Ofatumumab monoclonal antibody affinity maturation through in silico modeling

Background: Ofatumumab, an anti-CD20 mAb, was approved in 2009 for the treatment of chronic lymphocytic leukemia. This mAb acts through immune-mediated mechanisms, in particular complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity by natural killer cells as well as antibody-dependent phagocytosis by macrophages. Apoptosis induction is another mechanism of this antibody. Computational docking is the method of predicting the conformation of an antibody-antigen from its separated elements. Validation of the designed antibodies is carried out by docking tools. Increased affinity enhances the biological action of the antibody, which in turn improves the therapeutic effects. Furthermore, the increased antibody affinity can reduce the therapeutic dose of the antibody, resulting in lower toxicity and handling cost

Methods: Considering the importance of this issue, using in silico analysis such as docking and molecular dynamics, we aimed to find the important amino acids of the Ofatumumab antibody and then replaced these amino acids with others to improve antibody-binding affinity. Finally, we examined the binding affinity of antibody variants to antigen

Results: Our findings showed that variant 3 mutations have improved the characteristics of antibody binding compared to normal Ofatumumab antibodies. Conclusion: The designed anti- CD20 antibodies showed potentiality for improved affinity in comparison to commercial Ofatumumab

evaluation of nerve growth factor over expression on neural lineage specific genes in human mesenchymal stem cells

Objective: Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer's disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells [MSCs] as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor [NGF] and assessed their neural lineage genes

Materials and Methods: In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein [GFP] gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 [HEK-293] packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction [PCR]

Results: We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor [GDNF], glial fibrillary acidic protein [GFAP] and Microtubule-associated protein 2 [MAP2] genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor [BDNF] genes reduced

Conclusion: Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may be the utilization of differentiated neural stem cells

Different methylation patterns of RUNX2, OSX, DLX5 and BSP in osteoblastic differentiation of mesenchymal stem cells

Runt-related transcription factor 2 [RUNX2] and osterix [OSX] as two specific osteoblast transcription factors and distal-less homeobox 5 [DLX5] as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein [BSP], osteopontin and collagen type I?1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells [MSCs]. In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR [MSP]. Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction [PCR]. MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role

Zoledrinic acid induces steoblastic differentiation of mesenchymal stem cells without change in hypomethylation status of osterix promoter

Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells [MSCs] has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix [OSX] promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs. In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5 micro M Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection [MOI] treatment, gene specific methylation of OSX was analyzed by methylation specific PCR [MSP]. The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation [p<0.05], but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards. MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases

[Epigenetic changes of RUNX2 and DLX5 genes in osteoblastic differentiation induced by zoledronic acid in mesenchymal stem cells]

Zoledronic acid as a main treatment for osteoporosis has an important role in differentiation of mesenchymal stem cells. However, mechanism of osteoblastic differentiation induction by zoledronic acid is not well understood until now. In this research we evaluate zoledronic acid effect on methylation status of RUNX2 and DLX5 promoters. After isolation and expansion of hMSCs from BM, they were pulse treated with 5 micro M ZA for 3h, and incubated in osteogenic differentiation medium for 3 weeks. DNA was extracted after first, second and third weeks of culture and also from undifferentiated MSCs. After SBS treatment, gene specific methylation analysis for RUNX2 and DLX5 were carried out by MSP using methylated and unmethylated primers. In the genes RUNX2 and DLX5, M and U primers of MSP amplified promoter regions of undifferentiated and osteoblastic differentiated MSCs. Therefore, methylation status in RUNX2 and DLX5 promoters present incomplete methylation. Methyltion patterns of RUNX2 and DLX5 don't change during zoledronic acid osteoblastic differentiation of MSCs. Our findings show that zoledronic acid does not induce osteoblastic differentiation via epigenetic mechanisms. Therefore, zoledronic acid can induce osteoblastic differentiation in a manner independent from DNA epigenetic changes

[Differentiation of cord blood stem cells into erythroid progenitor cells in semisolid culture media containing SCF, IL-3, IL-6 and EPO]

The study of erythropoiesis needs to develop the methods for erythroid progenitor cells [EPCs] culture using stem cell potency to differentiate into variety of hematopoietic cells lineages. In this study, we induced differentiation of cord blood stem cells into erythroid progenitor cells in a semisolid culture media. Cord blood mononuclear cells were isolated and cultured in liquid culture media consist of erythroid differentiation factors [phase I]. Non-adherent cells were cultured in semisolid media containing SCF, IL-3, IL-6 and EPO [phase II]. After one week, the appeared erythroid colonies were picked up. Characterization of differentiated cells was performed by assessment of CD235a and CD36 using flowcytometry, giemsa cytological staining and gene expression analysis of GATA-1, EKLF, -globin genes by RT-PCR. Flowcytometry analysis to detect CD235a and CD36 positive cells showed that 94.3% and 95.5% of differentiated cells have erythroid specific cell markers, respectively. Morphology of the cells in giemsa stained slides demonstrated erythroid progenitor cells, ranged from proerythroblast to orthochromatic erythroblast. The RT-PCR results, confirmed the precursor state of erythroid differentiated cells by expression of GATA-1, EKLF, -globin genes. Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies

[3D culture of umblical cord blood-drived CD34 + cells on a DBM scaffold coated by cord blood-derived unrestricted somatic stem cells [USSC]]

Allogeneic transplantation with umbilical cord blood [UCB] in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell [MPC] -unrestricted somatic stem cells [USSC] -was incorporated in an attempt to expand CD34+ cells from UCB. To provide a similar environment in vitro, we coated DBM scaffold with USSC cells as the matrix for support UCB-CD34+ cells growth. Human placenta USSC was isolated and characterized by morphologic and immunophenotypical analysis. UCB CD34+ cells were expanded by coculture with placental USSC in 2D and 3D environment. Suitable aliquots of cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture [LTC-IC] output. Ex vivo expansion of UCB hematopoietic cells, when cultured in different 2D conditions and 3D condition for 3 weeks, was significantly enhanced, the total cell count increased within the 28-day period. For total CFC, the highest CFC expansion was observed at day 14.Flow cytometry analysis of the percentage of CD34+ cells showed a decline in USSC cocultures in 2D and 3D condition at 3 weeks. These results strongly suggest that human USSC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro and USSC-coated DBM can therefore provide an ex vivo mimicry of bone marrow by enhancing of surface/ volume ratio and feeder layers, recapitulate the desired niche, and provide a suitable environment for stem cell expansion and differentiation

[Glucose 6-phosphate dehydrogenase deficiency in Tehran, Zanjan and Sistan-Balouchestan provinces: prevalence and frequency of Mediterranean variant of G6PD]

Glucose-6-phosphate dehydrogenase [G6PD] deficiency is an enzymopathy affecting about 400 million people worldwide. The distribution of G6PD deficiency and the molecular genetics of this enzyme vary widely among different ethnic groups. The aim of this study was to find out the frequency of G6PD deficiency and characterize the Mediterranean type mutation in deficient individuals in Turk, Balouch and Fars ethnic groups in Zanjan, Iranshahr and Tehran provinces. 1500 unrelated male individuals from Zanjan and 305 unrelated male students from Iranshahr were screened for G6PD deficiency by fluorescent spot test. Genomic DNA was extracted from deficient individuals and also from 64 G6PD deficient individuals from Tehran city. PCR was used to amplify flanking regions of exons 6 and 7 of this gene. The PCR products were digested by the MboII enzyme and electrophoresed on 3% agarose gel. Thirty-three [2.2%] individuals were shown to be deficient for G6PD from Zanjan population. Twenty-four out of 33 [72.8%] of the deficient individuals showed a mutation at nt 563 which is characteristic of Mediterranean type of mutation. Nine individuals were negative for this mutation. Fifty nine [19.3%] individuals of Iranshahr were shown to be deficient for G6PD. At the molecular level, 50 [85%] of the individuals showed Mediterranean type of mutation and 15% were negative for this mutation. Our results from Tehran showed that 47/64 [73.4%] of deficient individuals had Mediterranean type mutation and 26.6% were negative for this mutation. Despite different frequencies exist for deficiency of G6PD in Turk, Balouch and Fars populations, the results of the present study and others have shown that the predominant mutation of G6PD in Iran is of Mediterranean type

[ combination effect of dexamethasone with either metoclopramide or propofol on nusea and vomiting after general anesthesia]

A common complication after general anesthesia is nausea and vomiting followed by different problems such as spasm, hypoxia and pulmonary aspiration. This complication is more common in patients with full stomach, Eye injury, head trauma, cesarean and laparoscopy. Propofol and metoclopramide are two common drugs to prevent nausea and vomiting after operation. On the other hand adding dexamethasone to the above drug, has an important effect on decreasing nausea and vomiting. In this study, the effect propofol and metoclopramide associated with dexamethasone on nausea and vomiting after operation was investigated. In this clinical trial study, 100 patients with ASA I, II classes, aged 16-60 years with selective orthopedic surgery randomly have divided into two groups. In group one, 48 patients received metoclopramide [10 mg] with dexamethasone [8 mg] and in group two, 52 patients received propofol [20 mg] with dexamethasone [8 mg], five minutes before the end of operation. Prevalence of nausea and vomiting in both groups was considered after 4 hours and results were analyzed by Chi-Square, t-student and Fisher exact tests. The rate of nausea in group 1 and 2 was 35.4% and 11.5% respectively [P<0.05]. The rate of vomiting was 27.7% and 7.7% in group 1 and 2 respectively [P<0.05]. This showed that the antiemetic effect of propofol with dexamethasone is more effective to prevent nausea and vomiting that metoclopromide with dexamthasone

Multilineage differentiation activity by the human umbilical vein-derived mesenchymal stem cells

Mesenchymal stem cells [MSC] are a very promising transplantable stem cell source for a variety of cell replacement therapies. As the main source of MSC is bone marrow [BM], most of studies have been done on BM-derived MSC [BM-MSC]. Umbilical cord [UC]-derived MSC [UC-MSC] which are recently introduced, is one of the good alternative source for these cells. The objective of this study was to isolate and characterize UC-MSC from human UC veins and studying of their potential to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this way, a cell population was isolated from 20 UC veins using a solution of 0.1% collagenase type IV. After identification of isolated cells by immunocytochemical and flow cytometry methods, these cells were exposured with adipogenic, osteogenic, chondrogenic and neurogenic agents. Resulted differentiated cells were detected using specific staining for each lineage and room temperature [RT]-PCR. Immunophenotypically, this cell population was positive for CD73, CD 166, CD 105 markers and alpha-smooth muscle actin and negative for CD31, CD34, CD49d markers, von Willebrand factor and smooth muscle myosin. Exposure of these cells to adipogenic, osteogenic, chondrogenic and neurogenic agents resulted in morphological changes followed by lineage-specific staining for each differentiated cell type. RT-PCR analysis showed that these differentiated cells express fat, bone, cartilage and neuronal markers, respectively. Altogether, these findings indicate that UC-MSC possess morphological, immunophenotypical and cell differentiation capacities similar to BM-MSC and so they can be used more extensively in cell based therapy protocols and in vitro differentiation study models

Increase of CXCR4 expression on expanded non-enriched cord blood CD34+cells using MSCs

A number of potential cell adhesion molecules, which mediate essential cell-to-cell or cell-to-matrix interactions, are expressed on the surface of CD34+ hematopoietic progenitor cells [HPCs], including integrins, CD44, and CXCR4. These molecules are essential for homing process. In this study, we compared the changes of expression of CD44 and CXCR4 on the CD34+ hematopoietic progenitor cells expanded on MSCs in the presence of cytokines. Cord blood CD34+ cells were expanded using human bone marrow mesenchymal stem cells and cytokines [TPO, SCF, FLt-3, IL-6, and IL-3], and then expression of CD44 and CXCR4 on CD34+ cells were evaluated by flow cytometric analysis. After 2 weeks of serum free culture of CD34+ cells in the presence of cytokines, the expression of CXCR4 on CD34+ cells was decreased 3.4 fold [p<0.05]. In contrast, the expression of CXCR4 on CD34+ cells expanded on hMSCs was increased [p<0.05]. The expression of CD44 on expanded CD34+ cells in both methods did not differ significantly. Our results indicated that co-culture of cord blood stem cells on hMSCs significantly increased CXCR4 expression on cord blood CD34+ cells

Isolation, culture and characterization of postnatal human umbilical vein-derived mesenchymal stem cells

On the basis of reports that mesenchymal stem cells [MSCs] can be isolated from the placenta/umbilical cord stroma, the present study was undertaken to isolate and characterize MSCs from the human umbilical cord veins. In this investigation, a cell population was isolated which was derived from the endothelium/subendothelium layers of 20 umbilical cord veins obtained from term deliveries using a solution of 0.1% collagenase type IV. Results suggest that these cells possess morphological, immunophenotypical and cell differentiation capacities similar to the bone marrow-derived mesenchymal stem cells [MSCs]. The isolated cell population has fibroblastoid morphology which upon proper stimulation gives rise to adipocytes, osteocytes and chondrocytes in culture. Immunophenotypically, this cell population is positive for CD54, CD29, CD73, CD49e, CD166, CD105, CD13, and CD44 markers and alpha-smooth muscle actin and negative for CD31, CD45, CD49d, and CD34 markers, von Willebrand factor [vWF] and smooth muscle myosin [MySM]. Altogether, these findings indicate that umbilical cord obtained from term deliveries is an important source of MSCs which could have an important application in cell therapy protocols