Comparative Analysis of Apigenin-3 Acetate versus Apigenin and Methyl-Prednisolone in Inhibiting Proliferation and Gene Expression of Th1 Cells in Multiple Sclerosis.

OBJECTIVE: In spite of the advances in therapeutic modalities, morbidity, due to multiple sclerosis (MS), still remains high. Therefore, a large body of research is endeavouring to discover or develop novel therapies with improved efficacy for treating MS patients. In the present study, we examined the immunomodulatory effects of apigenin (Api) on peripheral blood mononuclear cells (PBMCs) isolated from MS patients. We also developed an acetylated form of Api (apigenin- 3-acetate) to improve In its blood-brain barrier (BBB) permeability. Additionally, we compared its anti-inflammatory properties to original Api and methyl-prednisolone-acetate (a standard therapy), as a potential option in treating MS patients. MATERIALS AND METHODS: The current study was an experimental-interventional research. The half maximal inhibitory concentration (IC50) values for apigenin-3-acetate, apigenin, and methyl-prednisolone-acetate were determined in healthy volunteers' PBMCs (n=3). Gene expressions of T-box transcription factor (TBX21 or T-bet) and IFN-γ, as well as proliferation of T cells isolated from MS patients' PBMCs (n=5), were examined in co-cultures of apigenin-3-acetate, Api and methyl-prednisolone-acetate after 48 hours of treatment, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Our findings showed that apigenin-3-acetate, apigenin, and methyl-prednisolone-acetate at concentrations of 80, 80, and 2.5 M could inhibit Th1 cell proliferation after 48 hours (P=0.001, P=0.036, and P=0.047, respectively); they also inhibited T-bet (P=0.015, P=0.019, and P=0.022) and interferon-γ (IFN-γ) gene expressions (P=0.0001). CONCLUSION: Our findings suggested that Api may have anti-inflammatory properties, possibly by inhibiting proliferation of IFN-producing Th1 cells. Moreover, comparative immunomodulatory effects were found for the acetylated version of apigenin-3-acetate versus Api and methyl-prednisolone-acetate.

Blood IgMs from healthy donors and patients with systemic lupus erythematosus reduce the inflammatory properties of platelets from healthy donors.

Uncontrolled inflammation is the underlining mechanism of many human diseases and the increasing prevalence of such diseases mandate to develop new anti-inflammatory treatments. Utilizing the anti-inflammatory properties as well as other protective/beneficial features of natural IgMs (nIgMs) for treatment of human disorders seems as an easily accessible goal by the use of blood-purified IgMs as an alternative for polyclonal nIgMs. Despite the other blood cells, the functions of platelets have not been inspected under the influence of blood IgMs adequately. However, platelets, the second most numerous blood cells, are involved in the pathology of many inflammatory disorders through the production/expression of many inflammatory molecules. Thus, in the present study, we purified IgMs from serum of healthy donors and plasma of patients with systemic lupus erythematosus (SLE). Subsequently, we carried out comparative analysis of the inflammatory functions of normal platelets (P-selectin expression, GPIIb/IIIa activation, and secretion of soluble CD40L and TNF-α) that were stimulated by SLE microparticles (as key endogenous inflammation-drivers) in the presence or absence of the two IgM preparations; one with normal level of nIgMs (healthy blood IgMs) and the other with likely altered nIgM content (SLE blood IgMs). Both blood IgM preparations could suppress the elevated activation parameters of platelets in response to SLE microparticles. Additionally, the impact of SLE blood IgMs on the platelets was not superior to that of normal blood IgMs. The anti-inflammatory effects of blood IgMs on the activated platelets have been shown for the first time in the present study. Thus, this study provides evidence in favor of use of healthy blood IgMs as an anti-inflammatory therapy in clinical settings.

The impact of neutrophil extracellular trap from patients with systemic lupus erythematosus on the viability, CD11b expression and oxidative burst of healthy neutrophils.

BACKGROUND: NET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured. RESULTS: The level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET. CONCLUSION: The decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

Modulation of Th17 Proliferation and IL-17A Gene Expression by Acetylated Form of Apigenin in Patients with Multiple Sclerosis.

The presence of Th17 cells in CNS lesion of MS patients due to their inflammatory cytokines secretion is in line with the deterioration of the disease. Currently, the use of natural compounds with anti-inflammatory properties such as flavonoids have been considered to reduce inflammation in these patients, but the remaining issue is how deliver these compounds to the site of inflammation. Acetylation is a way to better uptake compound by cells and cross through cellular layers with tight junctions. This study aimed to investigate the in vitro effects of the Apigenin 3-Acetate on Th17 cells of MS patients and compare its efficacy with Apigenin and Methyl Prednisolone Acetate. IC50 for Apigenin 3-Acetate, and Methyl Prednisolone Acetate were determined using three healthy volunteers. The peripheral blood mononuclear cells (PBMCs) of five MS patients were isolated and co-cultured with a selected dose of Apigenin, Apigenin 3-Acetate, and Methyl Prednisolone Acetate for 48 hr, and then theproliferation of Th17 cells in isolated PBMCs was assessed by flow cytometry. The levels of RAR-related orphan receptor (RORC) and IL-17A expression were also determined by quantitative real-time PCR. The results showed that Apigenin 3-Acetate inhibited Th17 cells proliferation (P value: 0.018) at 80 µM concentration after 48 hr. Additionally, IL-17A gene expression significantly (P value≤ 0.0001) inhibited by Apigenin, Apigenin 3-Acetate and Methyl Prednisolone Acetate in 80 µM, 80 µM and 2.5 µM (selected dose in IC50 determination) respectively These results demonstrate that Acetate increases anti-inflammatory effects of Apigenin on Th17 cells.

Human Amniotic Epithelial Cells Affect the Functions of Neutrophils.

BACKGROUND AND OBJECTIVES: As a stem cell group, Human amniotic epithelial cells (HAECs) have numerous advantages over their embryonic and adult counterparts for therapeutic utility. They are closer to clinical applications compared to other stem cell types. Additionally, the anti-inflammatory and immunoregulatory properties of HAECs toward several immune cells have been shown previously. Nevertheless, despite the ever-increasing importance of neutrophils in the immune and non-immune processes, a few studies investigated the interaction of neutrophils and HAECs. To increase the current knowledge of HAECs immunology which is necessary for optimizing their future clinical applications, here we explored the effect of HAECs on two chief neutrophil functions; respiratory burst and phagocytosis. METHODS AND RESULTS: Freshly isolated human blood neutrophils were co-cultured with different number of HAECs for about 24 or 48 hours, then the oxidative burst and phagocytosis of stimulated neutrophils were assessed and compared. The results demonstrated a substantial elevation in the phagocytosis percentage, conversely a significant reduction in the oxidative burst of HAECs-cocultured neutrophils. These effects were dose-dependent, but did not show similar patterns. Likewise, the elongation of coculture period inversely influenced the HAECs-induced effects on the two neutrophil functions. CONCLUSIONS: The present study, for the first time, investigated the HAECs-mediated effects on the two main neutrophil functions. The findings suggest that HAECs by enhancement of phagocytic ability and simultaneously, attenuation of oxidative burst capacity of neutrophils protect the fetus from both microbial treats and oxidative stress and their consequent inflammation; thus corroborate the current anti-inflammatory vision of HAECs.

Autologous plasma versus fetal calf serum as a supplement for the culture of neutrophils.

OBJECTIVE: Currently, the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of the cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have a relatively equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 h of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry. RESULTS: Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.

Evaluation of polyacrylonitrile electrospun nano-fibrous mats as leukocyte removal filter media.

Removal of leukocytes from blood products is the most effective means for elimination of undesirable side effects and prevention of possible reactions in recipients. Micro-fibrous mats are currently used for removal of leukocytes from blood. In this study, samples of electrospun nano-fibrous mats were produced. The performance of the produced electrospun nano-fibrous mats as means of leukocytes removal from fresh whole blood was both evaluated and compared with that of commercially available micro-fibrous mats. In order to produce the samples, polyacrylonitrile (PAN) nano-fibrous mats were made under different electrospinning conditions. Mean fiber diameter, pore characterization and surface roughness of the PAN nano-fibrous mats were determined using image processing technique. In order to evaluate the surface tension of the fabricated mats, water contact angle was measured. The leukocyte removal performance, erythrocytes recovery percent and hemolysis rate of the nano-fibrous mats were compared. The effectiveness of nano-fibrous mats in removing leukocyte was established using both scanning electron microscope and optical microscope. Results showed that for given weight, the fabricated nano-fibrous mats were not only more efficient but also more cost-effective than their commercial counterparts. Results confirmed that changes in mean fiber diameter, the number of layer and weight of each layer in the absence of any chemical reaction or physical surface modification, the fabricated nano-fibrous mats were able to remove 5-log of leukocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1759-1769, 2018.

Expression of microRNA-370 in human breast cancer compare with normal samples.

BACKGROUND: Breast cancer is the second leading cause of deaths from cancer in the woman. MicroRNAs (miRNAs) are endogenous noncoding RNAs that are known critical player in carcinogenesis. The role of miR-370 in malignancies remains controversial because of its levels varying in different cancers according to its targets while the role of miR-370 in breast cancer has not been addressed so far. The aim of this study was to identify the expression pattern of miR-370 in human breast cancer tissue compared to adjacent healthy tissue. MATERIALS AND METHODS: Twenty-two fresh frozen tissues (normal and malignant) from patients with breast cancer were examined for miR-370 by quantitative real-time polymerase chain reaction method at 2013. RESULTS: We observed up-regulation (six-fold higher) of miR-370 in breast cancer tissue compared with normal adjacent tissue. Tumor samples in stage III, invasive ductal type, larger tumor size, human epidermal growth-factor receptor 2+, estrogen receptor/progesterone receptor-, P53 - status showed significantly increased expression in miR-370. CONCLUSION: Together, miR-370 may acts as an onco-miRNA, and it may have a novel role in breast cancer. Detection of miR-370 and its targets could be helpful as a diagnostic biomarker and therapeutic target.

In silico and in vitro studies of cytotoxic activity of different peptides derived from vesicular stomatitis virus G protein.

OBJECTIVES: This study aims at exploring cytotoxic activity of different peptides derived from VSVG protein against MCF-7 and MDA-MB-231 breast cancer cell lines and human embryonic kidney normal cell (HEK 293). MATERIALS AND METHODS: The ANTICP web server was used to predict anticancer peptides. The cytotoxic activity of peptides with high score (P26, P7) and low score (P19) was examined by MTT and DNA fragmentation assays. RESULTS: The results obtained from ANTICP web server demonstrated that 4 out of 48 peptides (P26, P7, P10, and P16) had anticancer activity. P26 and P7 peptides of these 4 peptides were detected to have high cytotoxic activity against MCF-7 cells with CC50 values of 98,280 µg/ml and MDA-MB231 cells with CC50 100,550 µg/ml, respectively. In addition, the results showed that amino acid residues of these 4 peptides were located near fusion domain. CONCLUSION: The results confirmed that P26 and P7 peptides might induce membrane damage and initiate apoptosis. The present study suggested that P26 and P7 peptides could be appropriate candidates for further studies as cytotoxic agents and modifications in the residue at positions 70-280 might potentially produce a more efficient VSVG protein in gene therapy.

Analysis of human papillomavirus and herpes simplex virus genus -2 from patients with cervical cancer in isfahan, iran.

BACKGROUND: Although cervical uterine cancer is a second form of cancer among women, but it occupies fifth form among all cancer types. METHODS: In the present study, human papillomavirus (HPV) and herpes simplex virus 2 (HSV-2) in cervical cancer patients by using real time polymerase chain reaction (PCR) technique and the relation between their viral loads were investigated. 156 cervical carcinoma tissues were collected from married women in health centers in Isfahan, Iran. RESULTS: The results showed that among 156 specimens, 58.97%, 45.51% and 7.05% were positive for HPV DNA, HPV-16 and HPV-18 respectively. Only in 2.3% specimens, HSV-2 and HPV-16 were positively detected where viral load HSV-2 in conjunction with HSV-16 dramatically increased. CONCLUSION: Thus the present study not only confirmed that viral load of HPV-16 is more than other HPV types, but also in possible conjunction with HSV-2, both rates will significantly increase.

Epidemiology of herpes human virus 6 and 7 infections in salivary gland neoplasms in isfahan, iran.

BACKGROUND: The previous studies showed that herpes human virus-6 (HHV-6) and HHV-7 exist in salivary glands. One of the important areas in oral and maxillofacial pathology field is tumors of the salivary glands. In this study, to declare the major sites of persistent infection with HHV-6 and HHV-7, the existence of HHV-6 and HHV-7 genomes in formalin-fixed paraffin embedded tissue samples of salivary gland tumors. METHODS: This analytical study was performed in 60 paraffin blocks samples of malignant and benign neoplasms of both major and minor salivary glands. This study performed with highly sensitive real time PCR method. RESULTS: Among 60 paraffin blocks salivary gland tumors with equal chances of presence of the HHV-7 and HHV-6 in the samples, 34% were positive for both HHV-7 and HHV-6 while 47.2% were only positive for HHV-7, 18.9% samples were positive for HHV-6. A relationship was noticed between HHV-7 and HHV-6 genomes. CONCLUSION: In conclusion, this study showed no relation between virus and diseases with P=0.953. Also it could be inferred that there is a relationship between HHV-6 and 7 in salivary glands neoplasms.

Biased Treg/Th17 balance away from regulatory toward inflammatory phenotype in relapsed multiple sclerosis and its correlation with severity of symptoms.

The opposing immune functions of Treg and Th17 lymphocytes and the plasticity of Treg/Th17 differentiation, has led us to investigate the effects of their fluctuations and counterbalance in autoimmune condition of multiple sclerosis (MS). Evaluation of Treg and Th17 frequency in peripheral blood of a group of relapsed MS patients, showed a decrease in Treg/Th17 ratio compared to that of healthy controls. A reverse correlation between these subsets was observed in controls but not in patient groups. Both Treg frequency and Treg/Th17 ratio were negatively correlated with severity of symptoms. There was shown to be an enduring increase in Treg frequency associated with MS disease.

The effect of platelet rich plasma on chondrogenic differentiation of human adipose derived stem cells in transwell culture.

OBJECTIVE(S): Platelet-rich plasma (PRP) has recently emerged as a promising strategy in regenerative medicine due to its multiple endogenous growth factors. Little is known about the role of PRP as a promoter in chondrogenesis of human adipose derived stem cells (hADSCs). The aim of this study was to determine whether PRP may be considered as a natural and easy achievable source of growth factors to promote the chondrogenic differentiation of hADSCs in Transwell culture. Materials and Methods : Biochemical, immunohistological and molecular assays were used to evaluate the effect of different concentrations (5%, 10%, and 15%) of PRP on chondrogenic differentiation of hADSCs in Transwell culture. Results : The cells in the presence of 10% PRP produced markedly higher amounts of GAG and DNA, in comparison to the control group. PRP also increased chondrogenic markers in these cells, such as sox-9, aggrecan and collagen type II. A high expression level of collagen type X as a hypertrophic marker was observed in cartilage produced by using either PRP or TGF-ß1. Conclusion : Our findings indicate that autologous PRP at an optimum concentration had beneficial effects on differentiation of hADSCs in Transwell culture. Further, in vivo studies are necessary to fully define the clinical implications of PRP.

The effect of different blood components on exchange transfusion outcomes.

BACKGROUND: Exchange transfusion (ET) has been known as an effective treatment in sever neonatal jaundice. Prescribing appropriate blood group makes an important role in patient's outcome and no single component is unequivocally the best. The purpose of this study was to evaluate the effect of ABO compatible packed cell, dried O, and routine O groups on exchange transfusion outcomes. METHODS: This multicenter clinical trial study is the combination of two studies which were conducted at three university hospitals (Isfahan University of Medical Sciences, Isfahan, Iran). A hundred full term infants with more than 2.5 kg body weight, serum bilirubin > or = 20 mg/dl and confirmed ABO-Hemolytic Disease of the Newborn (HDN) were participated in first study. Among 40 infants, 20 underwent the exchange transfusion with O packed cell (group 1) and other 20 were transfused with O dried packed cell (Hematocrit = 90%) (group 2). In the second study with the same eligibility criteria with first study, among the 60 infants, 30 had exchange transfusion with O packed cell (group 3) and the rest were transfused with infant isogroup (group 4). Serum bilirubin and hemoglobin (Hb) were evaluated before and 6, 12, 24 and 48 hours after the exchange transfusion. RESULTS: The means of Hb after the exchange transfusion were 14.3 mg/dl in group 1, 15.62 mg/dl in group 2, 14.98 mg/dl in group 3 and 14.30 mg/dl in group 4 with significantly higher in group 2 compared with others (p = 0.02). The mean of the bilirubin after the exchange transfusion had no statistical significant difference between the four groups (p > 0.05). The mean of Hb and bilirubin before exchange transfusion had no statistically difference between all groups (p > 0.05). The mean of bilirubin before the exchange transfusion in infants who had two transfusion was significantly higher than the mean of the bilirubin before the exchange transfusion in infants with one time transfusion (p = 0.05). There was no significant difference between four groups in exchange transfusion frequency (p > 0.05). DISCUSSION: This study indicated that the level of bilirubin before exchange transfusion is the only important factor which sometimes causes the necessity of second or third exchange.

Red cell antigens: Structure and function.

Landsteiner and his colleagues demonstrated that human beings could be classified into four groups depending on the presence of one (A) or another (B) or both (AB) or none (O) of the antigens on their red cells. The number of the blood group antigens up to 1984 was 410. In the next 20 years, there were 16 systems with 144 antigens and quite a collection of antigens waiting to be assigned to systems, pending the discovery of new information about their relationship to the established systems. The importance of most blood group antigens had been recognized by immunological complications of blood transfusion or pregnancies; their molecular structure and function however remained undefined for many decades. Recent advances in molecular genetics and cellular biochemistry resulted in an abundance of new information in this field of research. In this review, we try to give some examples of advances made in the field of 'structure and function of the red cell surface molecules.'