Epigenetic modulation of cytokine expression in Mycobacterium tuberculosis-infected monocyte derived-dendritic cells: Implications for tuberculosis diagnosis.

BACKGROUND: To delineate alterations in DNA methylation at high resolution within the genomic profile of monocyte-derived-dendritic cells (mo-DCs) in connection with Mycobacterium tuberculosis (MTB) infection, with particular emphasis on pro/ anti-inflammatory genes. METHODS: In the context of this investigation, mo-DCs were infected by various active strains of MTB (Rifampicin-resistant [RIFR], H37Rv, multidrug-resistant [MDR], and extensively drug-resistant [XDR]). Subsequently, the pro/anti-inflammatory hub gene expression levels within the IL-6, IL-12, IFN-γ, IL-1ß, TNF-α, and IL-10 pathways were evaluated employing real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, the effects of MTB infection on mo-DC protein expression were examined through western blot analysis. The methylation status (%) of TNF-α and IL-10 was considered through Methylation Sensitive-High Resolution Melting (MS-HRM). RESULTS: The results revealed an up-regulation of all pro-inflammatory genes among all groups, with TNF-α exhibiting the highest expression level. Conversely, the anti-inflammatory gene (IL-10) showed a down-regulated expression level. Furthermore, the DNA methylation status (%) of TNF-α decreased significantly among all the groups (P < 0.001), although there were no notable distinctions in the DNA methylation status (%) of IL-10 when compared to the control group (P > 0.05). CONCLUSION: MTB infection induces DNA methylation changes in mo-DCs. The hypo-methylation of TNF-α may induce the up-regulation of this gene. This correlation revealed that the more resistant the MTB strain (XDR) is, the lower the methylation status (%) in the TNF-α gene.

Downregulation of ACE, AGTR1, and ACE2 genes mediating SARS-CoV-2 pathogenesis by gut microbiota members and their postbiotics on Caco-2 cells.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a complex infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause also gastrointestinal symptoms. There are various factors that determine the host susceptibility and severity of infection, including the renin-angiotensin system, the immune response, and the gut microbiota. In this regard, we aimed to investigate the gene expression of ACE, AGTR1, ACE2, and TMPRSS2, which mediate SARS-CoV-2 pathogenesis by Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, and Bacteroides fragilis on Caco-2 cells. Also, the enrichment analysis considering the studied genes was analyzed on raw data from the microarray analysis of COVID-19 patients. MATERIALS AND METHODS: Caco-2 cells were treated with live, heat-inactivated form and cell free supernatants of A. muciniphila, F. prausnitzii, B. thetaiotaomicron and B. fragilis for overnight. After RNA extraction and cDNA synthesis, the expression of studied genes was assessed by RT-qPCR. DNA methylation of studied genes was analyzed by Partek® Genomics Suite® software on the GSE174818 dataset. We used GSE164805 and GSE166552 datasets from COVID-19 patients to perform enrichment analysis by considering the mentioned genes via GEO2R, DAVID. Finally, the related microRNAs to GO terms concerned on the studied genes were identified by miRPath. RESULTS: The downregulation of ACE, AGTR1, and ACE2 genes by A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis in live, heat-inactivated, and cell-free supernatants was reported for the first time. These genes had hypomethylated DNA status in COVID-19 patients' raw data. The highest fold enrichment in upregulated RAS pathways and immune responses belonged to ACE, AGTR1, and ACE2 by considering the protein-protein interaction network. The common miRNAs targeting the studied genes were reported as miR-124-3p and miR-26b-5p. CONCLUSION: In combination with our experimental data and bioinformatic analysis, we showed the potential of A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis and their postbiotics to reduce ACE, ATR1, and ACE2 expression, which are essential genes that drive upregulated biological processes in COVID-19 patients. Accordingly, due to the potential of studied bacteria on the alteration of ACE, AGTR1, ACE2 genes expression, understanding their correlation with demonstrated miRNAs expression could be valuable. These findings suggest the importance of considering targeted gut microbiota intervention when designing the possible therapeutic strategy for controlling the COVID-19.

Aberrant methylation of host macrophages induced by tuberculosis infection.

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P < 0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.

In silico design, cloning, expression and immunologic evaluation of ED fusion protein of NT H. influenzae.

Infections due to nontypeable Haemophilus influenzae (NTHi) are important causes of child mortality throughout the world. Given the lack of effective vaccines for these strains and the spread and prevalence of these infections in the world, it is necessary to design novel vaccine candidates against these strains. D and E proteins are conserved membrane-specific lipoproteins among encapsulated and non-encapsulated H. influenza strains, which, according to the exposure surface and conservation degree between both strains, can be considered as vaccine candidates suitable for studies. This research was conducted to design a recombinant truncated fusion protein ED. Vaccination of BALB/c mice with recombinant truncated fusion protein ED showed high level of protective responses against NTHi. There were also strong responses of IgG and its subclasses (especially IgG1) as well as high titer levels of IL-4. A mixture of responses was observed considering IgG2a and INF-γ antibody titers, but the dominant response was toward Th2. According to the obtained results and the importance of humoral immunity in the immune system and vaccines production, it could be concluded that the produced recombinant construct can be used as a suitable vaccine candidate against NTHi or together with other carrier proteins.

Apoptotic effects of two COX-2 inhibitors on breast adenocarcinoma cells through COX-2 independent pathway.

Recently, much effort has been directed toward the search for compounds that influence apoptosis and to understand their mechanisms of action. Cyclooxygenase (COX)-2 inhibitors may induce apoptosis through the COX-2-independent mechanism via a mitochondrial pathway. In view of the reported antiproliferative activities of two COX-2 inhibitor derivatives (1, 2) in breast cancer cells (MCF-7), the present study was undertaken to evaluate the potential of these compounds to induce apoptosis and unravel the associated mechanisms. The apoptotic activities of the two compounds were assessed using flow cytometry, fluorescence microscope, and Western blot analysis. Compounds 1 and 2-treated MCF-7 cells revealed the apoptotic cell death, as confirmed by the changes in nuclear morphology and the increased annexin-V/PI staining. Elevation of Bax to Bcl-2 ratio and activation of caspase-3 were found to be associated with the initiation of apoptosis induced by compound 1. Further investigation showed that compounds 1 and 2 inhibited NF-κB, FHC, and ERK activation, while no dramatic change was revealed in c-Myc and EGR-1 levels. Our data suggest that induction of apoptosis by compounds 1 and 2 is not associated with COX-2 expression and occurs through the NF-κB pathway, which sequentially inhibits P-ERK and FHC expression.

Antiproliferative effects of copper(II)-polypyridyl complexes in breast cancer cells through inducing apoptosis.

Although cisplatin has been used for decades to treat human cancer, some toxic side effects and resistance are observed. Previous investigations have suggested copper complexes as a novel class of tumor-cell apoptosis inducers. The present study aimed to evaluate the anti-breast cancer activities of two polypyridyl-based copper(II) complexes, [Cu(tpy)(dppz)](NO3)2 (1) and [Cu(tptz)2](NO3)2 (2) (tpy = 2,2':6',2″-terpyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine, tptz = 2,4,6-tris(2-pyridyl)-1,3,5-triazine), using human breast adenocarcinoma cell line (MCF-7). The ability of the complexes to cleave supercoiled DNA in the presence and absence of external agents was also examined. The apoptotic activities of the complexes were assessed using flow cytometry, fluorescence microscope and western blotting analysis. Our results indicated the high DNA affinity and nuclease activity of complexes 1 and 2. The cleavage mechanisms between the complexes and plasmid DNA are likely to involve a singlet oxygen or singlet oxygen-like entity as the reactive oxygen species. Complexes 1 and 2 also significantly inhibited the proliferation of MCF-7 cells in a dose-dependent manner (IC50 values = 4.57 and 1.98 µM at 24 h, respectively). Complex 2 remarkably induced MCF-7 cells to undergo apoptosis, which was demonstrated by cell morphology, annexin-V and propidium iodide staining. The caspase cascade was activated as shown by the proteolytic cleavage of caspase-3 after treatment of MCF-7 cells with complex 2. Additionally, complex 2 significantly increased the expression of the Bax-to-Bcl-2 ratio to induce apoptosis. In conclusion, these results revealed that complex 2 may be a potential and promising chemotherapeutic agent to treat breast cancer.

Comparison of the efficiency of ultrafiltration, precipitation, and ultracentrifugation methods for exosome isolation.

Extracellular vesicles (EVs) are enclosed by a lipid-bilayer membrane and secreted by all types of cells. They are classified into three groups: apoptotic bodies, microvesicles, and exosomes. Exosomes play a number of important roles in the intercellular communication and crosstalk between tissues in the body. In this study, we use three common methods based on different principles for exosome isolation, namely ultrafiltration, precipitation, and ultracentrifugation. We use field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) analyses for characterization of exosomes. The functionality and effect of isolated exosomes on the viability of hypoxic cells was investigated by alamarBlue and Flow-cytometry. The results of the FESEM study show that the ultrafiltration method isolates vesicles with higher variability of shapes and sizes when compared to the precipitation and ultracentrifugation methods. DLS results show that mean size of exosomes isolated by ultrafiltration, precipitation, and ultracentrifugation methods are 122, 89, and 60 nm respectively. AlamarBlue analysis show that isolated exosomes increase the viability of damaged cells by 11%, 15%, and 22%, respectively. Flow-cytometry analysis of damaged cells also show that these vesicles increase the content of live cells by 9%, 15%, and 20%, respectively. This study shows that exosomes isolated by the ultracentrifugation method are characterized by smaller size and narrow size distribution. Furthermore, more homogenous particles isolated by this method show increased efficiency of the protection of hypoxic cells in comparison with the exosomes isolated by the two other methods.

MiR-17-92 cluster: an apoptosis inducer or proliferation enhancer.

Study of the non-coding RNA roles in the regulation of adaptive immune responses through T cells could be the basis of novel therapeutic applications. MicroRNAs (miRNAs) are a class of short non-coding RNAs that control the cell's functions and destination. To investigate the role of miRNAs in T cell activation, herein the expressions of miR-17-92 cluster and its paralogs were studied in naïve CD4(+)T cells that were activated by anti-CD2, -CD3, -CD28 microbeads and induced with or without IL-2. Proliferation and apoptosis rate of the cultured cells were determined by BrdU incorporation assay (ELISA) and propidium iodide staining, respectively. In continuation the expressions of eight miRNAs of the mentioned clusters were analyzed quantitatively. In addition their potential targets were predicted using multiple algorithms; as a confirmation, the transcription of PIK3R3 (a putative target of modulated miRNAs) was evaluated. Stimulation index (SI) of activated cells was decreased on day 6; whereas, the IL-2 induced cells showed increase in SI in the assay time. Evaluation of eight members of the aforementioned cluster showed upregulation of miR-92a-2* (~15 times) in IL-2 un-induced (activated) cells relative to the IL-2 induced cells. In silico investigations revealed that the suggested miRNAs targeted genes that were involved in cell proliferation, survival, and apoptosis. Transcriptional analysis of PIK3R3 illustrated decrease in activated cells relative to IL-2 induced cells. According to our findings, it seems that multiple members of miR-17-92 families in activated CD4(+)T cells inhibited negative regulators of IL-2 such as DUSP, PTPN, and SOCS families after IL-2 induction. According to our findings, it seems that multiple genes of cell proliferation-related families such as MAPK, E2F, AKT, STAT, and JAK as well as PIK3R3 are inhibited by miR-17-92 cluster in activated cells. As FASL is a putative target of over-expressed miRNAs in activated cell, antigen-induced cell death (AICD) might be occurred in FASL-independent manner. Altogether this study suggested that clonal expansion through IL-2 signaling pathway does not depend on the members of miR-17-92 family; while, it appears that AICD in activated CD4(+)T cells without IL-2 induction is affected by these miRNA clusters.

Immunological evaluation of predicted linear B-cell epitopes of human CD20 antigen.

The importance of B-lymphocyte-restricted differentiation antigen Bp35 (CD20) as a target for immunotherapeutic depletion of B cells is irrefutable. Several anti-human CD20 (anti-hCD20) monoclonal antibodies are expressed at different stages of development. However, resistance to anti-CD20 therapy has made the search for new alternatives imperative. Identification of B-cell epitopes within hCD20 using in silico tools can provide new opportunities to develop monoclonal antibodies with different binding sites. Furthermore, identification of the relationship between amino acid sequences of predicted B-cell epitopes and immune responses facilitates the determination of immunogenic regions of proteins by using their primary structure. Experimental evaluation of predicted linear B-cell epitopes as candidate peptides and bioinformatics allows us to explore this relationship. In this study, we selected three candidate epitopes within the extra membrane loop of hCD20 with the aid of five immunoinformatics predictor web servers and evaluated mouse humoral response to keyhole-limpet-hemocyaninconjugated peptides, and P4 and P5 peptides (the extracellular loop of hCD20 without and with a disulfide bond, respectively). Injection of the peptides yielded results that confirmed the prediction and selection of candidates. ELISA and flow cytometry corroborated the in silico selections. The B-cell epitopes P1, P2, and P3 were effective for immunization of mice.

The relationship of surface roughness and cell response of chemical surface modification of titanium.

Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Previous researches proved that chemical surface modification of titanium implants could be used to improve Bone-to-implant contact. In this study, the surface topography, chemistry and biocompatibility of polished titanium surfaces treated with mixed solution of three acids containing HCl, HF and H(3)PO(4) with different etched conditions for example concentration, time and addition of calcium chloride were studied. Osteoblast cells (MG-63) were cultured on different groups of titanium surfaces. In order to investigate titanium surfaces, SEM, AFM and EDS analyses were carried out. The results showed that surfaces treated with HCl-HF-H(3)PO(4) had higher roughness, lower cytotoxicity level and better biocompatibility than controls. Moreover, addition of calcium chloride into mixed solution of three acids containing HCl, HF and H(3)PO(4) is an important, predominant and new technique for obtaining biofunction in metals for biomedical use including dentistry.

Molecular imprinting as a simple way for the long-term maintenance of the stemness and proliferation potential of adipose-derived stem cells: an in vitro study.

Cells are smart creatures that respond to every signal after isolation and in vitro culture. Adipose-derived stem cells (ADSCs) gradually lose their characteristic spindle shape, multi-lineage differentiation potential, and self-renewal ability, and enter replicative senescence after in vitro expansion. This loss of cellular function is a serious impediment to clinical applications that require huge numbers of cells. It has been proven that substrates with cell imprints can be applied for stem cells' differentiation into desired cells or to re-culture any cell type while maintaining its ordinary activity. This study demonstrated the application of cell-imprinted substrates as a novel method in the long-term expansion of ADSCs while maintaining their stemness. Here we used molecular imprinting of stem cells as a physical signal to maintain stem cells' stemness. First, ADSCs were isolated and cultured on the tissue culture plate. Then, cells were fixed, and stem cell-imprinted substrates were fabricated using PDMS. Afterward, ADSCs were cultured on these substrates and subjected to osteogenic and adipogenic differentiation signals. The results were compared with ADSCs cultured on a polystyrene tissue culture plate and non-patterned PDMS. Morphology analysis with optical and fluorescence microscopy and SEM images illustrated that ADSCs seeded on imprinted substrates kept ADSC morphology. Alizarin Red S and Oil Red O staining, flow cytometry, and qPCR results showed that ADSC-imprinted substrates could reduce the differentiation of stem cells in vitro even if the differentiating stimulations were applied. Also, cell cycle analysis revealed that ADSCs could maintain their proliferation potential. So this method can maintain stem cells' stemness for a long time and reduce the unwanted stem cell differentiation that occurs in conventional cell culture on tissue culture plates.

Efficacy and antitumor activity of a mutant type of interleukin 2.

Interleukin-2 (IL-2) is an important cytokine in survival, expansion, function of CD8+ T cells and natural killer cells in immunotherapy of melanoma and renal cell carcinomas. Its severe toxicity following binding to its high affinity IL-2 receptor alpha (IL-2Rα) has restricted its application in cancer patients. In the present study, we investigated the antitumor efficacy and cytotoxicity of a mutated human IL-2 previously designed by selective amino acid substitutions, and its reduced affinity towards high-affinity IL-2Rα (CD25) was approved compared to the wild type IL-2 (wtIL-2). Furthermore, their ability to induce PBMC cell proliferation, and interferon-gamma secretion was compared. The mutant IL-2 also represented higher antitumor activity and more efficient cytotoxicity than wild type hIL-2. The developed mutant IL-2 can be an alternative tool in IL-2 associated immunotherapy of various cancers.

Optimization of a PDMS-Based Cell Culture Substrate for High-Density Human-Induced Pluripotent Stem Cell Adhesion and Long-Term Differentiation into Cardiomyocytes under a Xeno-Free Condition.

Despite the numerous advantages of PDMS-based substrates in various biomedical applications, they are limited by their highly hydrophobic surface that does not optimally interact with cells for attachment and growth. Hence, the lack of lengthy and straightforward procedures for high-density cell production on the PDMS-based substrate is one of the significant challenges in cell production in the cell therapy field. In this study, we found that the PDMS substrate coated with a combination of polydopamine (PDA) and laminin-511 E8 fragments (PDA + LME8-coated PDMS) can support human-induced pluripotent stem cell (hiPSC) attachment and growth for the long term and satisfy their demands of differentiation into cardiomyocytes (iCMs). Compared with prior studies, the density of hiPSCs and their adhesion time on the PDMS surface were increased during iCM production. Although the differentiated iCMs beat and produce mechanical forces, which disturb cellular attachments, the iCMs on the PDA + LME8-coated PDMS substrate showed dramatically better attachment than the control condition. Further, the substrate required less manipulation by enabling one-step seeding throughout the process in iCM formation from hiPSCs under animal-free conditions. In light of the results achieved, the PDA + LME8-coated PDMS substrate will be an up-and-coming tool for cardiomyocyte production for cell therapy and tissue engineering, microfluidics, and organ-on-chip platforms.

Enhancement of the human factor IX expression, mediated by an intron derived fragment from the rat aldolase B gene in cultured hepatoma cells.

Combinations of a liver-specific rat aldolase B intronic enhancer (rABE) with either of the hepatocyte-specific human α1-antitrypsin promoter (hAATp) and cytomegalovirus enhancer/promoter (CMVp) were used to construct a number of plasmids expressing non-viral human factor IX (hFIX). The efficacies of the plasmids were evaluated in a hepatocyte cell line (HepG2). Potential of the rABE was evidenced, by 300%--and 800% increase of the hFIX expression levels when it was combined with the CMVp and hAATp, respectively. The highest hFIX expression level was obtained when the rABE was combined with the CMVp for which the maximum intracellular accumulation of hFIX was also evidenced. Therefore, the rABE is suggested as a suitable cis-acting element for protein expression in hepatocytes. Considering the potential of introns during post-transcriptional processes, the function of the human ß-globin (hBG) intron-II, within the hFIX coding region, in the second generations of the hFIX expressing plasmids was also examined, which leaded to reduction of the hFIX expression level, probably due to improper splicing of the hBG intron-II.

Carvacrol and Thymol Attenuate Cytotoxicity Induced by Amyloid ß25-35 via Activating Protein Kinase C and Inhibiting Oxidative Stress in PC12 Cells.

Background: Our previous findings indicated that carvacrol and thymol alleviate cognitive impairments caused by Aß in rodent models of Alzheimer's disease (AD). In this study, the neuroprotective effects of carvacrol and thymol against Aß25-35-induced cytotoxicity were evaluated, and the potential mechanisms were determined. Methods: PC12 cells were pretreated with Aß25-35 for 2 h, followed by incubation with carvacrol or thymol for additional 48 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. A flurospectrophotometer was employed to observe the intracellular reactive oxygen species (ROS) production. Protein kinase C (PKC) activity was analyzed using ELISA. Results: Our results indicated that carvacrol and thymol could significantly protect PC12 cells against Aß25-35-induced cytotoxicity. Furthermore, Aß25-35 could induce intracellular ROS production, while carvacrol and thymol could reverse this effect. Moreover, our findings showed that carvacrol and thymol elevate PKC activity similar to Bryostatin-1, as a PKC activator. Conclusion: This study provided the evidence regarding the protective effects of carvacrol and thymol against Aß25­35-induced cytotoxicity in PC12 cells. The results suggested that the neuroprotective effects of these compounds against Aß25-35 might be through attenuating oxidative damage and increasing the activity of PKC as a memory-related protein. Thus, carvacrol and thymol were found to have therapeutic potential in preventing or modulating AD.

Size effect of human epidermal growth factor-conjugated polystyrene particles on cell proliferation.

Conjugation of growth factors to a carrier is a favorable method to improve their efficacy as therapeutic molecules. Here, we report the carrier size effect on bioactivity of human epidermal growth factor (hEGF) conjugated to polystyrene particles. BALB/3T3 cells were treated with hEGF-conjugated particles (hEGF-conjs) sized from 20 to 1000 nm. At hEGF concentrations less than 0.5 ng ml-1, free hEGF was more potent than the hEGF-conjs at inducing cell proliferation. However, cell proliferation was size-dependent at higher concentrations of hEGF i.e. hEGF-conjs sized equal to or less than 200 nm displayed lower cell proliferation, compared to free hEGF, but larger particles showed increased cell proliferation. This is in agreement with previous studies showing accumulation of activated-EGFRs in early endosomes triggers apoptosis of A431 and HeLa cells. The confocal microscopy and co-localization fluorescence staining showed the 500 and 1000 nm hEGF-conjs exclusively remained on the cell surface, probably enabling them to activate EGF receptors for a longer time. Conversely, smaller particles were mostly inside the cells, indicating their rapid endocytosis. Similarly, A431 cells treated with 20 nm hEGF-conj, endocytosed the particles and experienced decreased cell proliferation, while the 500 and 1000 nm hEGF-conjs were not internalized, and induced partial cell proliferation. Moreover, we showed multivalency of hEGF-conjs is not the cause of enhanced cell proliferation by large particles, as the degree of EGFR phosphorylation by free EGF was higher, compared to hEGF-conjs. Our results suggest the potential of micron-sized particles as a carrier for hEGF to enhance cell proliferation, which could be explored as a promising approach for topical application of growth factors for accelerating wound healing.

Effectiveness of Plasmocure™ in Elimination of Mycoplasma Species from Contaminated Cell Cultures: A Comparative Study versus other Antibiotics.

OBJECTIVE: Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problems associated with cell culture in biology research centers or biotechnological companies. Mycoplasma are also resistant to several antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported. InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasma contamination. The aim of this study was the selection of the best antibiotics for treatment of Mycoplasma in cell cultures. MATERIALS AND METHODS: In this experimental study, a total of 100 different mammalian cell lines contaminated with different Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochrome staining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods. In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. The treatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BMcyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. RESULTS: Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those in Plasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma spp. A moderate toxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells were fully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. However, the outcome of the treatment process (i.e. the frequency of complete cure, regrowth or cell death) varied among different antibiotics. CONCLUSION: The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowest regrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.

Two Novel Tri-Aryl Derivatives Attenuate the Invasion-Promoting Effects of Stromal Mesenchymal Stem Cells on Breast Cancer.

BACKGROUND: The concept of Epithelial-Mesenchymal Transition (EMT) to promote carcinoma progression has been recognized as a venue for research on novel anticancer drugs. Triaryl template-based structures are one of the pivotal structural features found in a number of compounds with a wide variety of biological properties including anti-breast cancer. Among the various factors triggering EMT program, cyclooxygenase-2 (COX-2), NF-κB as well as the transforming growth factor-beta (TGF-ß) have been widely investigated. OBJECTIVE: Here, we aim to investigate the effect of two novel compounds A and B possessing triaryl structures, which interact with both COX-2 and TGF-ß active sites and suppress NF-κB activation, on EMT in a co-culture system with breast cancer and stromal cells. METHODS: MDA-MB-231 and bone-marrow mesenchymal stem (BM-MS) cells were co-cultured in a trans-well plate. Migration, matrigel-based invasion and colony formation in soft agar assays along with Real- time PCR and Western blot analysis were performed to examine the effect of compounds A and B on the invasive properties of MDA-MB-231 cells after 72 hours of co-culturing with BM-MSCs. In addition, TGF-beta interaction was investigated by Localized Surface Plasmon Resonance (LSPR). RESULTS: BM-MSCs enhanced migration, invasion and anchorage-independent growth of the co-cultured MDAMB- 231 cells. A reduction in E-cadherin level concomitant with an increase in vimentin and N-cadherin levels following the co-culture implied EMT as the underlying process. Compounds A and B inhibited invasion and anchorage-independent growth of breast cancer cells co-cultured with BM-MSCs at 10µM. The observed inhibitory effects along with an increase in E-cadherin and a reduction in vimentin and ZEB2 levels suggest that the anti-invasive properties of compounds A and B might proceed through the blockade of stromal cell-induced EMT, mediated by their interaction with TGF-beta. CONCLUSION: These findings introduce compounds A and B as novel promising agents, which prevent EMT in invasive breast cancer cells.

A developed antibody-drug conjugate rituximab-vcMMAE shows a potent cytotoxic activity against CD20-positive cell line.

Rituximab is a chimeric monoclonal antibody directed against B-lymphocyte specific antigen CD20, which is used for the treatment of B-cell malignancies. However, the effectiveness of rituximab is limited partly due to treatment resistance. The aim of this study was to develop rituximab-based antibody drug conjugate (ADC) to enhance rituximab activity. In this study, monomethyl auristatin E (MMAE) was covalently conjugated to dithiothreitol -reduced rituximab via a valine-citrulline peptide linker (rituximab-vcMMAE). The conjugates were then characterized by using nonreducing sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and cell-based enzyme-linked immunosorbent assay (ELISA). The cytotoxic activity of the ADC was evaluated against Raji (human B-cell lymphoma; CD20-positive) and MOLT-4 (T lymphoblast; acute lymphoblastic leukemia; CD20-negative) cell lines. In addition, the colony formation assay was used to identify the propagation ability of ADC-treated cells in vitro. Results from nonreducing SDS-PAGE revealed various species of rituximab-MC-Val-Cit-PABC-MMAE (rituximab-vcMMAE), as compared with unconjugated rituximab. The binding capacity of rituximab-vcMMAE to the CD20-positive cell was similar to that of the parental rituximab. Most importantly, our results revealed that rituximab-vcMMAE was highly potent against the CD20-positive cell line, but not against the CD20-negative cell. At the same time, rituximab-vcMMAE was able to inhibit colony formation in CD20-positive cells. These data indicate that rituximab-vcMMAE may be a highly effective and selective therapy for the treatment of B-cell lymphoma.

Comparative study of pathogenic and non-pathogenic Escherichia coli outer membrane vesicles and prediction of host-interactions with TLR signaling pathways.

OBJECTIVE: The intestine is the major defensive barrier in the body by having more than 60% of the immune cells in the intestinal mucosa. The aim of this study was to evaluate the Toll like receptor (TLR) signaling pathways and immune response profiles, against outer membrane vesicles (OMVs) in pathogenic and non-pathogenic strains of Escherichia coli. RESULTS: Our results demonstrated that despite inducing inflammatory and regulatory responses to OMVs released by both strains, there is a remarkable difference in the nature and severity of these responses between the two strains. Following the production and release of OMV by the pathogenic strain, the expressions of the pro-inflammatory cytokines were significantly elevated, in comparison to the non-pathogenic strains. Eventually, our findings suggest that OMV released by the pathogen strain might be colonized, causing inflammation, eliminating the tight junctions of epithelial cells and damaging underlying cells, without the presence of IL-17 at the inflammation site. This could have happened to prevent the development of more severe inflammation, which could lead to the inhibition of colonization. The production of IL-10 is also preventing such inflammations. On the other hand, OMV released by non-pathogenic E. coli appears to influence intestinal homeostasis by causing more anti-inflammatory responses and mild inflammation.