Iranian

---

Antiproliferative protein (APP) isolated from conditioned media of two androgen-independent prostatic carcinoma cell lines, PC3 and Du-145 was shown to inhibit selectively cell proliferation of androgen-dependent prostate cancer cell line LNCaP in a dose dependent manner. This protein was further purified with HPLC using hydrophobic interaction column (phenyl 5PW) and was used to study the modulation of protein phosphorylation of LNCaP cells. The data indicated that antiprolif-erative protein could partially change the cytosolic protein phosphorylation. When compared with control LNCaP cells, in APP-treated cells 4 new proteins (88, 46, 30 and 18 kDa) were phosphory-lated, while a 72 kDa phosphoprotein was de-phosphorylated. The same results were obtained when two types of protein kinase activators were used. Protein kinase activators showed that the above changes of protein phosphorylation are not due to the protein kinase C or DNA protein kinase activ‌ity. These data may indicate the inhibition of LNCaP cell's proliferation by APP may be is due to the modulation of protein kinases and as a result due to interference on second messenger pathway.

---

A murine IgM monoclonal antibody (MA-2C6) with κ-light chains directed against an antigenic determinant of outer surface protein A (OspA) of the Lyme disease spirochete, Borreliaburgdorferi, is produced. This antibody could bind specifically to OspA antigen of several isolates of B. burgdorferi, but not to the non-Lyme disease bacteria such as T. pallidum and B. hermsii. Antibody MA-2C6 was purified by ion-exchange chromatography and used for purification of OspA antigen from Borreliaburgdorferi cell lysate. This antibody together with an IgG1 monoclonal antibody specific for OspA, that was previously characterized, were used to test whether these antibodies recognize different epitopes on OspA antigen of Borreliaburgdorferi. For this test, ELISA double antibody binding was used. Two antibodies were added to the antigen either separately or simultaneously, and the amount of bound antibody was quantitatively measured by the use of rabbit anti-mouse IgG conjugated with alkaline phosphatase. Additivity of the bound enzymatic activity was observed when the monoclonal antibodies bind to distinct epitopes. With this test, two distinct epitopes were recognized on the OspA molecule. This antibody can be used not only for the purification and subtyping of OspA, but also for neutralization and immunotherapy.

---

Clinical studies have shown that in pathological conditions such as endometriosis and reproductive tract infection (male and female) there is an activation status of macrophages that produce large quantities of nitric oxide (NO) in addition to other effector molecules. Large amounts of NO may have embryotoxic roles and produce infertility. This study was designed to evaluate the effect of different concentrations of NO on mouse pre-implantation embryo development in vitro. Mouse embryos (2-cell stage) were cultured in media containing different concentrations of sodium nitroprusside (SNP), an NO donor, or L-arginine methyl ester (L-NAME), an NO syntase (NOS) inhibitor. At the end of culture, cell apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique. The results showed that development of preimplantation embryos were inhibited by high concentration of SNP (1 and 10 μM). In contrast, 0.1 μM of SNP stimulated the embryo development. Similarly, the inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development, but the addition of 0.1 μM SNP with L-NAME reversed the inhibitory effect of L-NAME. TUNEL technique showed that high concentration of NO could induce apoptosis in the embryo, but at low concentration, it decreased apoptotic cell death

---

In human fertilization, the sperm centrosome nucleates a radial array of microtubules called the sperm aster. The sperm aster is responsible for apposition of male and female pronuclei, and later gives rise to the first meiotic spindle. The objective of this study was to determine microtubule assembly and chromatin configuration in rabbit oocytes following intracytoplasmic injection with human sperm by piezo-driven pipette. Oocytes were collected from superovulated dose 14-15 h after hCG injection and were fertilized by injection of a single human sperm into the ooplasm of each oocyte without additional activation treatment. Four hours post heterologous intracytoplamic sperm injection (ICSI), rabbit eggs were fixed and microtubule organization and chromatin configuration were examined by immunofluorescence microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle. Following human sperm injection, an aster of microtubules formed adjacent to the sperm head, around mid-piece, and sperm aster was enlarged and assembled around male and female pronuclei. During pronuclear centration, male and female pronuclei were surrounded by a microtubule array without nucleation sites. With fertile human sperm, the sperm aster formation rate was 54.6%. From our data we concluded that human spermatozoa can be injected successfully into rabbit oocytes, resulting in a reasonable survival rate, and that rabbit oocytes provide a reliable tools for assessing human sperm centrosomal function using the Piezo-ICSI system

---

Recent scientific evidence indicates that distinct patterns of susceptibility in BALB/c mice to Leishmania major infection are attributable to the differential expansion of distinct CD4+ T-cell subsets and their cytokines production. Production of the Th1 cytokine IFN-g is associated with resistance, whereas production of the Th2 cytokines IL-4 and IL-10 are associated with extreme susceptibility. The major host immune defense mechanism against Leishmania is activation of macrophages by INF-γ derived from T cells. The inability of susceptible hosts to mount the immune response necessary to activate macrophage and destroy the parasites may be due to the parasite-specific proteins that are able to suppress the immune system. In the present study, we have semi-purified the excreted antigens of Leishmania major promastigote and amastigote by column chromatography. The isolated fraction showed a potent immunosuppressive activity on normal BALB/c mice lymphocytes stimulated with mitogens. Fifteen microgram of the isolated fraction caused 81% suppression of lymphocyte proliferation. These data may suggest that the parasite by secreting immunosuppressive factor down regulate the immune system and as a result survive in the body

---

The aim of this study was to evaluate changes that occur in motility parameters of progesterone treated mouse spermatozoa during course of hyperactivation. Mouse spermatozoa treated with different doses of progesterone were videotaped after 10 min and 90 min of incubation. For each sperm, one second of movement of the head-midpiece junction was traced from the videotape and for each tracing seven motility parameters were studied using computer assisted image analysis. For all progesterone treated spermatozoa, motility rate differed significantly from control group after 90 min of incubation. Motility parameters for high doses of progesterone 10 and 100 µg/ml showed hyperactivation occurred during 10 min of incubation. With treatment of 1 µg/ml progesterone, hyperactivated motility pattern of spermatozoa occurred 90 min after incubation similar to the control group showing that low dose of progesterone is unable to induce hyperactivation. In conclusion, progesterone induces hyperactivation in mouse sperm and reduces the motility rate during the time

---

Background: Recent research on several DNA fragments covering open reading frames (ORF) 1-37 shows a new genetic marker in ORF 6 which is specific for differentiating wild-type varicella-zoster virus (VZV) strains from Oka varicella vaccine strain. On the other hand, herpes simplex virus (HSV) genome analysis by restriction enzymes is used to differentiate types one and two of the virus and even strains of each type. Previous studies using PCR-sequencing technique have shown that the thymidine kinase (TK) gene of HSV-1 is polymorphic. Methods: In this study, TK gene and DNA binding protein (UL29) gene of HSV-1 were selected. Both genes were analyzed with restriction endonucleases in order to identify a genetic marker for differentiating native strains of HSV-1 from the foreign strain. Three isolates of HSV-1 as well as standard strain of KOS were propagated in Vero cells. Initially, a pair of specific primers for each gene was designed to amplify UL29 and TK genes of these isolates. Subsequently, PCR products of these genes were digested separately with five restriction enzymes and subjected to polyacrylamide gel electrophoresis. Results: Using PCR-RFLP (Restriction fragment length polymorphisms) technique, results indicate that the patterns of restriction endonuclease digestion of UL29 and TK genes of the three isolates show no differences when compared to KOS strain. Conclusion: The genotypes of Iranian isolates are the same as KOS genotype and both genotypes are derived from a common ancestor. Hence, it can be postulated that in the process of random population flow among Iran, Europe and USA, the original KOS strain infected the Iranian population at some point in time. Iran. Biomed. J. 10 (3): 157-161, 2006

---

Background: The therapy of leishmania infection is difficult and each year 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated, therefore, there is a need for an effective vaccine. Monoclonal antibody (mAb) is one of the suitable methods for isolation and purification of leishmania antigens. In this report, we produced several mAb against leishmania infantum antigens for antigen purification to be used as candidate vaccine. Methods: BALB/c mice were injected with freeze-thawed promastigote twice together with Freund adjuvant. Three days before fusion, antigen in saline was injected into the tail vain and then mice were killed and the spleen lymphocytes were fused with myeloma SP2/0. Results: Five mAb against promastigote form of Leishmania infantum parasite were obtained. Western-blot analysis showed that these mAb recognize a band of 57- kDa protein either in parasite lysate or on whole L. infantum, L. tropica, L. major and L. donovani. It seems that the 57 kDa-protein is the major surface leishmania antigen (gp63) that is neither stage-specific nor differentially regulated. These mAb do not recognize the recombinant gp63 antigen and seems recognizing only the native form of a gp63 isoform. The IgG1 mAb was purified by affinity column and was used to purify 57 kDa antigens from Leishmania lysate. Conclusion: Since these antibodies recognizing one specific protein band in 4 different strains of leishmania, they could be used for leishmania diagnostic kits and also for purification of antigen to be tested for its protective effect against leishmania infection.

---

Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of a polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies.

---

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.

---

We were greatly saddened to hear the news of Professor Fereydoun Malekzadeh's death at age of 79 in September 9, 2012. Professor Malekzadeh was a distinguished and highly respected Iranian Microbiologist and a member of the editorial board of the Iranian Biomedical Journal since October 1997. He was born in 1933 in city of Tabriz in Province of Azarbaijan, Iran. After graduated from high school, he moved to Tehran and attended at Tehran University and obtained his BS in Biology in 1956 and subsequently his MSc degree in Mycology in 1959. Professor Malekzadeh received “Fulbright Scholarship” and obtained his Ph.D. degree in Bacteriology in 1961 from Louisiana State University. He returned home and since then taught microbiology in Biology Department of Faculty of Science, in Tehran University and later in Azad University. His contribution to microbiology was immense and he played a major role in establishing the Doctoral degree in the Microbiology Department of Faculty of Science in Tehran University and in Azad University. He was also a key funder of Iranian Society for Biology and a member of "American Society for Microbiology". Dr. Malekzadeh wrote several reference books, such as Introduction to Microbiology, Medical Microbiology, Infectious Diseases, etc., and also authored several papers in international peer-reviewed journals. His many academic awards included two best scientific books of the year, Botany and Introduction to Microbiology co-authored with Dr. Moghaddam and Dr. Shahamat, respectively. He also received "UNESCO Science Prize" in 1999, and "International Kharazmi Award" for discovering two new strains of bacteria, "Cellulomonas Persia" and "Cellulomonas iranesis" that have been established in Bergey's Manual of Systematic Bacteriology and deposited in ATCC. Dr. Malekzadeh was visiting professor at University of Illinois, State University of Arizona, University of Göttingen, University of Alberta and Institute of Marine Biotechnology in University of Maryland. Dr. Malekzadeh loved to work at the bench and continued to do so long after his official retirement. He was extraordinarily hard-working, and held very high standards not only in his work but also in his personal life. He loved interacting with young people. He also spotted talent, and went to extraordinary lengths to promote young, talented scientists. Professor Malekzadeh was diagnosed with a neurodegenerative disease in 2006, which caused deterioration in his health over a long period of time. He demonstrated great courage, determination and dignity during this very difficult time, seeming more concerned about others than about himself. He showed clearly strength of character during his illness. He is survived by his wife and two daughters, Dr. Shirin and Katayoun Malekzadeh.

---

Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region (DMR) in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies heve shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception microenvironments often occur by assisted reproduction techniques (ART). This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART. Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. Result: Results showed that the overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85±4.87% and 43.75±5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined (49.52±1.86% and 50%, respectively). Conclusion: Considering the absence of in vivo produced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks.

---

Background: Reduction/alkylation is one of the leading strategies for the development of antibody drug conjugates (ADCs). Precise control of the reduction process would not only yield a defined number of free thiols per antibody but also result in development of more homogenous conjugates. Methods: In the present study, we investigated the effect of various dithiothreitol (DTT) concentrations, temperature conditions, and DTT exposure times on antibody reduction. After antibody reduction, the Ellman's test and SDS-PAGE analysis were used to evaluate free thiols produced and confirm the reduction process, respectively. Results: DTT concentration seems to be a potential factor in the reduction process. Concentrations of 0.1, 1, 5, 10, 20, 50, and 100 mM DTT at 37°C for 30 minutes resulted in approximately 0.4, 1.2, 5.4, 7, 8, 8, and 8 thiols per antibody, respectively. Conclusion: Optimized site-specific conjugation can provide better process control and reproducibility for the development of disulfide-based ADCs.

---

Background: The majority of male patients with spinal cord injury (SCI) suffer from infertility. Nucleotide-binding oligomerization domain-like receptors NOD-like receptors (NLRs) are a kind of receptors that corporate in the inflammasome complex. Recent studies have introduced the inflammasome as the responsible agent for secreting cytokines in semen. Reactive oxygen species (ROS) is one of the elements that trigger inflammasome activation. Genital infections in SCI can lead to ROS generation. We investigated the relation between lipid peroxidation and inflammasome complex activity in testicular tissue of SCI rats. Methods: Adult male rats (n=20), weighting 200-250 g, were included and divided into four groups: three experimental groups, including SCI1, SCI3, and SCI7, i.e. the rats were subjected to SCI procedure and sacrificed after one, three, and seven days, respectively and a control group. We performed a moderate, midline spinal contusion injury at thoracic level 10. The animals were anesthetized, and testes were collected for measurement of gene expression by real-time PCR. Caudal parts of epididymis were collected for malondialdehyde (MDA) measurement. Results: No NLRP1a mRNA overexpression was seen in the testes of control and SCI groups. After seven days from SCI surgery, NLRP3 mRNA expression was significantly increased in SCI7 animals (p ≤ 0.05). There was a significant difference in MDA level in SCI7 versus control group, as well as SCI1 and SCI3 animals (p ≤ 0.05). Conclusion: NLRP3 overexpression occurs due to the increased ROS production in testis tissue of SCI rats. 

---

Background:  Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications and  acting as carriers of enzymes, antibodies or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale. Methods: Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes. Results: Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly.  The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium.  The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures. Conclusion: This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

---

Background: Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct. Methods: In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation. Results: Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA⁺), as compared with DU145 (PSMA^-). Conclusion: Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.

---

Background: Pasteurella multocida is a Gram-negative, non-motile, non-spore forming, and aerobic/anaerobic cocobacillus known as the causative agent of human and animal diseases. Humans can often be affected by cat scratch or bite, which may lead to soft tissue infections and in rare cases to bacteremia and septicemia. Commercial vaccines against this agent include inactivated, live attenuated, and non-pathogenic bacteria. Current vaccines have certain disadvantages such as reactogenicity or reversion to virulence. Therefore, the aim of this study was to reach a multi-epitope vaccine candidate that could be serotype independent and covers most incident serotypes of P. multocida. Methods: In this study, reverse vaccinology strategy was used to identify potentially immunogenic and protective epitopes. First, multiple alignments of different sequences of Pasteurella lipoprotein E (PlpE) from various serotypes of P. multocida were analyzed to identify the conserved regions. Bioinformatics tools were then applied to predict and select epitopes for further studies. Results: Three different conserved immunogenic regions were selected according to the selected criteria, and their various sequential orders were evaluated structurally by in silico tools to find the best order. Conclusion: In searching the epitopes of PlpE to design a new vaccine candidate against pasteurellosis, we found the region 1 + region 2 + region 3 (without any linker between regions) of epitope, including the regions of PlpE protein of P. multocida, as the appropriate serotype independent vaccine candidate against pasteurellosis.

---

Introduction: Multiple Sclerosis (MS) is currently a leading cause of neurologic disabilities, especially among young adults. Various disease-modifying drugs (DMDs) are available, aimed to slow down the progression of symptoms. Despite the success of DMDs through the last decade, treatment adherence has remained a challenge in MS care and management. Although there are self-reported, quantitative measures for treatment adherence, they cannot predict and determine the underlying causes of non-adherence. Therefore, self-reported questionnaires are more informative in clinical settings. The Multiple Sclerosis Treatment Adherence Questionnaire (MS-TAQ) is the only specifically designed instrument to assess treatment adherence in MS patients. This study evaluated the validity and reliability of the Persian translation of MS-TAQ. Methods and Materials: The back-translation method was employed for proper translation with maximum precision in the first phase. In the second phase, face validity was evaluated by qualitatively distributing the questionnaire to 10 MS patients. Quantitative face validity was analyzed using the impact score. The content validity was assessed by a panel of 20 faculty members, qualitatively by interview and quantitatively using both content validity ratio and content validity index. The final validated version was given to another 10 MS patients to evaluate internal consistency by Cronbach's alpha.
Results: The Persian translation was carried out by an English translator, the backward translation to English was performed by a native English speaker who was also fluent in Persian, and a third English expert approved the original English version of MS-TAQ. In the face validity assessment, 10 MS patients were interviewed, and their comments were applied. No item was removed in face validity, as no items had an impact score of less than 1.5. The panelists' comments in the content validity assessment were also applied, with six items added in the qualitative section and five removed in the quantitative assessment. The Cronbach's alpha was 0.8 for the whole questionnaire.
Conclusion and Discussion: MS-TAQ is a reliable and validated instrument for treatment adherence assessment in Iranian MS patients. Further evidence is needed to validate the construct of MS-TAQ, which is an ongoing part of this study in selected hospitals affiliated with Isfahan University of Medical Sciences.

---