplotype (51.7%) after three years.

    CONCLUSIONS:

    The absence of quintuple mutations in the examined isolates supports the continued use of SP as the treatment of choice for uncomplicated malaria as a partner drug to artemisinin combination therapy in Iran. However, the increase in the triple pfdhfr/pfdhps (R(59)N(108)/G(437)) mutant haplotypes indicates that the P. falciparum parasite populations have the potential to evolve into dhfr/dhps quintuple mutants in the near future. Therefore, monitoring the status of dhps alleles as a predictor of the development of clinical resistance to sulfadoxine should be a high priority in this region.

    Authors: Zakeri S, Farahani MS, Afsharpad M, Salehi M, Raeisi A, Djadid ND
    Keywords: 437G mutation, sulfadoxine resistance, Plasmodium falciparum, clinical isolates, sulfadoxine-pyrimethamine
  • Summary:

    BACKGROUND:

    Analysis of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations in Plasmodium vivax wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the dhfr and dhps genes in P. vivax clinical isolates circulating in two malaria endemic areas in Afghanistan.

    METHODS:

    P. vivax clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of the pvdhps genes using PCR-RFLP methods.

    RESULTS:

    All 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of pvdhfr and pvdhps haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I13P33F57S58T61N 117I173/A383A553 (6.4%).Double (I13P33S57R58T61N117I173/A383A553) and triple mutant haplotypes (I13P33S57R 58T61N117I173/G383A553) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates.

    CONCLUSION:

    The present study shows a limited polymorphism in pvdhfr from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the dhfr and dhps genes in both P. vivax and Plasmodium falciparum isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of P. vivax and P. falciparum molecular markers are needed to monitor the development of resistance to SP in the region.

    Authors: Zakeri S, Afsharpad M, Ghasemi F, Raeisi A, Safi N, Butt W, Atta H, Djadid ND
    Keywords: Molecular surveillance, Plasmodium vivax dhfr and dhps, mutations, Afghanistan
  • Summary:

    Leptospirosis is the most common zoonotic disease, which is transmitted to humans through contaminated water or direct exposure to the urine of infected animals. In this study, the presence and prevalence of Leptospira species in the infected samples of human (n=369) and sheep (n=75) sera and also dogs' urine (n=150), collected from four provinces of Iran, were investigated by using nested-PCR/RFLP assay followed by sequencing analysis. Nested-PCR assay detected that 98/369 (26.5%) human, 13/75 (17.33%) of sheep's sera and 33/150 (22%) dogs' urine samples were positive for Leptospira DNA. RFLP assay detected that all positive cases had either pathogenic or intermediate Leptospira species. By sequence analysis, Leptospira interrogans was the most prevalent species among the examined samples of human (53/82, 64.6%) and sheep (11/13, 84.6%). However, in dog samples, Leptospira wolffii (27/29, 93.1%) was detected for the first time and was the dominant species. The presence of L. wolffii with 100% identity in clinical human samples and animals suspected with Leptospira may provide evidence for circulation of L. wolffii and its role in transmission cycle within human and animal hosts. In addition, this species can be potentially pathogenic to human and probably animal hosts. A large epidemiology survey would be needed to define the presence and the prevalence of this species in global endemic regions.

    Authors: Zakeri S, Khorami N, Ganji ZF, Sepahian N, Malmasi AA, Gouya MM, Djadid ND
    Keywords: Leptospira wolffii, pathogenic Leptospira species, human, sheep and dog
  • Summary:

    The current study aimed to provide further evidence on the status of species composition, insecticide resistance, and vectorial capacity within the members of Anopheles (Anopheles) Hyrcanus Group in Ardebil, Giulan, and Khuzestan provinces of Iran. Sequencing the internal transcribed spacer 2 (ITS2) of ribosomal DNA gene led to identification of two members of Hyrcanus complex: Anopheles hyrcanus Pallas and a new species/form, hereafter called Anopheles hyrcanus sp(IR) as a world record. Furthermore, we identified and compared partial sequences of exons I and II and the whole intron I region of insecticide resistance-related voltage-gated sodium channel (vgsc) gene in populations of Hyrcanus Group and other main old world Anopheles species. The ITS2 and vgsc sequences in members of Hyrcanus Group and other Anopheles species were used for construction of phylogenetic tree, which demonstrated the evolutionary relatedness among Western and Eastern Palearctic taxa within the Hyrcanus Group. A nested polymerase chain reaction assay for detection of Plasmodium species revealed the infection of Plasmodium falciparum within An. hyrcanus collected from Fooman district in Guilan province. The data from this study led to the introduction of a new member/form within the Hyrcanus Group, identification and definition of the status of knockdown resistance related to pyrethroids and DDT in their vgsc gene, detection of Plasmodium infection, and further evidence on genetic relatedness within these taxa. The overall results may suggest reconsidering the role ofAn. hyrcanus in malaria transmission, which would be useful for implementation and evaluation of malaria control programs in Western Palearctic region.

    Authors: Djadid ND, Jazayeri H, Gholizadeh S, Rad ShP, Zakeri S
    Keywords: Anopheles Hyrcanus Group, molecular identification, diagnosis, phylogeny, status of kdr resistance,Plasmodium infection
  • Summary:

    In this study, the nature and extent of genetic diversity of Plasmodium vivax populations circulating in Afghanistan have been investigated by analyzing three genetic markers: csp, msp-1, and msp-3 alpha. Blood samples (n=202) were collected from patients presenting with vivax malaria from south-western (Herat) and south-eastern (Nangarhar) parts of Afghanistan, and analysed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 revealed type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant in parasites in both study areas. The sequence analysis of 57 P. vivax isolates identified a total of 26 distinct alleles. Genotyping pvcsp gene showed that VK210 type (86.6%) is predominant in Afghanistan. Moreover, three major types of the pvmsp-3 alpha locus: type A, type B and type C were distinguished among Afghani isolates. The predominant fragments among Nangarhar and Herat parasites were type A (70.8% and 67.9%, respectively). PCR/RFLP products with Hha I and Alu I were detected 52 and 38 distinct variants among Nangarhar and Herat isolates, respectively. These results strongly indicate that the P. vivax populations in Afghanistan are highly diverse.

    Authors: Zakeri S, Safi N, Afsharpad M, Butt W, Ghasemi F, Mehrizi AA, Atta H, Zamani G, Djadid ND
    Keywords: Genetic structure, Plasmodium vivax isolates, malaria endemic areas, Afghanistan
  • Summary:

    BACKGROUND:

    The identification of key molecules is crucial for designing transmission-blocking vaccines (TBVs), among those ookinete micronemal proteins are candidate as a general class of malaria transmission-blocking targets. Here, the sequence analysis of an extra-cellular malaria protein expressed in ookinetes, named von Willebrand factor A domain-related protein (WARP), is reported in 91 Plasmodium vivax isolates circulating in different regions of Iran.

    METHODS:

    Clinical isolates were collected from north temperate and southern tropical regions in Iran. Primers have been designed based on P. vivax sequence (ctg_6991) which amplified a fragment of about 1044 bp with no size variation. Direct sequencing of PCR products was used to determine polymorphism and further bioinformatics analysis in P. vivax sexual stage antigen, pvwarp.

    RESULTS:

    Amplified pvwarp gene showed 886 bp in size, with no intron. BLAST analysis showed a similarity of 98-100% to P. vivax Sal-I strain; however, Iranian isolates had 2 bp mismatches in 247 and 531 positions that were non-synonymous substitution [T (ACT) to A (GCT) and R (AGA) to S (AGT)] in comparison with the Sal-I sequence.

    CONCLUSION:

    This study presents the first large-scale survey on pvwarp polymorphism in the world, which provides baseline data for developing WARP-based TBV against both temperate and tropical P. vivax isolates.

    Authors: Gholizadeh S, Djadid ND, Basseri HR, Zakeri S, Ladoni H
    Keywords: von Willebrand factor, A domain-related protein (WARP), polymorphism temperate and tropical Plasmodium vivax, field isolates
  • Summary:

    In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3alpha (msp-1 and msp-3alpha) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n=187) and Iran (n=150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3alpha, three major types: type A (1800bp), type B (1500bp) and type C (1200bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3alpha with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.

    Authors: Zakeri S, Raeisi A, Afsharpad M, Kakar Q, Ghasemi F, Atta H, Zamani G, Memon MS, Salehi M, Djadid ND
    Keywords: Molecular characterization, Plasmodium vivax, clinical isolates, Pakistan, Iran, pvmsp-1, pvmsp-3alpha, pvcsp genes, molecular markers
  • Summary:

    The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1(19)) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1(19) were evaluated in individuals (n=94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1(19) and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1(19), the frequency of antibodies was determined in the infected subjects (n=74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1(19) was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1(19). These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1(19)-based vaccine.

    Authors: Mehrizi AA, Zakeri S, Salmanian AH, Sanati MH, Djadid ND
    Keywords: IgG, antibody, C-terminal region, merozoite surface, protein 1 of Plasmodium vivax, unstable hypoendemic region, Iran
  • Summary:

    BACKGROUND:

    In Iran, co-infections of Plasmodium vivax and Plasmodium falciparum are common and P. vivax infections are often exposed to sulphadoxine-pyrimethamine (SP). In the present study, the frequency distribution of mutations associated to SP resistance was investigated in pvdhfr and pvdhps genes from field isolates.

    METHODS:

    Clinical isolates of P. vivax were collected in two different malaria endemic regions in northern and south-eastern Iran, between 2001 and 2006. All 189 collected isolates were analysed for SNP/haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of pvdhps genes using nested PCR-RFLP methods

    RESULTS:

    All 189 examined isolates were found to carry wild-type amino acids at positions 13, 33, 61 and 173, while 57L and 58R and 117N mutations in pure form was detected among 1.1%, 17.5% and 26% examined samples, respectively, with no polymorphisms in different loci of dhps genes. Based on size polymorphism of pvdhfr genes at repeat region, among northern isolates, the frequency distribution for type A and B were 2.2% and 97.8% respectively. However, in southern samples the prevalence of type A, B and C were 7%, 89.5% and 7.7%, respectively. Mixed genotype infections (type B and C) were detected in only 4.2% (6/143) of southern, but in none of the northern isolates. The combination of pvdhfr and pvdhps haplotypes among all 189 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were I13P33F57S58T61S117I173/A383A553 (65.6%) and I13P33F57S58T61N117I173 (16.4%). Two other alleles with one point mutation I13P33F57R58T61S117I173/A383A553 and two mutations I13P33F57R58T61N117I173/A383A553 accounted for 7.4% and 9.5% of the total isolates.

    CONCLUSION:

    The present molecular data provide important information for making decisions on population based drug use in Iran. In addition, since October 2005, with more availability of SP as first-line treatment, P. vivax isolates are more exposed to SP and the selection or spread of resistant pvdhfr and pvdhps alleles might increase in the near future in this region.

    Authors: Zakeri S, Motmaen SR, Afsharpad M, Djadid ND
    Keywords: Molecular characterization, antifolates resistance-associated genes, Plasmodium vivax isolates, Middle East
  • Summary:

    The region II of Plasmodium vivax Duffy binding protein (PvDBP-II) contains the critical binding residues, which is a major target for development of naturally acquired immunity. Several studies showed sequence polymorphisms in PvDBP-II, which may inhibit antibodies recognition. Therefore, in this study the level of PvDBP-II polymorphism within and among P. vivax populations from re-emerged areas in north and endemic areas in south of Iran were evaluated by sequencing analysis in 75 isolates for the first time. Fourteen non-synonymous and one synonymous mutations were identified and none of the amino acid substitutions were directly involved in erythrocyte binding. Only 6 out of 14 detected mutations have been found among northern isolates, including D384G, R390H, N417K, L424I, W437R, and I503K. In total, two and nine different variants have been identified among northern and southern isolates, respectively. High association of the amino acid frequencies for codons 417, 437, and 503 were found among northern (85% for trio association and 100% for N417K with W437R), and southern (36% for trio association and 98% for N417K with W437R) samples. Polymorphisms at positions R308S, K371E, D384G, K386N, R390H, N417K, L424I, W437R, and I503K were identified from Iran and diverse geographic areas; however, mutation at position F306L was only reported from Asian malaria endemic areas. It is suggested that to develop polyvalent vaccine against P. vivax infection, it is better to incorporate the common and high prevalent allelic variants of the antigen that were reported from different malaria endemic regions.

    Authors: Babaeekho L, Zakeri S, Djadid ND
    Keywords: Genetic mapping, the duffy binding protein (DBP), ligand domain, Plasmodium vivax from unstable malaria region in the Middle East
  • Summary:

    A rapid and specific nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to detect and differentiate pathogenic and nonpathogenic Leptospira spp. Leptospiral genomic DNA was extracted from suspected human sera using an improved method of standard phenol-chloroform, and specific primers have been used to amplify 16S ribosomal RNA from all pathogenic and nonpathogenic Leptospira spp. The PCR products of all nonpathogenic species were digested with ApoI enzyme, but not pathogenic. To evaluate this assay, we analyzed 283 serum samples collected from suspected patients with leptospirosis. Nested PCR assay confirmed that 42 (14.8%) of 283 samples harbored Leptospira infection, and RFLP assay confirmed 38 (90.5%) of 42 and 4 (9.5%) of 42 positive cases had pathogenic and nonpathogenic Leptospira spp., respectively. Based on sequencing results, Leptospira interrogans, Leptospira kirschneri, and Leptospira wolffii and nonpathogenic Leptospira biflexa and Leptospira genomospecies 3 have been detected among analyzed samples. The nested PCR-RFLP assay developed in this study fulfills this requirement in the early stage of infection.

    Authors: Djadid ND, Ganji ZF, Gouya MM, Rezvani M, Zakeri S
    Keywords: nested polymerase, polymorphism technique, pathogenic and nonpathogenic Leptospira spp
  • Summary:

    The leading candidates for a Transmission Blocking Vaccine (TBV) in Plasmodium vivax parasite are the ookinete surface protein 25 (Pvs25) and Pvs28, which their phase I clinical trial is ongoing. Therefore, we carried out survey of polymorphisms of the pvs25 and pvs28 genes in P. vivax populations that are circulating in the two malaria areas of contrasting endemicity in Iran, before field application of the TBV. To characterize the polymorphisms of pvs25 and pvs28 genes, 50 isolates were analyzed by sequencing method and their gene structure was compared with parasite populations from India, Bangladesh, Indonesia, Thailand, Mexico and Brazil. Three mutations were detected in pvs25 and pvs28 including Q87K, E97Q, I130T and M52L, T65K, T140S with two and four distinct haplotypes, in comparison with the Sal I sequence type, respectively. Both haplotypes of Pvs25 were found among northern and southern P. vivax isolates; however, only two and three of the Pvs28 variants were observed among the northern and southern isolates, respectively. In conclusion, the present results show the limited sequence polymorphism of the pvs25 and pvs28 genes among field P. vivax population in Iran. These results highly encourage with respect to applicability of Pvs25 and Pvs28-based vaccine against P. vivax infection in the region, where these parasites are prevalent, whether these occur in the temperate or tropical zones.

    Authors: Zakeri S, Razavi S, Djadid ND
    Keywords: Genetic diversity, transmission blocking, vaccine candidate (Pvs25 and Pvs28) antigen, Plasmodium vivax, clinical isolates, Iran
  • Summary:

    This study was designed to analyze the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) mutations as markers of chloroquine (CQ) resistance in 200 blood samples collected from malaria patients in south-eastern Iran during 2002-2005. Among these, 25 (post-treatment) fulfilled the 28-day follow-up study. A high number of Iranian P. falciparum (97%) strains harbored quadruple mutations at codons 76T, 220S, 326D, and 356L. All post-treatment isolates harbored the mutant allele 76T, but low rates of the mutant allele 86Y (44%) of the pfmdr1 gene were detected. No wild haplotype of pfcrt (72-CVMNKAQNIR-371) was found in post-treatment samples; however, 56% of clinical "failure" samples carried the wild type of pfmdr1 (NYSND). The present results suggest a strong association between pfcrt 76T, but not pfmdr1 86Y mutation and in vivo CQ resistance. Furthermore, we found the CQ resistance-associated SVMNT haplotype, which previously had been seen in South American isolates. Although Iran is located more proximally to Southeast Asia than to South America, no CQ resistance-associated CVIET haplotye has been observed in this region. Therefore, these results were not consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. In conclusion, P. falciparum mutations associated with resistance to CQ are abundant in south-eastern Iran and this finding strongly supports that CQ as the first line drug is inadequate for treatment of uncomplicated falciparum malaria in Iran.

    Authors: Zakeri S, Afsharpad M, Kazemzadeh T, Mehdizadeh K, Shabani A, Djadid ND
    Keywords: pfcrt, chloroquine resistance, Iranian isolates, Plasmodium falciparum
  • Summary:

    C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1) isolated from different parts of the world revealed sequence variability, however no data exist on sequence heterogeneity of this region from Iran. To address this question, DNA encoding the carboxyl (C)-terminal region of PfMSP-1 was amplified in 144 Iranian P. falciparum clinical isolates, using allele type-specific primers. In this study both MAD20 (88.2%) and K1 (7.6%) types were detected. Sequence analysis of 33 and 92 fragments corresponding to pfmsp-1(42) and pfmsp-1(19) revealed eight (15MAD1-15MAD7 and 15KCH) and five [A1 (E/TSR/L), A2 (Q/KNG/F), A3 (E/KNG/F), A4 (E/TSG/L), and A5 (Q/KNG/L)] distinct haplotypes, respectively. E/TSG/L variant type was the predominant haplotype, and reported only from Thailand and India, but E/KNG/L is widespread in Africa, Asia, and Latin America; but not found among Iranian isolates. In summary, result of this study indicates limited antigenic diversity, and thus support the potential utility of the C-terminal region of PfMSP-1 in designing polyvalent vaccine constructs.

    Authors: Mehrizi AA, Zakeri S, Salmanian AH, Sanati MH, Djadid ND
    Keywords: Plasmodium falciparum, sequence analysis, gene, C-terminus region, merozoite surface protein-1, malaria vaccine antigen, Iranian clinical isolates
  • Summary:

    BACKGROUND:

    Anopheles culicifacies is a main malaria vector in southeastern part of Iran, bordring Afghanistan and Pakistan. So far, resistance to DDT, dieldrin, malathion and partial tolerance to pyrethroids has been reported in An. stephensi, but nothing confirmed on resistance status of An. culicifacies in Iran.

    METHODS:

    In current study, along with WHO routine susceptibility test with DDT (4%), dieldrin (0.4%), malathion (5%), permethrin (0.25%), lambadacyhalothrin (0.1%), and deltamethrin 0.025, we cloned and sequenced segment VI of domain II (SII6) in voltage-gated sodium channel (vgsc) gene of An. culicifacies specimens collected in Sistan and Baluchistan province (Iran).

    RESULTS:

    A 221-bp amplified fragment showed 91% and 93% similarity with exon I and exon II of An. gambiae. The size of intron II in An. culicifacies is 62 bp, while in An. gambiae is 57 bp. The major difference within An. culicifacies specimens and also with An. gambiae is in position 29 of exon I, which led to substitution of Leu to His amino acid.

    CONCLUSION:

    This data will act as first report on partial sequence of vgsc gene and its polymorphism in An. culicifacies. A Leu to His amino acid substitution detected upstream the formerly known knockdown resistance (kdr) mutation site could be an indication for other possible mutations related to insecticide resistance. However, the result of WHO susceptibility test carried out in Baluchistan of Iran revealed a level of tolerance to DDT and dieldrin, but almost complete susceptibility to pyrethroids in An. culicifacies. We postulate that the molecular diagnostic tool developed for detection and identification of kdr-related mutations in An. culicifacies, could be useful in monitoring insecticide resistance in Iran and neighbouring countries such as Pakistan and Afghanistan. A phylogenetic tree also constructed based on the sequence of exon I and II, which readily separated An. culicifacies populations from An. stephensi, An. fluviatilis and An. gambiae.

    Authors: Djadid ND, Forouzesh F, Karimi M, Raeisi A, Hassan-Zehi A, Zakeri S
    Keywords: Monitoring pyrethroid, insecticide resistance, malaria vector Anopheles culicifacies, molecular tools, conventional susceptibility test
  • Summary:

    BACKGROUND:

    This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran.

    METHODS:

    The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003-2005.

    RESULTS:

    All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas.

    CONCLUSION:

    Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria.

    Authors: Zakeri S, Afsharpad M, Raeisi A, Djadid ND
    Keywords: Prevalence of mutations, antimalarial drugs, Plasmodium falciparum isolates, sulphadoxine-pyrimethamine, first-line treatment, Iran
  • Summary:

    Parasite genotyping studies have indicated that the Plasmodium falciparum populations circulating in Iran are genetically diverse and that multiple genotype infections are observed regularly. We wished to extend the analysis to the Pfcsp gene, coding for the dominant sporozoite surface antigen on which the leading malaria vaccine candidate RTS,S is based. Infected blood samples were collected mainly from Iranian, as well as Afghani and Pakistani, patients on admission with falciparum malaria. DNA was purified from 90 isolates, and from these, 21 fragments corresponding to Pfcsp and 69 fragments corresponding to the 3'-end conserved domain were amplified and sequenced. Overall diversity was low. Six patterns were noted for the repeat region, but mixed genotypes were not observed in any of the isolates. T cell epitopes also displayed limited diversity, with only five haplotypes (combined Th2R/Th3R epitopes) noted, and of these, three were dominant, accounting for 94% of the 90 sequences. These observations are akin to those observed in Thai P. falciparum isolates, where a particular Pfscp Th2R/Th3R haplotype seems to be maintained in an otherwise genetically diverse parasite population. The data imply that the selective pressure that maintains a restricted T cell epitope is caused by factors outside the mammalian host immune responses. Furthermore, they sustain the notion that protective responses induced by RTS,S vaccination are not strain-specific.

    Authors: Zakeri S, Avazalipoor M, Mehrizi AA, Djadid ND, Snounou G
    Keywords: Restricted T-cell, epitope diversity, circumsporozoite protein, Plasmodium falciparum populations, Iran
  • Summary:

    BACKGROUND:

    Members of Anopheles maculipennis complex are effective malaria vectors in Europe and the Caspian Sea region in northern Iran, where malaria has been re-introduced since 1994. The current study has been designed in order to provide further evidence on the status of species composition and to identify more accurately the members of the maculipennis complex in northern Iran.

    METHODS:

    The second internal transcribed spacer of ribosomal DNA (rDNA-ITS2) was sequenced in 28 out of 235 specimens that were collected in the five provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan in Iran.

    RESULTS:

    The length of the ITS2 ranged from 283 to 302 bp with a GC content of 49.33-54.76%. No intra-specific variations were observed. Construction of phylogenetic tree based on the ITS2 sequence revealed that the six Iranian members of the maculipennis complex could be easily clustered into three groups: the An. atroparvus-Anopheles labranchiae group; the paraphyletic group of An. maculipennis, An. messeae, An. persiensis; and An. sacharovi as the third group.

    CONCLUSION:

    Detection of three species of the An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as shown as new records in northern Iran, is somehow alarming. A better understanding of the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to the correct identification of species complexes, to the detection of Plasmodium composition in Anopheles vectors and to the status of insecticide resistance by looking to related genes.

    Authors: Djadid ND, Gholizadeh S, Tafsiri E, Romi R, Gordeev M, Zakeri S
    Keywords: Molecular identification, Palearctic members, Anopheles maculipennis, northern Iran
  • Summary:

    Glutathione S-transferases (GSTs) are soluble dimeric proteins that are involved in the metabolism, detoxification, and excretion of a large number of endogenous and exogenous compounds such as insecticides from the cell. In the current study, field specimens of Anopheles stephensi Liston, Anopheles fluviatilis James, and Anopheles culicifacies Giles collected from Sistan and Baluchistan province in Iran and subjected to World Health Organization susceptibility test. Only An. stephensi was resistant to 4% DDT. DNA extraction and rDNA-ITS2-polymerase chain reaction (PCR) for correct species identification, followed by amplification of GSTe2 gene, including exon I and II and full sequence of intron I, identified a 500-bp fragment in these three species. These fragments were purified and sequenced from both ends. The comparison of coding sequence of GSTe2 gene between these species and with Anopheles gambiae Giles showed 82 to 86% similarity at nucleic acid levels and identified nucleotide polymorphisms within An. culicifacies and An. stephensi populations. Species-specific differences have been detected in intron I of GSTe2 gene. This is in concordance with the previous studies and confirmed the conserved nature of intron sequence in GSTe2 gene of each species, probably useful as a molecular marker for species-specific identification. Phylogenetic analysis based on rDNA-ITS2, and coding (exon I and II) and noncoding sequences of GSTe2, showed the systematic relatedness between Iranian malaria vectors and the possibility of using these sequences in both differentiation of Anopheles species and defining their evolutionary relationship with the only available GSTe2 sequence of An. gambiae. These data may be useful for implementation and evaluation of malaria control programs in aspects of population genetics and molecular resistance.

    Authors: Djadid ND, Barjesteh H, Raeisi A, Hassanzahi A, Zakeri S
    Keywords: Identification, sequence analysis, comparative study, GSTe2 insecticide, resistance gene,malaria vectors, Anopheles stephensi, Anopheles culicifacies, Anopheles fluviatilis
  • Summary:

    BACKGROUND:

    The knowledge on population structure of the parasite isolates has contributed greatly to understanding the dynamics of the disease transmission for designing and evaluating malaria vaccines as well as for drug applications. msp-1 and msp-3alpha genes have been used as a genetic marker in population studies of Plasmodium vivax isolates. In this study, msp-3alpha was compared and assessed with msp-1 marker in order to find whether msp-3alpha is a reliable genetic marker for P. vivax population studies.

    METHODS:

    This comparative study was designed and carried out as the first assessment of diversity in Pvmsp-3alpha gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the 50 northern and 94 southern P. vivax isolates from Iran, which had been analysed before for msp-1 gene.

    RESULTS:

    Three allele size as, Type A (1.8 kb), Type B (1.5 kb) and Type C (1.2 kb) have been detected among both northern and southern isolates based on PCR results. Type C (70%) and Type A (68.7%) were the predominant fragments among northern and southern parasites, respectively. 99 distinct Pvmsp-3alpha fragments defined by the size were detected in the 94 southern samples by PCR analysis. However, no mixed genotype infections have been detected among northern isolates. Based on restriction pattern from digestion with Hha I and Alu I 12 and 49 distinct allelic variants have been detected among 50 northern and 94 southern isolates. However, based on msp-1 gene, 30 distinct variants identified in all 146-sequenced Iranian P. vivax isolate.

    CONCLUSION:

    The results suggested that PCR-RFLP on msp-3alpha gene is an adequate, applicable and easily used technique for molecular epidemiology studies of P. vivax isolates without the need for further sequencing analysis.

    Authors: Zakeri S, Barjesteh H, Djadid ND.
    Keywords: Merozoite surface protein-3alpha, population, genetic analysis, Plasmodium vivax
  • Summary:

    To date, there is no information on the genetic diversity of the circumsporozoite protein (CSP), a leading vaccine candidate, in Plasmodium vivax populations circulating in Iran. The gene for this protein, Pvcsp, was amplified from 374 P. vivax isolates collected in the temperate northern, and in the tropical southern endemic areas. PCR-RFLP analysis of the repeated central region revealed that the parasites collected in the northern area were almost exclusively of the VK210 type. Parasites collected in the south-eastern areas were of both VK210 and VK247 types. We detected VK210 parasite in 70.5% of the samples, VK247 parasites in 17.5% and mixed type infections in 12% of the isolates. Sequence analysis of 137 isolates obtained from both areas identified a total of 25 distinct genotypes. The degree of genetic diversity was generally higher for the tropical (21 genotypes) than the temperate (7 genotypes) P. vivax populations, a difference possibly reflecting the high cross-border exchanges between Afghanistan and Pakistan and southern Iran. Interestingly, all but two VK210 type isolates sequenced harboured a 36-bp post-repeat insert previously only observed in North Korea and China. This large-scale survey of parasite diversity in the Eastern Mediterranean Region provides a set of baseline data suitable for future molecular epidemiological studies of P. vivax.

    Authors: Zakeri S, Abouie Mehrizi A, Djadid ND, Snounou G
    Keywords: Circumsporozoite protein, gene diversity, temperate and tropical Plasmodium vivax isolates, Iran
  • Summary:

    To obtain the genetic structure of Plasmodium vivax populations in the northern and southern malaria-endemic areas in Iran, which differ in endemicity, sequence diversity in the variable block 5 and the C-terminal part of P. vivax merozoite surface protein 1 (Pvmsp 1) was analyzed. The variable block 5 fragment from 52 northern and 94 southern isolates was amplified and sequenced. Type 1, type 2, and recombinant type 3 allelic variants were found in both northern and southern isolates, with type 1 predominant in parasites from the north and type 2 in those from the south. A total of 7 and 27 distinct variants were detected among northern and southern isolates, respectively. A single variant predominated (71%) in the northern isolates, whereas variants were evenly distributed among southern isolates, with only two exceeding 10%. Thus, parasites from the southern malaria-endemic area were more polymorphic than those circulating in the northern area, where malaria is a re-emerging disease. Sequence alignments showed that although some variants were found only in northern or southern isolates, some were common to both and had also been observed in parasites from Azerbaijan, Turkey, Thailand, Bangladesh, and China. The Pvmsp 1 fragment corresponding to the C-terminal region was also amplified and the sequences derived from 20 northern and 50 southern isolates were identical. This high degree of conservation reinforces the potential of this polypeptide fragment for inclusion in synthetic vaccines being developed against P. vivax.

    Authors: Zakeri S, Mehrizi AA, Mamaghani S, Noorizadeh S, Snounou G, Djadid ND
    Keywords: Population structure analysis, Plasmodium vivax, iran, malaria endemicity
  • Summary:

    This study compared basic microscopy with molecular detection of Plasmodium species. According to thick-film microscopy, 100% of 142 malaria cases in Pars-Abad, Ardebil province, were infected with a single species, P vivax. However, nested polymerase chain reaction (PCR) detected mixed species infections of both P. vivax and P. falciparum in 7.0%. In Mazanderan province, 2/20 blood films were diagnosed with only P. falciparum and 18/20 with only P. vivax. However, nested PCR detected 17/20, 2/20 and 1/20 with P. vivax only, P. falciparum only and mixed species respectively. The unexpected presence of P. falciparum urges prompt investigation and immediate treatment of malaria cases in this region.

    Authors: Zakeri S, Mamaghani S, Mehrizi AA, Shahsavari Z, Raeisi A, Arshi S, Dinparast-Djadid N
    Keywords: mixed P. vivax, P. falciparum infections, northern Islamic Republic of Iran
  • Summary:

    Anopheles stephensi is one of the most important malaria vectors in the Middle-East, the Indian subcontinent, the Far-East and is the main malaria vector in south of Iran. This vector is thought to be a single but polytypic species, despite its enormous geographical range. To examine this hypothesis, we analyzed the rDNA-ITS2 and RAPD loci in different populations of An. stephensi from Iran. rDNA-ITS2 region in all sequenced specimens of An. stephensi contained a (CA)7 microsatellite sequence. Construction of phylogenetic tree based on rDNA-ITS2 sequences revealed that there only is a minor polymorphism between the different populations, despite their vast geographical distances. RAPD-PCR could differentiate rural and urban populations of An. stephensi, but it is unclear whether these two samples represent mysorensis and the type form. Further characterization of interested RAPD fragments by cloning; have shown the nature of inverted repeats and the presence of microsatellite region (GT) in both ends near to inverted repeat sequences of primers. These results showed that An. stephensi in Iran could be considered a single species with different biological and ecological forms in different zoogeographical zones. Further studies are needed to demonstrate the relation between RAPD and microsatellite sequences and the differences seen in the field for this species. This data will serve as first report on the sequence of rDNA-ITS2 and a microsatellite-containing RAPD region, which could be used for species-specific diagnosis and differentiation of urban and rural populations in An. stephensi.

    Authors: Djadid ND, Gholizadeh S, Aghajari M, Zehi AH, Raeisi A, Zakeri S
    Keywords: Genetic analysis, rDNA-ITS2, RAPD loci, malaria vector, Anopheles stephensi (Diptera: Culicidae), control program in Iran
  • Summary:

    In Iran, malaria transmission mainly occurs in south-eastern regions through both Plasmodium falciparum and P. vivax. The genetic diversity of P. falciparum isolates was analysed in 108 patients attending the regional hospital in Chabahar District, using the molecular markers msp1 and msp2. Multiple genotypes were detected in 87% of patients and the mean numbers of msp1 and msp2 genotypes were 2.51 (95% CI: 2.29-2.73) and 2.61 (95% CI: 2.39-2.83) respectively. Various allelic types of msp1 and msp2 were found, with msp2 3D7/IC type detected in 94% of infections. Plasmodium falciparum infections in south-east Iran appear to have a higher genetic diversity than expected for an area of low transmission. A situation of higher transmission in this area may be emerging, possibly because of reduced efficacy of first-line treatments.

    Authors: Zakeri S, Bereczky S, Naimi P, Pedro Gil J, Djadid ND, Färnert A, Snounou G, Björkman A
    Keywords: Multiple genotypes, merozoite surface proteins 1 and 2, Plasmodium falciparum infections, hypoendemic area, Iran
  • Summary:

    The merozoite surface protein of Plasmodium vivax (PvMSP-1) has been considered as a vaccine candidate, which exhibits antigenic diversity among isolates. We investigated the extent of sequence variation in the polymorphic region 5 of PvMSP-1 in order to characterize the genetic structure and composition of P. vivax in clinical isolates from Iranshahr and Chahbahar districts of Sistan and Baluchistan province, Iran. The PvMSP-1 gene amplification revealed size variation among the isolates, ranging from 430 to 550 bp. Sequences were obtained for 15 Iranian and one Pakistani isolates and 14 different alleles were detected. Results also showed three distinct sequence types of the polymorphic region. Sequence analysis has shown several single nucleotide polymorphisms to occur in this block of PvMSP-1, creating different alleles in the progeny and also microheterogeneity in the region. Thus, this study provides preliminary evidence of sequence heterogeneity in the Iranian P. vivax population.

    Authors: Zakeri S, Dinparast Djadid N, Zeinali S
    Keywords: Sequence heterogeneity, merozoite surface protein-1 gene, Plasmodium vivax, wild isolates, southeastern Iran
  • Summary:

    BACKGROUND:

    Rapid diagnosis and correct treatment of cases are the main objectives of control programs in malaria-endemic areas.

    METHODS AND RESULTS:

    To evaluate these criteria and in a comparative study, blood specimens were collected from 120 volunteers seeking care at the Malaria Health Center in Chahbahar district. One hundred and seven out of 120 Giemsa-stained slides were positive for malaria parasites by microscopy. Eighty-four (70%) and 20 (16.7%) were identified as having only Plasmodium vivax and Plasmodium falciparum infections, respectively, while only 3 (2.5%) were interpreted as having mixed P. vivax-P. falciparum infections. The target DNA sequence of the 18S small sub-unit ribosomal RNA (ssrRNA) gene was amplified by Polymerase Chain Reaction (PCR) and used for the diagnosis of malaria in south-eastern Iran. One hundred twenty blood samples were submitted and the results were compared to those of routine microscopy. The sensitivity of PCR for detection of P. vivax and P. falciparum malaria was higher than that of microscopy: nested PCR detected 31 more mixed infections than microscopy and parasite positive reactions in 9 out of the 13 microscopically negative samples. The results also confirmed the presence of P. vivax and P. falciparum.

    CONCLUSIONS:

    These results suggest that, in places where transmission of both P. vivax and P. falciparum occurs, nested PCR detection of malaria parasites can be a very useful complement to microscopical diagnosis.

    Authors: Zakeri S, Najafabadi ST, Zare A, Djadid ND
    Keywords: malaria parasites, nested PCR, south-eastern Iran, highly mixed infections
  • Summary:

    The treatment of cancer usually involves lethal effect on normal body cells as side effects. Cheminformatics methodology can play a significant role in biomed/clinical scientific research. Similarity searching is a standard cheminformatics tool in drug discovery area and database design. In this study, five novel herbal extracts in combination with doxorubicin and cisplatin have been used to sensitize ACHN and A2780/cp cells. These herbal extracts have been selected on the basis of novel cheminformatics methodology and assayed for the first time. The findings confirmed predicted outcomes from the in silico research and the results introduced may bring to use the effects of these herbs in reversing of multidrug resistance (MDR) phenomenon.

    Authors: Ghavami G, Sardari S, Ali Shokrgozar M
    Keywords: Cheminformatics, selection and synergism,herbal extracts, anticancer agents, drug resistance tumor cells-ACHN,A2780/cp cell lines
  • Summary:

    Cancer is a multi-factorial disease resulting in uncontrolled division of body cells, with difficult and complex suppression. The main treatment strategy for this disease depends on killing of tumor cells that usually exist in the proximity of normal body cells. During the course of treatment, the healthy cells should not be affected by the toxic doses of the drug. Therefore, it seems that combination drug therapy is a suitable solution to address the mentioned concern. Indeed the use of multiple drugs with different actions and/or targets, in order to overcome the tumor cells, can lead to decreased drug effective dose and increased protection of normal cells against antitumor drugs. This review is focused on informatics applications in cancer combination drug therapy. At first, a brief description of recent findings on biology of resistance to cytotoxic agents is covered. Then, combinational drug therapy in cancer treatment, cheminformatics applications of synergistic compounds in cancer therapy, strategies used to overcome MDR (Multi Drug Resistance) and combinational drug therapy in cancer treatment have been discussed in the continuation. Natural compounds synergistic with anticancer agents have been reviewed in the following topics and lastly, the recent patents in the related area of combinational therapy are briefed.

    Authors: Ghavami G, Kazemali MR, Sardari S
    Keywords: Informatics, drug synergism, anticancer agents
  • Summary:

    The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.

    Authors: Fahham N, Sardari S, Ostad SN, Vaziri B, Ghahremani MH
    Keywords: C-terminal domain, p16(INK4a),cell cycle arrest, growth inhibition, CDK4/6 interaction, full length protein, HT-1080 fibrosarcoma cells
  • Summary:

    Bioinformatics and traditional medicine can be used in discovery and design of novel candidate drugs to efficient cancer chemotherapy. In this study, similarity search tools employed to screen and introduce novel herbs with antitumor property. Several novel herbs have been selected by using logical computational algorithms and assayed on six cancerous cell lines. Complementary assays involved hemolytic and antifungal MIC tests have been performed to determine selectivity and their biocompatibility with RBC of herbal extracts. Final findings may point at selective activity of herbal extracts Rheum ribes, Ficus bengalensis, Morus alba, Musa sapientum, Arnebia decumbens, Citrus limon, Fraxinus excelsior, Rumex acetosella, Arnebia echioides in inducing cytotoxicity on cancerous cell lines. In the present research, in vitro results confirmed predicted findings from our in silico work. Complementary assays including antifungal MIC and hemolytic tests were carried out also to determine selectivity of herbal extracts. Findings resulted from hemolytic test showed that candidate herbal extracts did not induce hemolysis similar to negative control, also antifungal test results indicated that six herbal extracts had antifungal activity in concentration of 250 microg/ml.

    Authors: Sardari S, Shokrgozar MA, Ghavami G
    Keywords: Cheminformatics, selection and cytotoxic effects, herbal extracts
  • Summary:

    Protein-protein interactions studies can greatly increase the amount of structural and functional information pertaining to biologically active molecules and processes. The information obtained from such studies can lead to design and application of new modification in order to obtain a desired bioactivity. Many application packages and servers performing docking, such as HEX, DOT, AUTODOCK, and ZDOCK are now available for predicting the lowest free energy state of a protein complex. In this study, we have focused on cyclin-dependent kinase 4 (Cdk4), a key molecule in the regulation of cell cycle progression at the G(1)-S phase restriction point and p16(INK4a), a tumor suppressor which inhibits Cdk4 activity. Truncated structures were created to find the more critical regions of p16 for interaction. The tertiary structures were determined by ProSAL, GENO3D Web Server. We evaluated their interactions with Cdk4 using two docking systems, HEX 4.5 and DOT 1. Calculations were performed on a high-speed computer. Minimizations and visualizations were carried out by PdbViewer 3.7. Considering shape and shape/electrostatic total energy, structures containing ANK II, III and IV motifs that lack the N-terminal region of the full length p16 molecule showed the best fit complexes among the p16 truncated forms. The free energies were compatible with that of p16 full length original form, the full length. It seems that the N-terminal of the molecule is not crucial for the interaction since the truncated structure containing only this region did not show a good total energy.

    Authors: Fahham N, Ghahremani MH, Sardari S, Vaziri B, Ostad SN.
    Keywords: Simulation of different truncated p16(INK4a) forms and in silico study of interaction with Cdk4
  • Summary:

    Curettage and packing with polymethylmethacrylate cement is a routine treatment for giant-cell tumour (GCT) of bone. We performed an in vitro evaluation of the cytotoxic effect of a combination of cement and methotrexate, doxorubicin and cisplatin on primary cell cultures of stromal GCT cells obtained from five patients. Cement cylinders containing four different concentrations of each drug were prepared, and the effect of the eluted drugs was examined at three different time intervals. We found that the cytotoxic effect of eluted drugs depended on their concentration and the time interval, with even the lowest dose of each drug demonstrating an acceptable rate of cytotoxicity. Even in low doses, cytotoxic drugs mixed with polymethylmethacrylate cement could therefore be considered as effective local adjuvant treatment for GCTs.

    Authors: Savadkoohi DG, Sadeghipour P, Attarian H, Sardari S, Eslamifar A, Shokrgozar MA.
    Keywords: Cytotoxic effect, drugs, polymethylmethacrylate, stromal giant-cell tumour cells, in vitro study
  • Summary:

    The existing chemical data such as those created by high throughput screening (HTS), structure-activity relationship (SAR) studies are converted into information as a result of storage and registration. Accessibility, manipulation, and data mining of such information make up the knowledge for drug development. Cheminformatics, exploiting the combination of chemical structural knowledge, biological screening, and data mining approaches is used to guide drug discovery and development and would assist by integrating complex series of rational selection of designed compounds with drug-like properties, building smarter focused libraries. This paper presents cheminformatics approaches and tools for designing and data mining of chemical databases and information. Many examples of success in lead identification and optimization in the area of anti-infective therapy have been discussed.

    Authors: Sardari S, Dezfulian M
    Keywords: Cheminformatics, anti-infective agents, discovery
  • Summary:

    Abstract

    AIMS:

    A novel chimeric-truncated form of tissue-type plasminogen activator (t-PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed-batch processes.

    METHODS AND RESULTS:

    The expression cassette for the novel t-PA was prepared in pET-28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase(®) Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t-PA Assay Kit. The fed-batch fermentation mode, coupled with a Ni-NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg(-1) ) compared to traditional batch mode (35·8 IU mg(-1) ).

    CONCLUSIONS:

    Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed-batch cultivation methods showed the potential to replace miss-folded formats of protein with proper folded, soluble form with improved potency.

    SIGNIFICANCE AND IMPACT OF THE STUDY:

    Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post-translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.

    Authors: Mahboudi F, Barkhordari F, Godarzi RM, Enayati S, Davami F
    Keywords: cultivation, Escherichia coli, chimeric-truncated, tissue plasminogen activator
  • Summary:

    Abstract

    Background: Development of an effective vaccine is highly needed in order to restrict the AIDS pandemic. DNA vaccines initiate both arms of immunity without the potential of causing disease. HIV-1 p24 and gp41 (gag and env) proteins play important roles in viral pathogenesis and are effective candidates for immune induction and vaccine design. Objective: In this study, new DNA vaccine candidates constructed from HIV-1 fused p24-gp41 or gp41 alone were evaluated in Balb/c mice for induction of cellular and humoral immune responses. Methods: Recombinant plasmids, pcDNA3.1/Hygro expression vector containing immunogenic sequences of fused p24-gp41 or gp41alone were produced. Dendrosome used as a system for carrying vectors in laboratory animals, and an IL-12 containing vector (pCAGGS-IL-12) was co-immunized with the p24-gp41 vector as a genetic adjuvant. Induction of effective immune responses against the designed vectors as DNA vaccine candidates in Balb/c mice was evaluated. Levels of total antibodies, IgG isotypes (IgG2a and IgG1); IFN-γ and IL-4 were measured by ELISA. MTT assay was used to evaluate lymphoproliferation. Results: The results confirmed that the immunogenic epitopes of both p24 and gp41 genes are highly effective inducers of immune responses, and administration of fused p24-gp41 alone or along with IL-12 resulted in further enhancement of immune responses. Group 4 that received fused fragments (p24-gp41) along with an IL-12 expressing vector demonstrated a significantly higher Stimulation Index (SI) and IFN-γ production (p<0.0001) with a significant increase in IgG2a/IgG1 ratio, indicating the stimulation of CMI towards Th1. Although gp41 containing vector (group 6) also showed significant increases in both proliferation and IFN-γ production, the responses were persistently lower than that of p24-gp41 containing vectors. Total antibody production was highest in group 6 as expected. Conclusion: Dendrosome proved to be an efficient carrier of recombinant plasmids constructed in this study. Further studies are necessary to evaluate these constructs as HIV vaccine candidates.

    Authors: Fatemeh Roodbari, Farzaneh Sabahi, Mohamad Nabi Sarbolouki, Farzaneh Barkhordari, Ahmad Adeli, Amel Jamedar, Fereidoun Mahboudi
    Keywords: Dendrosome, DNA Vaccine, gp41, HIV-1, IL-12, p24