Anti-Mucin1 aptamer-conjugated chitosan nanoparticles for targeted co-delivery of docetaxel and IGF-1R siRNA to SKBR3 metastatic breast cancer cells

Background: Targeted co-delivery of siRNA and a chemotherapeutic drug is an attractive approach to cancer drug design and treatment. This study was carried out to design an anti-Mucin1 aptamer [Apt]-conjugated chitosan nanoparticle [NP] for targeted co-delivery of insulin-like growth factor receptor 1 [IGF-1R] Silencer siRNA and docetaxel [DTX] to SKBR3 cells

Methods: Characterization of nano-drugs, cellular uptake of NPs, cell viability, and gene expression studies were evaluated based on metastatic breast cancer cells

Results:The results of this study showed that NPs had spherical and smooth morphology with 110-118 nm in size and had positive zeta potential [12-14 mV]. siRNA and DTX were considerably loaded into NPs. The appropriate conjugation of the Apt to the NPs was affirmed by gel electrophoresis. The Apt-conjugated NPs were observed to enhance the cellular uptake of NPs into the SKBR3 cells. Although the combination treatment significantly decreased the cell viability of SKBR3 cells, the augmentative effect was observed when Apt was conjugated to NPs. Furthermore, Apt-conjugated NPs dramatically reduced the genetic expression of IGF-1R, signal transducers and activators of transcription 3 [STAT3], matrix metalloproteinases [MMP9], and vascular growth factor [VEGF]

Conclusion: The targeted NPs may augment the targeting of pathways involved in tumorigenesis and metastasis of breast cancer. Therefore, more animal model experiments are needed to further clarify the efficacy and safety of this functionalized nanodrug

[ simple method for purification of mouse igg2a by ion exchange and affinity chromatography]

Background: Purified mouse IgG2a [a product that could be used in medical research] subclass could be used for animal immunization to production of polyclonal Antibody and to obtain hybridomas in Monoclonal Antibody Production Procedures. The goal of this study was to purify the mouse IgG2a

Methods: In one step, Ion exchange chromatography was carried out for purification of mouse IgG and then in second step, ProA affinity chromatography was used for IgG2a purification .The chosen method for determination of purity was reduced and non-reduced SDS-PAGE. ELISA method was used for titer and isotype determination

Results: Mouse Igs with a protein concentration of 27mg/ml [volume: 3CC] was applied on Ion exchange column. Purification by Ion exchange chromatography yielded about 28mg of mouse IgG. Eight mg mouse IgG2a was obtained by ProA affinity chromatography. In reduced SDS-PAGE analysis of purified antibody, two bands were seen in 25and 50 KDa MW positions. Isotype determination of purified mouse IgG2a with mouse isotyping Kit showed the presence of mouse IgG2a isotype with a kappa light chain in related fraction

Conclusion: Purified mouse IgG2a subclass was obtained with purity more than 95%. Due to the obtained high purity we concluded that Ion exchange chromatography following by ProA affinity chromatography could be a suitable method for purification of mouse IgG subclasses with high quality. Our product is an economical and suitable product that takes a step towards self-sufficiency of the country

Effect of anger patterns and depression on serum IgA and NK cell frequency

Background: there are conflicting findings about relationship between depression and anger with immunological parameters

Objective: to investigate the relationship between anger patterns and immune system in depressed patients

Methods: thirty-five patients with major depressive disorder were selected according to DSM-IV criteria. The Hamilton Depression Scale and Spielberger Anger questionnaires were used to determine severity of depression and "anger expression pattern", respectively. The control group without a previous history of mental illness was also selected. In the group of patients with moderate depression, serum IgA levels and NK cell percentage were measured

Results: mean differences of all types of "anger expression pattern", including; "state-trait anger", "anger expression out", "anger expression in", "anger control out" and "anger control in", between study and control groups, were statistically significant [p<0.05]. Difference in mean serum levels of IgA in either group was not significant [p=0.9], but the mean difference was significant in terms of NK-cell percentage in both groups [p=0.04]. There was no significant relationship between IgA levels and percentage of NK-cell with all types of "anger expression pattern" in both groups. Only in the control group, IgA had significant correlation with Anger control out [p=0.04]

Conclusion: moderately depressed patients versus control group had higher Spielberger scores in all types of anger expression pattern except anger controlout and anger control-in. We found no evidence supporting the relationship between" anger expression pattern" and IgA levels and NK cell percentage; however, it seems that depression itself causes reduced number of NK cells and increased IgA levels

Frequency of null allele of Human Leukocyte Antigen-G [HLA-G] locus in subjects to recurrent miscarriage

Background: Human leukocyte antigen-G [HLA-G] is a non-classical class I molecule highly expressed by extravillous cytotrophoblast cells. Due to a single base pair deletion, its function can be compensated by other isoforms. Investigating the frequency of null allele in Recurrent Miscarriage [RM] subjects could be useful in understanding the relationship between frequency of this allele and RM in a given population

Objective: This study aimed to determine the frequency of HLA-G*0105N null allele and its potential association with down-regulation of HLA-G in subjects with RM

Materials and Methods: Western blotting was used to assess the level of HLA-G protein expression. For investigating the frequency of HLA-G*0105N null allele in RM subjects, PCR-RFLP method was used. Exon 3 of HLA-G gene was amplified by polymerase chain reaction [PCR]. Subsequently, PpuM-1 enzyme was employed to digest the PCR products and fragments were analyzed using gel electrophoresis

Results: Digestion using restriction enzyme showed the presence of heterozygous HLA-G*0105N null allele in 10% of the test population. Western blotting results confirmed the decrease in expression of HLA-G in the placental tissue of subjects with RM compared to subjects who could give normal birth

Conclusion: The frequency of heterozygous HLA-G*0105N null allele was high to some extent in subjects with RM. The mutation rate in subjects suggested that there is a significant association between RM and frequency of mutations in this allele

Correlation between major depressive disorder and circulating natural killer cells

Background: major depressive disorder is a mental disorder characterized by a pervasive and persistent low mood that is accompanied by low self-esteem and loss of interest or pleasure in normally enjoyable activities. Depression is associated with multiple immunological disorders. Aim of the present study was to determine correlation between percentage of circulating NK cells and major depressive disorder

Materials and Methods: patients older than 18 years with the desire to participate were enrolled in this study. For depression evaluation, we used the Hamilton Depression Rating Scale and for determination of percentage of NK cells in peripheral blood, flow cytometry method was used

Results: our results showed that in patients with major depressive disorder, numbers of circulating NK cells have significantly reduced

Conclusion: according to our findings, depression is associated with "immune suppression". NK cells are important in early phase of immunological surveillance versus viral infections and tumors. Indeed, depressive patients are susceptible to cancers and infections

Development of a stable cell line, overexpressing human T-cell immunoglobulin mucin 1

Background: Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 [TIM-1] glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases

Objectives: In this study, we aimed to express TIM-1 protein on Human Embryonic kidney [HEK] 293T cell line in order to have an available source of the TIM-1 antigen

Materials and Methods: The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells [PBMC] and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA[TM]3.1/Hygro [+] and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR

Results: The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells

Conclusions: Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1

Cloning and expression of the variable regions of anti-EGFR monoclonal antibody in E.coli for production of a single chain antibody

Epidermal growth factor receptor [EGFR] overexpression is a characteristic of several malignancies and could be considered as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smaller sizes and short circulation half-lives. The aim of this study was cloning, expression and purification of variable regions of anti-EGFR monoclonal antibody in E. coli for production of single chain antibodies. The RNA, extracted from the C225 hybridoma cells, was reverse transcribed into cDNA and used for PCR amplification of genes encoding light and heavy chains from the variable regions. The PCR products were cloned and expressed in E. coli BL21 for production of a single chain antibody. The expressed protein was analyzed by SDS-PAGE and purified by Ni-NTA affinity chromatography. The reactivity of purified C225-scFv with EGFR-expressing A431 tumor cell line was tested by western blotting and enzyme-linked immunosorbent assays. The results indicated that C225-scFv was highly expressed in E. coli and appeared as a protein with a mass of 27 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE] analysis of the induced cell lysate. Reactivity analysis of the purified C225-scFv with A431 tumor cell line by western blotting and enzyme linked immune sorbant assay [ELISA] revealed high binding affinity of the recombinant C225-scFv to the target cells. The results of this study indicated that C225-scFv is capable of binding to EGFR and could be considered as a useful tool for diagnosis and treatment of EGFR-overexpressing tumor cells

Generation and characterization of anti-CD34 monoclonal antibodies that react with hematopoietic stem cells

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies [mAbs] directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin [KLH]-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine [HAT] selective medium and cloned by the limiting dilution [L.D] method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay [ELISA] and Western blotting analyses. One of the mAbs [3D5] was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells [UCB] in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies [mAb] can be a useful tool for isolation and purification of human hematopoietic stem cells [HSCs]

Protective properties of nontoxic recombinant exotoxin A [domain I-II] against pseudomonas aeruginosa infection

Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. In this study, recombinant exotoxin A [domains I and II], [ExoA I-II] protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A [Domains I, II]. Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 [7.5 x 10[7] CFU] of clinical strain of mucoid P. aeruginosa. Sequencing of the cloned gene showed that the sequence of ExoA one-two gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA one-two increased survival times against intra-peritoneal challenge with an approximate 7.5 x 10[7] CFU [2 x LD50] of clinical strain of P. aeruginosa. Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa

Cloning, expression and characterization of recombinant exotoxin A-flagellin fusion protein as a new vaccine candidate against pseudomonas aeruginosa infections

Background: infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A [exo A] and killed whole cell. However, none of them are currently available clinically

Methods: in this research, recombinant exoA-flagellin [fliC] fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated

Results: expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections

Conclusion: these results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Iran. Biomed. J. 17 [1]: 1-7, 2013

Study on serum homocysteine level in Alzheimer's disease and its relationship with the stage of this disease

To investigate homocysteine levels in Alzheimer's disease and its relationship with the severity of disease. This investigation was performed as a case-control study on 40 Alzheimer patients and 40 non-Alzheimer patients in Tabriz, Iran from May 2006 to September 2007. Alzheimer patients were selected based on the criteria of the American Psychological Association. The severity of illness was determined based on Reisberg scale. Mental status of the patients was evaluated by Mini Mental State Examination [MMSE]. The serum levels of homocysteine were measured by enzyme-linked immunosorbent assay method. The average serum homocysteine level in the 40 patient group was 23.01 +/- 14.40mmol/L, and in the 40 patient control group was 15.40 +/- 6.23 [p=0.003]. The average serum homocysteine level in the first group of patients was 21.7 +/- 12.7mmol/L, in the second group 22.3 +/- 13.8, and in the third group 24.9 +/- 17.2. The relationship between MMSE score and serum homocysteine level of patients was not significant [p=0.4]. The average serum homocysteine level in Alzheimer patients was higher than in the control group, however, it did not show a significant relationship with the severity of illness

Evaluation of hemostatic factors in children with nephrotic syndrome

Thromboembolism [TE] is one of the serious complications of nephrotic syndrome [NS]. The aim of this study was to evaluate the haemostatic factors in children with nephrotic syndrome. Plasma Level of protein C, Protein S, fibirinogen and antithrombin III [AT III] were evaluated in thirty nephrotic children at relapse and remission period and the results were compared with those of 30 healthy children. The mean age of patients was 5.38 +/- 3.07 years. Plasma Level of protein S and AT III during relapse were significantly lower than their level in remission period and in control group. The mean fibrinogen level during relapse was significantly higher than its level in remission period and in control group. There was no significant difference in protein C levels at relapse with remission period and with control group. Serum albumin levels during relapse were positively correlated with AT III levels. There was no correlation between urinary protein excretion and the haemostatic factors. Despite reduced levels of AT III and protein S and increased levels of fibrinogen, none of our patients revealed thromboembolism. It seems that coexistence of several factors is necessary to induce TE