Epitope Mapping of Tetanus Toxin by Monoclonal Antibodies: Implication for Immunotherapy and Vaccine Design

Tetanus as a life-threatening disease is characterized by muscle spasm. The disease is caused by the neurotoxin of Clostridium tetani. Active form of tetanus neurotoxin is composed of the light chain (fragment A) and the heavy chain. Fragment A is a zinc metalloprotease, which cleaves the neuronal soluble N-ethylmaleimide-sensitive attachment receptor (SNARE) protein, leading to the blockade of inhibitory neurotransmitter release and subsequent generalized muscular spasm. Two functional domains of the heavy chain are fragment C, which is required for neuronal cell binding of the toxin and subsequent endocytosis into the vesicles, and fragment B, which is important for fragment A translocation across the vesicular membrane into the neuronal cytosol. Currently, polyclonal immunoglobulins against tetanus neurotoxin obtained from human plasma of hyper-immunized donors are utilized for passive immunotherapy of tetanus; however, these preparations have many disadvantages including high lot-to-lot heterogeneity, possibility of transmitting microbial agents, and the adverse reactions to the other proteins in the plasma. Neutralizing anti-tetanus neurotoxin monoclonal antibodies (MAbs) lack these drawbacks and could be considered as a suitable alternative for passive immunotherapy of tetanus. In this review, we provide an overview of the literature discussing epitope mapping of the published neutralizing MAbs against tetanus toxin. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

The effect of Setarud (IMOD�) on angiogenesis in transplanted human ovarian tissue to nude mice

Background: One of the promising methods in fertility preservation among women with cancer is cryopreservation of ovarian cortex but there are many drawbacks such as apoptosis and considerable reduction of follicular density in the transplanted ovary. One solution to reduce ischemic damage is enhancing angiogenesis after transplantation of ovarian cortex tissue. Objective: The aim of this study was to investigate the effect of Setarud, on angiogenesis in transplanted human ovarian tissue. Materials and Methods: In this case control study, twenty four nude mice were implanted subcutaneously, with human ovarian tissues, from four women. The mice were randomly divided into two groups (n=12): the experimental group was treated with Setarud, while control group received only vehicle. Each group was divided into three subgroups (n=4) based on the graft recovery days post transplantation (PT). The transplanted fragments were removed on days 2, 7, and 30 PT and the expression of Angiopoietin-1, Angiopoietin-2, and Vascular endothelial growth factor at both gene and protein levels and vascular density were studied in the grafted ovarian tissues. Results: On the 2nd and 7th day PT, the level of Angiopoietin-1 gene expression in case group was significantly lower than that in control group, while the opposite results were obtained for Angiopoietin-2 and Vascular endothelial growth factor. These results were also confirmed at the protein level. The density of vessels in Setarud group elevated significantly on day 7 PT compared to pre-treatment state. Conclusion: Our results showed that administration of Setarud may stimulates angiogenesis in transplanted human ovarian tissues, although further researches are needed before a clear judgment is made. � 2015, Research and Clinical Center for Infertitlity. All rights reserved

Kinetics of murine decidual dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells (APC) capable of induction of primary immune responses as well as immunologic tolerance. Myeloid and lymphoid subsets of murine DCs are able to shift cytokine responses of T cells toward Th2 and Th1 profiles respectively. Thus, DCs would be suitable candidates to mediate the balance of maternal immune responses to conception. We analyzed pregnancy-related variations in uterus and splenic DCs in a murine model. C57BL/6-mated Balb/c female mice with vaginal plugs were scarified at early, middle, and late pregnancy. Frozen sections of uterus and spleen at each stage of pregnancy were immunostained with CD11c- and MHC-II-specific antibodies. Two-color immunohistochemistry was also carried out using anti-CD11c and one of the antibodies against CD11b, CD8α, CD86, and DEC-205. Using morphometric analysis, the average density of DCs and relative percentage of myeloid (CD11c+, CD11b+) and lymphoid DCs (CD11c+, CD8α+) were determined at each stage. Our results showed that DCs are present throughout the pregnancy in decidua. The average density of decidual DCs at early pregnancy was significantly higher relative to middle and late gestation or to those of endometrial DCs of non-pregnant mice. Interestingly, the average density of decidual and splenic DCs, followed the same variations at different stages of pregnancy. The relative percentage of decidual lymphoid DCs (LDC) was significantly higher at mid-gestation when compared with other stages of pregnancy or non-pregnant mice. Inversely, the frequency of myeloid DCs (MDC) and the MDC/LDC ratio were statistically lower at the middle stage of pregnancy. A majority of decidual DCs expressed MHC-II and CD86. At early pregnancy, DCs were more concentrated subadjacent to the luminal epithelial layers, whereas at mid- or late gestation, DCs were randomly distributed in the stroma and around the epithelium. Mid-pregnancy period was a critical point with regard to splenic DCs kinetics, as both the average density of DCs and the frequency of MDCs decreased significantly when compared with early or late pregnancy, although the relative percentage of splenic LDCs did not change. Our data suggest that the balance of MDC and LDC is finely tuned throughout pregnancy, pointing an eminent immunoregulatory role of DCs in the maintenance of pregnacy. © 2007 Society for Reproduction and Fertility

Effects of Chlamydia trachomatis Infection on Fertility; A Case-Control Study.

BACKGROUND: Nowadays, Chlamydia trachomatis is known as a causative agent of infertility. Because of, asymptomatic nature of infection, many may suffer from its lasting complications such as infertility. This study was performed in Tehran during April 2007 to April 2008 to compare the prevalence of Chlamydia trachomatis infection in fertile and infertile women using ELISA and PCR methods. METHODS: Overall, 234 infertile and 223 pregnant women, as the fertile group, participated in this hospital-based case-control study. After completing an informed consent form and the questionnaire, first catch urine and blood sample were obtained for PCR and ELISA (IgG, IgM) tests, respectively. Logistic regression analysis was used to control possible confounding factors, and determine adjusted odds ratio of infertility due to the infection. RESULTS: PCR results revealed that 29 (12.4%) of the infertile and 19 (8.5%) of the fertile women were positive for C. trachomatis infection (p = 0.440). IgG was positive in 21 (9.0%) of the infertile and 11 (5.0%) in the fertile group (p = 0.093). IgM assays identified that 2 (0.9%) of the infertile and 4 (1.8%) of the fertile women were positive for the micro-organism (p = 0.375). CONCLUSION: We found no significant differences among fertile and infertile women for Chlamydia trachomatis infection. Nevertheless, molecular techniques which are more sensitive, more specific and non-invasive can be used to detect C. trachomatis infection

Expression profiling of plac1 in murine cancer cell lines

Aim: Placenta-specific 1 (PLAC1) is among recently-discovered placental antigens which exerts fundamental role in placental function and development. Increasing body of literature shows that PLAC1 is frequently activated and expressed in a wide variety of human cancers and promote cancer progression. However, no data is available regarding the expression of mouse orthologue, plac1, in murine cancer cell lines. Materials and Methods: We investigated the expression of plac1 in a series of murine cell lines from different histological origins, mammary carcinoma (4T1), melanoma (B16F10), colorectal carcinoma (CT26), renal carcinoma (Renca), glioma (GL26), B-cell lymphoma (A20 and BCL1) and also two fibroblast cell lines (NIH3T3 and L929), using RT-PCR, Western blotting and flow cytometry. Results: Our data demonstrated that plac1 transcript and plac1 protein were expressed in all examined cell lines, as judged by RT-PCR and Western blot, respectively. The molecular weight of mouse plac1 was experimentally observed to be approximately 24 kD. Flow cytometric analysis showed surface expression of plac1 in aforesaid cell lines ranging from 2 to 42.5. Conclusion: Based on the ubiquitous expression of plac1, the investigated cancer cell lines or immortalized cell lines can be used to examine the role of plac1 in the process of immortalization. Copyright © Experimental Oncology 2019

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia: Association between CD38 expression and disease progression

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. Objectives: To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. Methods: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. Results: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5 +/CD19+/sIg+). The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4) and CD45RO (74/87, 83.9) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and intensity of CD38 expression in progressive group (40.5) compared to indolent (11.9) patients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. Conclusion: Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL

Optimization and efficient purification in production of Brucella melitensis recombinant HSP and TF proteins with low endotoxin contents

Background: The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important. Objectives: In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants. Materials and Methods: pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1 Triton X-114 in washing buffers. Results: An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80 yield. Conclusions: As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense. © 2013, Ahvaz Jundishapur University of Medical Sciences; Licensee Kowsar Ltd

Inhibitory Effect of Polyclonal Antibodies Against HER3 Extracellular Subdomains on Breast Cancer Cell Lines

OBJECTIVE: Human epidermal growth factor receptor 3 (HER3) is a unique member of the tyrosine kinase receptors with an inactive kinase domain and is the preferable dimerization partner for HER2 which lead to potent tumorigenic signaling. METHODS: In this study, the expression plasmids coding for the human HER3 subdomains were transfected into CHO-K1 cells. Produced proteins were characterized by ELISA and SDS-PAGE. Rabbits were immunized and produced polyclonal antibodies (pAbs) that were characterized by ELISA, Immunoblotting and flowcytometry and their inhibitory effects were assessed by XTT on BT-474 and JIMT-1 breast cancer cell lines. RESULT: The recombinant subdomains were highly immunogenic in rabbits. The pAbs reacted with the recombinant subdomains as well as commercial HER3 and the native receptor on tumor cell membranes and could significantly inhibit growth of Trastuzumab sensitive (BT-474) and resistant (JIMT-1) breast cancer cell lines in vitro. CONCLUSION: It seems that HER3 extra cellular domains (ECD) induce a strong anti-tumor antibody response and may prove to be potentially useful for immunotherapeutic applications.

Immunophenotypic subtyping of leukemic cells from Iranian patients with acute lymphoblastic leukaemia: Association to disease outcome

Background: Immunophenotypic characterization of the leukemic cells has been widely used as a tool for diagnosis, classification, stratification and prognosis of leukaemia. Objective: To investigate the immunophenotypic subtype profiles of Iranian patients with acute lymphoblastic leukemia (ALL) and its association to disease outcome. Methods: In this study, a total of 60 Iranian patients with ALL were immunophenotyped by flow cytometry using a panel of monoclonal antibodies specific for CD2, CD3, CD5, CD10, CD13, CD14, CD19, CD20, CD33, CD34, CD45, HLA-DR and TdT molecules. Results: The samples were initially categorized into T-ALL (n=9), B-ALL (n=50) and mixed lineage (n=1) based on the expression patterns of CD3 and CD19 molecules. B-ALL patients could further be classified into four subtypes, including Pro-B (n=7, 11.7), Pre-B I (n=28, 46.7), Pre-B II (n=13, 21.7) and immature/mature B cells (n=2, 3.3) on the basis of expression of CD10, CD19, CD20, HLA-DR and TdT. Clinical manifestations and laboratory findings of the patients did not reveal association with immunophenotypic subtypes of ALL, with the exception of mediastinal mass and WBC count at the time of diagnosis which were found to be significantly higher in patients with T-ALL compared with BALL (p=0.001 and 0.014), respectively. Conclusion: Our results indicate that overall the immunophenotypic profile of Iranian ALL patients is similar to previous reports and it might be used for monitoring of minimal residual disease and prognosis

Fc receptor-like 1-5 molecules are similarly expressed in progressive and indolent clinical subtypes of B-cell chronic lymphocytic leukemia

Fc receptor-like (FCRL) 1-5 molecules are exclusively expressed in B-cells and have recently been considered as potential targets for immunotherapy of B-cell malignancies. In this study, the expression pattern of FCRL1-5 molecules was investigated in Iranian patients with B-cell chronic lymphocytic leukemia (B-CLL). Our RT-PCR results have demonstrated that all FCRL molecules, except FCRL4, were expressed in the vast majority of the patients with B-CLL. However, comparison of the relative mRNA expression levels of FCRL between B-CLL (n = 86) and elderly normal subjects (n = 10) revealed significantly lower expression levels of FCRLl (p < 0.0001), FCRL3 (p = 0.01) and FCRL4 (p = 0.002), but not FCRL2 or FCRL5, in cases with B-CLL. No significant differences were observed between the indolent and progressive subtypes of patients with B-CLL. Comparison between the mutated and unmutated subtypes revealed a significantly higher expression level of FCRL3 (p = 0.017) in patients with mutated CLL. Surface and intracytoplasmic expression of FCRL1, 2, 4 and 5 in leukemic cells of 12 patients by flow cytometry revealed similar results to those obtained by RT-PCR with a few exceptions. Thus, while FCRL4 was expressed in only 2 samples at intracytoplasmic level, FCRL1 and 2 were expressed in the majority of samples, both at surface and intracytoplasm. FCRL5 protein was also detected in 10 samples, but surface expression was confirmed in only 2. Analysis of B-cells from 5 normal subjects by flow cytometry revealed higher expression levels of FCRL molecules compared to CLL. Our results indicate differential expression of FCRL molecules in B-CLL and suggest the potential implication of FCRL1 and 2 for immunotherapeutic interventions. © 2008 Wiley-Liss, Inc