Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ ² P > 0.05), including only one common haplotype (GenBank access no. {"type":"entrez-nucleotide","attrs":{"text":"EF413075","term_id":"151936076","term_text":"EF413075"}}EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran     

Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7

Background:

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin (I), with its own receptor translocated intimin receptor (Tir) and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7.

Methods:

A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT (rEIT) was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan (TMC), as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines.  Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT.

Results:

The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7.

Conclusion:

Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7.Key Words: EnterohemorrhagicEscherichia coli, Nanoparticles, Intranasal vaccination     

Association of Lecithin Cholesterol Acyltransferase rs5923 Polymorphism in Iranian Individuals with Extremely Low High-Density Lipoprotein Cholesterol: Tehran Lipid and Glucose Study

Background:

The serum concentration of high-density lipoprotein cholesterol (HDL-C) is one of the important heritable risk factors for cardiovascular disease and is a target for therapeutic intervention. In this study, we aimed to evaluate the effects of lecithin cholesterol acyltransferase (LCAT) gene polymorphism rs5923 on LCAT enzyme activity and serum HDL-C concentration.

Methods:

The study population was selected from consecutive individuals with HDL-C ≤ 5^th percentile (n = 73) and extremely high HDL-C ≥ 95^th percentile (n = 57) who had participated in the Tehran Lipid and Glucose Study. The rs5923 polymorphism was genotyped using direct sequencing. LCAT activity was measured by fluorometric assay kit, and lipid concentrations were measured using the enzymatic colorimetric method.

Results:

The genotype frequencies were significantly different between the high HDL-C group (CC 94.7%, CT 5.3%) and the low HDL-C group (CC 83.6%, CT 16.4%) (P = 0.048). The T-allele frequencies in subjects with low and high HDL-C were 0.082 and 0.026, respectively (P = 0.16). The association of the single-nucleotide polymorphism rs5923 with low HDL-C was not statistically significant after adjustment for age, sex, and BMI (odd ratio = 2.65, 95% confidence interval = 0.32-21.5, P = 0.36, regression logistic analysis). Also, the effects of LCAT enzyme activity did not depend on the HDL-C level (P = 0.24).

Conclusion:

rs5923 polymorphism is not associated with low HDL-C levels in Iranian population. Key Words: Polymorphism, Single nucleotide, Lipoproteins     

The role of Ile3434Thr XRCC7 gene polymorphism in Differentiated Thyroid Cancer risk in an Iranian population

Background

The aim of this study was to understand any association between differentiated thyroid carcinoma (DTC) and Ile3434Thr XRCC7 gene polymorphism (GenBank accession number: rs7830743). DTC is the most prevalent thyroid neoplasm, which includes papillary and follicular cell carcinoma. XRCC7 gene encodes a protein that functions in non-homologous end joining DNA repair pathway. Non-synonymous polymorphisms in this gene may alter DNA repair capacity of the cell and change the risk of developing cancers.

Methods

DTC patients (n = 173) and cancer free individuals (n = 204) were enrolled in a case-control study. The Ile3434Thr polymorphic alleles were discriminated by using amplification refractory mutation system-PCR method. The frequencies of this single nucleotide polymorphism in case and control groups were compared. Also, risk ratio for developing DTC in dichotomized genotypes was estimated by multivariate logistic regression analysis.

Results

Dichotomized genotypes into those with and without the 3434Thr allele showed that this allele was associated with DTC (OR [odd ratio]: 1.89, 95% confidence interval (CI) = 1.29-2.79, P<0.001). Also, TC genotype was significantly associated with increased risk of DTC (OR: 2.42, 95% CI = 1.55-3.81, P = 0.0001) in individuals carrying this genotype.

Conclusion

Allele 3434Thr in XRCC7 gene might be associated with differentiated thyroid cancer risk. Further studies with larger samples are needed to verify these initial findings. Key Words: DNA repair enzymes, Thyroid neoplasms, Genetic polymorphism     

Attenuation of Focal Cerebral Ischemic Injury Following Post-Ischemic Inhibition of Angiotensin Converting Enzyme (ACE) Activity in Normotensive Rat

Background

Central renin angiotensin system has an important role on the cerebral microcirculation and metabolism. Our previous work showed that inhibition of angiotensin converting enzyme () activity prior to induction of ischemia protected the brain from severe ischemia/reperfusion (I/R) injuries. This study evaluated the impacts of post-ischemic inhibition of , enalapril, on brain infarction in normotensive rats.

Methods

Rats were anesthetized with chloral hydrate (400 mg/kg). Focal cerebral ischemia was induced by 60-min intraluminal occlusion of right middle cerebral artery (MCA). Intraperitoneal injection of enalapril (0.03 or 0.1 mg/kg) was done after MCA reopening (reperfusion). Neurological deficit score (NDS) was evaluated after 24 h and the animals randomly assigned for the assessments of infarction, absolute brain water content (ABWC) and index of brain edema

Results

Severe impaired motor functions (NDS = 2.78 ± 0.28), massive infarction (cortex = 214 ± 19 mm³, striatum = 86 ± 5 mm³) and edema (ABWC = 83.1 ± 0.46%) were observed in non-treated ischemic rats. Non-hypotensive dose of enalapril (0.03 mg/kg) significantly reduced NDS (1.5 ± 0.22), infarction (cortex = 102 ± 16 mm³, striatum = 38 ± 5 mm³) and edema (ABWC = 80.9 ± 0.81%). Enalapril at dose of 0.1 mg/kg significantly lowered arterial pressure could not improve NDS (2.0 ± 0.45) and reduce infarction (cortex = 166 ± 26 mm³, striatum = 71 ± 11 mm³).

Conclusion

Post-ischemic ACE inhibition in the normotensive rats without affecting arterial pressure protects the brain from reperfusion injuries; however, this beneficial action is masked by hypotension. Key Words: Angiotensin converting enzyme (ACE) inhibitors, Enalapril, Brain edema, Cerebral infarction     

Prevalence of Class 1 Integrons and Extended Spectrum Beta Lactamases among Multi-Drug Resistant Escherichia coli Isolates from North of Iran

Background:

Extended spectrum beta lactamases (ESBLs) are an important cause of transferable multidrug resistance (MDR) in gram-negative bacteria. The most described ESBL genes are generally found within integron-like structures as mobile genetic elements. The aim of this study was to identify the accompanying of class 1 integrons and ESBLs in the MDR E. coli isolates.

Methods:

Susceptibility to antimicrobial agents was determined for 33 E. coli strains by the disk diffusion method. Double-disk synergy test was applied for screening ESBL. To identify the strains carrying integrons, the conserved regions of integron-encoded integrase gene intI1 were amplified. For detection of gene cassettes, 5′CS and 3′CS primers were used.

Results:

All E. coli isolates were identified as multi-drug resistant. More than 50% of the isolates were resistant to tetracycline, cephalothin, cefuroxime, amoxicillin-clavulanic acid, and third generation cephalosporines. Nearly all of the isolates displayed sensitivity to piperacillin. There was a significant correlation between production of ESBL and resistance to all antibiotics except for ciprofloxacin and piperacillin (P < 0.01). Thirty two MDR strains (97%) included class 1 integron, and some isolates that included integrons were similar in the size of gene cassettes. The isolates were different in the resistance profiles; however, some others had similar resistance profiles. Of eight ESBL positive isolates, seven (87.5%) carried class 1 integrons.

Conclusion:

Class 1 integrons were frequent in MDR and also ESBL-producing E. coli isolates. High prevalence of class 1 integrons confirms that integron-mediated antimicrobial gene cassettes are important in E. coli resistance profile. Key Words: Antibiotic, Integrons, Escherichia coli     

Impact of Methylenetetrahydrofolate Reductase C677T Polymorphism on the Risk of Gastric Cancer and Its Interaction with Helicobacter pylori Infection

Background

Attempts for early detection of gastric cancer have recently focused on host''s genetic susceptibility factors and gene-environment interactions. We have, herein, studied the association of MTHFR C677T single nucleotide polymorphism (SNP) and its interaction with Helicobacter pylori infection, smoking, age and gender on the risk of gastric cancer among an Iranian population.

Methods

Gastric cancer patients (n = 450) and cancer-free controls (n = 780) were studied for serum H. pylori-specific IgG antibodies by ELISA and MTHFR C677T polymorphism (SNP) by PCR-RFLP. Demographic and life style data were collected through patient interviews. Unconditional logistic regression model estimated odds ratio (OR) and the corresponding 95% confidence intervals (CI).

Results

The interactions of MTHFR genotype with H. pylori infection (P = 0.03), age (P = 0.049) and gender (P = 0.007) were statistically significant. Accordingly, MTHFR C677T carriers who were also positive for H. pylori infection exhibited 80% (OR = 1.8, 95% CI = 1.0-2.9) significant excess risk of non-cardia gastric cancer. Furthermore, subjects over the age of 50 or female subjects carrying MTHFR C677T SNP showed 40 (OR = 1.4, 95% CI = 1.0-2.0) and 100 (OR = 2.0, 95% CI = 1.2-3.2) percent increased risk of gastric cancer, respectively.

Conclusion

MTHFR C677T SNP seems to increase the risk of gastric cancer and the effect is significantly inflated by interactions with H. pylori infection, age and gender. Key Words: Helicobacter pylori, Smoking, Gender identity, Age group, Methylenetetrahydrofolate reductase     

Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect

Background:

The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage.

Methods:

Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105.

Results:

11.88±1.29% of human endometrial cells within tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (P<0.05). Oct4 and Nanog-expressing cells comprise 1.43±0.08 and 0.54±0.01% of endometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells.

Conclusion:

The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.Key Words: Endometrium, Immunohistochemistry, Mesenchymal stem cells     

Role of XmnIG Polymorphism in Hydroxyurea Treatment and Fetal Hemoglobin Level at Isfahanian Intermediate β-Thalassemia Patients

Background:

β-thalassemia is the most common monogenic disorder in human. The (CT) polymorphism at -158 upstream region of the γ^G-globin gene and pharmacological factors such as hydroxyurea have been reported to influence γ-globin gene expression and the severity of clinical symptoms of β-thalassemia.

Methods:

In the present study, 51 β-thalassemia intermediate patients were studied. Xmn1γ^G polymorphism genotype was determined using Tetra-Primer ARMS-PCR technique. Hemoglobin (Hb) and fetal hemoglobin (HbF) levels were determined by gel electrophoresis.

Results:

Of 51 patients, 35 (68.6%) patients were heterozygous (CT) and 16 (31.4%) patients were homozygous (CC). Of 30 patients under treatment by hydroxyurea, 20 (66.7%) patients were heterozygous (CT) and 10 (33.3%) patients were homozygous (CC). Our results demonstrated that in the heterozygous (CT) genotype, the Hb (9.58 ± 1.25 gm/dl) and HbF (89.30 ± 21.87) levels were significantly higher in comparison with homozygous (CC) genotype (7.94 ± 1.34 gm/dl and 70.32 ± 40.56, respectively). Furthermore, we observed that after drug usage, the Hb and HbF levels in patients with heterozygous (CT) genotype (0.7 ± 1.26 gm/dl and 5.95±14.8, respectively) raised more in comparison with homozygous (CC) genotype (0.26 ± 1.43 gm/dl and 0.8 ± 1.31, respectively).

Conclusion:

Hb and HbF levels in the patients carrying T allele are increased significantly, and they also response to hydroxyurea treatment.Key Words: Fetal hemoglobin (HbF), Hydroxyurea, Intermediate β-thalassemia     

Modulation of Lipopolysaccharide Stimulated Nuclear Factor kappa B Mediated iNOS/NO Production by Bromelain in Rat Primary Microglial Cells

Background:

Microglial cells act as the sentinel of the central nervous system .They are involved in neuroprotection but are highly implicated in neurodegeneration of the aging brain. When over-activated, microglia release pro-inflammatory factors, such as nitric oxide (NO) and cytokines, which are critical in eliciting neuroinflammatory responses associated with neurodegenerative diseases. This study examined whether bromelain, the pineapple-derived extract, may exert an anti-inflammatory effect in primary microglia and may be neuroprotective by regulating microglial activation.

Methods:

Following the isolation of neonatal rat primary microglial cells, the activation profile of microglia was investigated by studying the effects of bromelain (5, 10, 20, and 30 µg/ml) on the levels of NO, inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) in microglia treated with lipopolysaccharide (LPS) (1 µg/ml). Data were analyzed using Student''s t-test. P values less than 0.05 were considered to be statistically significant, compared with the LPS-treated group without bromelain.

Results:

Results showed that pretreatment of rat primary microglia with bromelain, decreased the production of NO induced by LPS (1 µg/ml) treatment in a dose-dependent manner. Bromelain (30 µg/ml) also significantly reduced the expression of iNOS at mRNA level and NF-κB at protein level. Moreover, the study of mitochondrial activity in microglia indicated that bromelain had no cytotoxicity at any of the applied doses, suggesting that the anti-inflammatory effects of bromelain are not due to cell death.

Conclusion:

Bromelain can be of potential use as an agent for alleviation of symptoms in neurodegenerative diseases.     

Genetic and physiological analysis of tolerance to acute iron toxicity in rice

Background

Fe toxicity occurs in lowland rice production due to excess ferrous iron (Fe²⁺) formation in reduced soils. To contribute to the breeding for tolerance to Fe toxicity in rice, we determined quantitative trait loci (QTL) by screening two different bi-parental mapping populations under iron pulse stresses (1,000 mg L⁻¹ = 17.9 mM Fe²⁺ for 5 days) in hydroponic solution, followed by experiments with selected lines to determine whether QTLs were associated with iron exclusion (i.e. root based mechanisms), or iron inclusion (i.e. shoot-based mechanisms).

Results

In an IR29/Pokkali F₈ recombinant inbred population, 7 QTLs were detected for leaf bronzing score on chromosome 1, 2, 4, 7 and 12, respectively, individually explaining 9.2-18.7% of the phenotypic variation. Two tolerant recombinant inbred lines carrying putative QTLs were selected for further experiments. Based on Fe uptake into the shoot, the dominant tolerance mechanism of the tolerant line FL510 was determined to be exclusion with its root architecture being conducive to air transport and thus the ability to oxidize Fe²⁺ in rhizosphere. In line FL483, the iron tolerance was related mainly to shoot-based mechanisms (tolerant inclusion mechanism). In a Nipponbare/Kasalath/Nipponbare backcross inbred population, 3 QTLs were mapped on chromosomes 1, 3 and 8, respectively. These QTLs explained 11.6-18.6% of the total phenotypic variation. The effect of QTLs on chromosome 1 and 3 were confirmed by using chromosome segment substitution lines (SL), carrying Kasalath introgressions in the genetic background on Nipponbare. The Fe uptake in shoots of substitution lines suggests that the effect of the QTL on chromosome 1 was associated with shoot tolerance while the QTL on chromosome 3 was associated with iron exclusion.

Conclusion

Tolerance of certain genotypes were classified into shoot- and root- based mechanisms. Comparing our findings with previously reported QTLs for iron toxicity tolerance, we identified co-localization for some QTLs in both pluse and chronic stresses, especially on chromosome 1.     

Protective Effects of Restricted Diet and Antioxidants on Testis Tissue in Rats Fed with High-Fat Diet

Background:

A high-fat diet (HFD) promotes the oxidative stress formation, which in turn has hazardous effects on reproductive system and fertility. The present study examines the potential positive effects of a restricted high-fat diet (RHFD) and antioxidants consumption on sperm parameters and testis tissue in rats.

Methods:

Male rats (n = 48) were divided into four groups (12 in each group): control group (Cont), HFD group, RHFD, and RHFD with astaxanthin and vitamins E and C group (RHFDA). After 12 weeks, serum analysis and sperm parameters study were performed. Sections of fixed testes were stained with Hematoxilin and Eosin to study the histological changes. A one-way ANOVA was used to compare the data.

Results:

HFD fed animals presented significant increase in weight load and serum low density lipoprotein (LDL-C) levels (P < 0.05). The sperm count in RHFD was lower than three other groups (P < 0.05) and sperm motility of RHFDA group was significantly higher than HFD and RHFD groups (P < 0.05). The histological study was showed a significant increase in spermatogonium number in RHFDA compared to three other groups (P < 0.05). The number of spermatocyte I and spermatid in RHFD was significantly (P < 0.05) lower than Cont and HFD groups.

Conclusion:

HFD and obesity can affect sperm parameters and spermatogenesis and antioxidants consumption may improve their quality. Although the RHFD is a benefit way in weight loss and decrease of LDL-C of serum, but it is suggested that is not effective on sperm quality improvement.Key Words: Spermatogenesis, high-fat Diet, Restricted fat diet, Antioxidants, Astaxanthin     

in vitro Anti-Proliferative Effect of Adiponectin on Human Endometriotic Stromal Cells through AdipoR1 and AdipoR2 Gene Receptor Expression

Background:

Endometriosis is a complex disorder in reproductive age women which consist of stromal and epithelial cells implantation outside the uterine cavity. Adiponectin is a member of cytokine family with various metabolic roles and proliferation inhibition of many cancer cells. The aim of the present research was to determine adiponectin effect on human endometriotic stromal cells (ESCs) proliferation and their expression of adiponectin receptors.

Methods:

In this experimental study, endometrial biopsies (n=7) were taken. ESCs isolation was done by enzymatic digestion and cell filtrations. ESCs of each biopsy were divided into four groups: 0 (control), 10, 100, and 200 ng/ml adiponectin concentrations in three different times (24, 48, or 72 h). The effect of adiponectin on ESC viability and expression of mRNA Adipo receptor1 (R1) and Adipo receptor2 (R2) was determined by Trypan blue staining and semi-quantitative RT-PCR, respectively. Data were analyzed by one-way ANOVA and unpaired student’s t-test, and P<0.05 was considered statistically significant.

Results:

Adiponectin inhibited human endometriotic stromal cell proliferation in time- and dose-dependent manners significantly (P=0.001). Expression of AdipoR1 and AdipoR2 gene receptors was increased in human ESCs significantly (P<0.05).

Conclusions:

Adiponectin can suppress endometriosis by inhibiting ESC proliferation and increased AdipoR1 and AdipoR2 expression.Key Words: Adiponectin, Adiponectin receptors, Endometriosis, Stromal cells     

Comparison of Antimicrobial Susceptibility of Campylobacter Strains Isolated from Food Samples and Patients with Diarrhea

Background:

Campylobacter infections may lead to serious conditions, including septicemia or other invasive forms of the disease, which require rapid and accurate laboratory diagnosis and subsequently appropriate antimicrobial therapy. The aim of this study was to compare the species distribution and antimicrobial susceptibility pattern of Campylobacter spp. strains isolated from patients and food samples.

Methods:

Biochemical identification was performed on 15 clinical and 30 food isolates of Campylobacter recovered onto Brucella agar containing 5% sheep blood. PCR was carried out to confirm the identity of Campylobacter spp. using primers for cadF, hipO, and asp genes of Campylobacter. To determine antibiotic sensitivity of isolates, Kirby-Bauer assay was carried out using 16 different antibiotic discs.

Results:

PCR assay and biochemical tests confirmed all 45 isolates as Campylobacter: 20 (44.44%) as C. jujeni, 10 (22.22%) as C. coli, and 15 (33.34%) as other Campylobacter strains. The maximum resistance was observed to cefotaxime and imipenem (each 86.49%) and the maximum sensitivity to erythromycin (48.65%).

Conclusion:

C. jujeni is dominant among isolates from clinical and food samples. In addition, tetracycline remains the first-line therapeutic agent against Campylobacter infections in Iran. Key Words: Campylobacter, Genetic diversity, Drug resistance     

Molecular Genetic Analysis of the Variable Number of Tandem-Repeat Alleles at the Phenylalanine Hydroxylase Gene in Iranian Azeri Turkish Population

Background:

The variable numbers of tandem-repeat (VNTR) alleles at the phenylalanine hydroxylase (PAH) gene have been used in carrier detection and prenatal diagnosis in phenylketonuria families. This study was carried out to analyze VNTR alleles at the PAH gene in Iranian Azeri Turkish population.

Methods:

In this study, 200 alleles from general population were studied by PCR.

Results:

The frequencies of VNTR alleles were 45%, 46%, 2%, 3%, 1%, and 3% in studied group regarding 3, 8, 9, 11, 12, and 13 repeat copies, respectively. Statistically significant differences were not found between expected and observed frequencies of VNTR genotypes (P > 0.05).

Conclusions:

VNTR alleles with three and eight repeats were frequent, and the VNTR alleles with 13 repeats showed 3% frequency in the tested group. This study is the first report on tested population genetic structure using VNTR alleles at the PAH gene. Key Words: Phenylalanine hydroxylase, Population genetics, Variable numbers of tandem-repeat     

Association Study of rs1333040 and rs1004638 Polymorphisms in the 9p21 Locus with Coronary Artery Disease in Southwest of Iran

Background:

Coronary artery disease (CAD) is a multifactorial and heterogenic disease. Recently, genome-wide association studies have reported that rs1333040 (C/T) and rs1004638 (A/T) single nucleotide polymorphisms (SNPs) in the 9p21 locus have very strong association with CAD. This study aimed to examine these associations in Southwest of Iran.

Methods:

Blood samples were collected from 200 CAD patients and 110 healthy individuals with no CAD. The association of two SNPs with CAD was evaluated by PCR and restriction fragment length polymorphism.

Results:

Chi-square test showed no association between rs1333040 SNP and CAD (X²: 4.66, df: 2, P=0.09). Also, there was no association between rs1004638 SNP and CAD (X²: 0.27, df: 2, P=0.88).

Conclusion:

No association was observed between rs1333040 and rs1004638 SNPs in the 9P21 region and CAD in Southwest of Iran. Key Words: Coronary artery disease, Single nucleotide polymorphisms, Genetic association study, Iran     

Level of Serum Enzymes and Electrocardiogram in Healthy Rabbits after Injection of ICD-85 as an Anticancer Agent

Background:

Our previous in vivo studies confirmed that ICD-85, as an anticancer agent, was able to prevent further growth of breast tumors and expand the life expectancy of mice with breast cancer.

Methods:

Blood collection was carried out before, 1, 3, and 6 hours after ICD-85 injection. Sera were used to determinate the cardio and hepatic enzymes levels, including ALT, AST, LDH, CPK, and Ck-MB. Coagulation factors such as PT and PTT were also assayed. ECGs of all rabbits were recorded during the experiment.

Results:

ECG results showed that the injection of 50 and 100 µg/kg ICD-85 into healthy rabbits has no significant effect on heart function while the injection of 150 to 200 µg/kg ICD-85 caused ECG wave changes and mild bradycardia without toxic effects on heart. After ICD-85 injection (concentrations below 100 µg/kg), no significant increase was observed in liver and cardiac enzymes (ALT, AST, LDH, CPK, and CK-MB). However, the concentration of 150 µg/kg and above caused a rise in the enzymes. Comparison of the PT and PTT before and after ICD-85 injection showed no significant clotting time at any concentrations below 200 µg/kg.

Conclusion:

Based on the results obtained in the present study as well as our previous reports, ICD-85 at concentrations below 100 µg/kg seems to have no significant effect on the serum enzymes as indicators of hepatotoxicity and cardiotoxicity in healthy rabbits. However, to confirm this conclusion, more detailed surveys on heart and liver is needed to be carried out. Key Words: ICD-85, Electrocardiogram, Anticancer     

Heat Shock Proteins Enriched-Promastigotes of Leishmania major Inducing Th2 Immune Response in BALB/c Mice

Background

Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined.

Methods

BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 10⁶ stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks.

Results

No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-γ, IL-4, TGF-β, IgG1 and IgG2b were increased in both groups, IFN-γ was significantly higher in SLA group and IgG2a in HSP-enriched SLA.

Conclusion

These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection. Key Words: Leishmaniasis, Heat shock proteins (HSP), Adjuvant     

Quality Control of Widely Used Therapeutic Recombinant Proteins by a Novel Real-Time PCR Approach

Background:

Existence of bacterial host-cell DNA contamination in biopharmaceutical products is a potential risk factor for patients receiving these drugs. Hence, the quantity of contamination must be controlled under the regulatory standards. Although different methods such as hybridization assays have been employed to determine DNA impurities, these methods are labor intensive and rather expensive. In this study, a rapid real-time PCR test was served as a method of choice to quantify the E. coli host- cell DNA contamination in widely used recombinant streptokinase (rSK), and alpha interferon (IFN-α) preparations.

Methods:

A specific primer pair was designed to amplify a sequence inside the E. coli 16S rRNA gene. Serial dilutions of DNA extracted from E. coli host cells, along with DNA extracted from Active Pharmaceutical Ingredients of rSK, and IFN-α samples were subjected to an optimized real-time PCR assay based on SYBR Green chemistry.

Results:

The test enabled us to detect a small quantity of genomic DNA contamination as low as 0.0002 pg in recombinant protein-based drugs. For the first time, this study showed that DNA contamination in rSK and IFN-α preparation manufactured in Pasteur Institute of Iran is much lower than the safety limit suggested by the US FDA.

Conclusion:

Real-time PCR is a reliable test for rapid detection of host-cell DNA contamination, which is a major impurity of therapeutic recombinant proteins to keep manufacturers’ minds on refining drugs, and provides consumers with safer biopharmaceuticals. Key Words: DNA contamination, Real-time PCR, Streptokinase, Interferon-alpha (IFN-α)