Presence Of Methotrexate Research Articles

Formulation and Evaluation of Albumin-Stabilized Silver Nanoparticles Loaded with Methotrexate: Anticancer Activity and DNA Binding Analysis with Molecular Modeling

Nano-metal technology has emerged recently as an exceptional biological targeted field with numerous advantages like small size and high surface charge. In this report, we described our research on the stabilization of prepared silver nanocores coated with bovine serum albumin and further loaded with methotrexate (MTX) drug. The prepared nanoparticles were characterized by using UV-Visible, X-ray diffraction (XRD), energy dispersive X-ray (EDX), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The UV spectra of AgNP showed a prominent peak at 400 nm. The presence of MTX was confirmed by the FT-IR and EDX analysis. The AgNP-BSA-MTX particles resembled elliptical shape like structures in the SEM images. In-vitro drug release for free drug MTX occurred rapidly in a period of 12 hrs (85%), whereas a sustained release of MTX greater than 85% from the AgNP-BSA-MTX nanoparticles was observed for 48 hrs. The AgNP-BSA-MTX demonstrated a five-fold higher cytotoxic activity compared to free MTX drug alone against PC-3 cell lines, performed by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay method. DNA binding studies were performed for the AgNP-BSA-MTX with calf thymus DNA (ct-DNA). In-silico studies were performed for MTX with DNA (Protein Data Bank ID-IZ3F) and a prominent interaction with the nucleotides was noted. Molecular Dynamics (MD) simulation studies were also performed for the docked MTX-DNA complex. The average of the Root mean square deviation (RMSD) values for the DNA backbone was calculated to be 5.71 ± 0.48 nm.

Astaxanthin Co-treatment with Low Dose Methotrexate Increases the Cell Cycle Arrest and Ameliorates the Methotrexate-induced Inflammatory Response in NALM-6.

Methotrexate (MTX), an antimetabolite agent, is widely used for acute lymphoblastic leukemia treatment, despite its association with significant organ dysfunction. Astaxanthin (AST) is a natural carotenoid which has recently been emerged as a promising anti-tumor and anti-inflammatory agent. In this study, we aimed to evaluate the effectiveness of astaxanthin and low-dose methotrexate co-treatment in acute lymphoblastic leukemia cell line. The expression of Dihydrofolate reductase (DHFR), Thymidylate synthase (TYMS), apoptotic, anti-apoptotic as well as inflammatory genes was investigated using qRT-PCR. Flow cytometry was performed for cell cycle quantitative evaluation. Clonogenic assay was used to assess NALM6 cells proliferation capacity following treatment with AST, MTX, and co-treatment. To compare the antioxidant property of each group, the ferric ion reducing anti-oxidant power assay was performed. A reduction in viability was observed in the presence of MTX, AST, and their combined treatment. Both AST alone and in combination with MTX caused cell cycle arrest and a reduction in the expression of DHFR and TYMS. While MTX, AST, and their combination could reduce STAT3 and BCL-XL gene expression, they could act as positive regulators for the expression of BAX and CASP3, TNFα, and IL6. AST and MTX co-treatment inhibited the colony formation ability. FRAP assay also revealed that AST and AST+MTX increased the antioxidant capacity. Our data suggests that AST can improve MTX treatment efficacy and their combination therapy can be considered as a promising strategy for the management of acute lymphoblastic leukemia.

Evolution of Gold and Iron Oxide Nanoparticles in Conjugates with Methotrexate: Synthesis and Anticancer Effects.

Au and Fe nanoparticles and their conjugates with the drug methotrexate were obtained by an environmentally safe method of metal-vapor synthesis (MVS). The materials were characterized by transmission and scanning electron microscopy (TEM, SEM), X-ray photoelectron spectroscopy (XPS), and small-angle X-ray scattering using synchrotron radiation (SAXS). The use of acetone as an organic reagent in the MVS makes it possible to obtain Au and Fe particles with an average size of 8.3 and 1.8 nm, respectively, which was established by TEM. It was found that Au, both in the NPs and the composite with methotrexate, was in the Au0, Au+ and Au3+ states. The Au 4f spectra for Au-containing systems are very close. The effect of methotrexate was manifested in a slight decrease in the proportion of the Au0 state-from 0.81 to 0.76. In the Fe NPs, the main state is the Fe3+ state, and the Fe2+ state is also present in a small amount. The analysis of samples by SAXS registered highly heterogeneous populations of metal nanoparticles coexisting with a wide proportion of large aggregates, the number of which increased significantly in the presence of methotrexate. For Au conjugates with methotrexate, a very wide asymmetric fraction with sizes up to 60 nm and a maximum of ~4 nm has been registered. In the case of Fe, the main fraction consists of particles with a radius of 4.6 nm. The main fraction consists of aggregates up to 10 nm. The size of the aggregates varies in the range of 20-50 nm. In the presence of methotrexate, the number of aggregates increases. The cytotoxicity and anticancer activity of the obtained nanomaterials were determined by MTT and NR assays. Fe conjugates with methotrexate showed the highest toxicity against the lung adenocarcinoma cell line and Au nanoparticles loaded with methotrexate affected the human colon adenocarcinoma cell line. Both conjugates displayed lysosome-specific toxicity against the A549 cancer cell line after 120 h of culture. The obtained materials may be promising for the creation of improved agents for cancer treatment.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

Methotrexate-resistance is associated with double minute chromosomes in lymphocytes from patients with prostate cancer

Human lymphocytes have long been used to assess the gene damage occurring in vivo as well as in vitro . In this study , peripheral blood lymphocytes from patients with prostate cancer were cultivated in vitro short-term culture in the presence of methotrexate (MTX) to investigate the possible mechanism of resistance to MTX in those cells. The results indicate that the resistance to MTX in lymphocytes from prostate cancer patients is associated with dihydrofolate reductase (DHFR) gene amplification . Metaphase chromosome preparations from those cells were examined and were shown to contain double minute chromosomes (DMs). We conclude, that the presence of DMs may support that the resistance to MTX in prostate cancer patients may be associated with DHFR gene amplification.

Spectrofluorimetric Method for Monitoring Methotrexate in Patients' Plasma Samples and Cell Lysates Using Highly Fluorescent Carbon Dots.

For the first time, nitrogen, sulfur, phosphorus, and boron-doped carbon dots (N, S, P, B-codoped CDs) were synthesized through a hydrothermal reaction. The produced CDs were utilized to develop an optical sensor to determine methotrexate (MTX) in cell lysates and patients’ plasma samples. Basically, in the presence of MTX, the fluorescence emission of the CD-based probe was quenched. Under optimum conditions, a good proportional relationship was obtained between the quenched fluorescence signal and MTX concentrations from 74.9 ng/mL to 99.9 µg/mL with a limit of detection of 74.9 ng/mL. The developed nanoprobe provided a wide linear range and high accuracy and was successfully utilized in the routine therapeutic drug monitoring of MTX in plasma samples. The obtained results proposed the developed nanoprobe for the on-time and specific detection of MTX in blood samples. As another application, N, S, P, B-codoped CDs were utilized for bioimaging MCF-7 cancer cells and could be proposed as efficient bioimaging agents for tumor cells.

One-Step Preparation of S, N Co-Doped Carbon Quantum Dots for the Highly Sensitive and Simple Detection of Methotrexate.

(1) Background: Carbon quantum dots (CQDs) are a new class of carbon nanomaterials with favorable features, such as tunable luminescence, unique optical properties, water solubility, and lack of cytotoxicity; they are readily applied in biomedicine. (2) Methods: S, N co-doped CQDs were prepared to develop a highly selective and sensitive fluorescence technique for the detection of methotrexate (MTX). For this purpose, citric acid and thiourea were used as C, N, and S sources in a single-step hydrothermal process to prepare the S, N co-doped CQDs, which displayed remarkable fluorescence properties. (3) Results: Two optimal emissions were observed at the excitation/emission wavelengths of 320/425 nm, respectively. The two emissions were significantly quenched in the presence of MTX. Under optimal conditions, MTX was detected in the linear concentration range of 1–300 μmol/L, with the detection limit of 0.33 μmol/L. The sensing mechanism was due to the fact that the effect of the inner filter on MTX and S, N-CQDs causes fluorescence quenching. The contents of MTX in real medicine samples were evaluated with acceptable recoveries of 98–101%. (4) Conclusions: This approach has great potential for detecting MTX in pharmaceutical analysis.

The Fast Track for Intestinal Tumor Cell Differentiation and In Vitro Intestinal Models by Inorganic Topographic Surfaces.

Three-dimensional (3D) complex in vitro cell systems are well suited to providing meaningful and translatable results in drug screening, toxicity measurements, and biological studies. Reliable complex gastrointestinal in vitro models as a testbed for oral drug administration and toxicity are very valuable in achieving predictive results for clinical trials and reducing animal testing. However, producing these models is time-consuming due to the lengthy differentiation of HT29 or other cells into mucus-producing goblet cells or other intestinal cell lineages. In the present work, HT29 cells were grown on an inorganic topographic surface decorated with a periodic pattern of micrometre-sized amorphous SiO2 structures for up to 35 days. HT29 cells on topographic surfaces were compared to undifferentiated HT29 in glucose-containing medium on glass or culture dish and with HT29 cells differentiated for 30 days in the presence of methotrexate (HT29-MTX). The cells were stained with Alcian blue for mucus, antibodies for mucus 2 (goblet cells), villin (enterocytes), lysozyme (Paneth cells), and FITC-labeled lectins to identify different cells, glycomic profiles, and cell features. We observed that HT29 cells on topographic surfaces showed more similarities with the differentiated HT29-MTX than with undifferentiated HT29. They formed islands of cell clusters, as observed for HT29-MTX. Already after 2 days, the first mucus secretion was shown by Alcian blue stain and FITC-wheat germ agglutinin. After 4–6 days, mucus was observed on the cell surface and in the intercellular space. The cell layer was undulated, and in 3D reconstruction, the cells showed a clear polarisation with a strong actin signal to one membrane. The lectins and the antibody-staining confirmed the heterogeneous composition of differentiated HT29 cells on topographic surfaces after 6–8 days, or after 6–8 days following MTX differentiation (30 days).

Analysis of chemical contamination by hazardous drugs with BD HD Check® system in a tertiary hospital.

The presence of contamination in the healthcare work environment by one of the types of hazardous drugs, cytostatics, has been found in multiple international studies. Recent studies and guidelines recommend surface monitoring for risk assessment of healthcare professionals' exposure. The availability of detection techniques is critical to successfully carry out this type of monitoring. The use of new semi-quantitative techniques allows quicker results. The main objective of this study was to determine the existence of hazardous drugs on the working surfaces in different locations of a tertiary hospital using the BD HD Check® semi-quantitative device. The presence of methotrexate, doxorubicin and cyclophosphamide was analysed at 80, 89 and 82 locations in 10, 13 and 11 clinical units, respectively. A total of 251 samples were analysed. The monitoring results were positive for 13.1% of the analysed samples, with 36.3% of the methotrexate samples, 0% of the doxorubicin samples and 4.9% of the cyclophosphamide samples. Mapping the presence of HD in our hospital has allowed us to evaluate the effectiveness of controls established in the hospital to minimise the exposure of healthcare professionals to hazardous drugs. The speed in obtaining results has enabled immediate corrective actions in cases where contaminated surfaces were detected.

Effect of methotrexate and its photodegradation products on the temperature induced denaturation of human serum albumin.

Effect of the photodegradation of chemotherapeutic agent methotrexate on the denaturation of human serum albumin has been studied by differential scanning calorimetry and fluorimetric measurements. Photoluminescence studies highlighted entropy driven binding of both methotrexate molecules and its degradation products to the albumin molecules. The calorimetric measurements evaluated by the Kissinger method show elevated activation energy in the presence of methotrexate. Similar but moderated enhancement of the activation energy was obtained in the presence of the photodegradation products of methotrexate. These results highlight stabilization of the human serum albumin by the methotrexate drug which finding may contribute to fine tuning of methotrexate applications in therapy.

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans.

While the folate biosynthetic pathway has provided a rich source of antibacterial, antiprotozoal, and anticancer therapies, it has not yet been exploited to develop uniquely antifungal agents. Although there have been attempts to develop fungal-specific inhibitors of dihydrofolate reductase (DHFR), the protein itself has not been unequivocally validated as essential for fungal growth or virulence. The purpose of this study was to establish dihydrofolate reductase as a valid antifungal target. Using a strain with doxycycline-repressible transcription of DFR1 (PTETO-DFR1 strain), we were able to demonstrate that Dfr1p is essential for growth in vitro Furthermore, nutritional supplements of most forms of folate are not sufficient to restore growth when Dfr1p expression is suppressed or when its activity is directly inhibited by methotrexate, indicating that Candida albicans has a limited capacity to acquire or utilize exogenous sources of folate. Finally, the PTETO-DFR1 strain was rendered avirulent in a mouse model of disseminated candidiasis upon doxycycline treatment. Collectively, these results confirm the validity of targeting dihydrofolate reductase and, by inference, other enzymes in the folate biosynthetic pathway as a strategy to devise new and efficacious therapies to combat life-threatening invasive fungal infections.IMPORTANCE The folate biosynthetic pathway is a promising and understudied source for novel antifungals. Even dihydrofolate reductase (DHFR), a well-characterized and historically important drug target, has not been conclusively validated as an antifungal target. Here, we demonstrate that repression of DHFR inhibits growth of Candida albicans, a major human fungal pathogen. Methotrexate, an antifolate, also inhibits growth but through pH-dependent activity. In addition, we show that C. albicans has a limited ability to take up or utilize exogenous folates as only the addition of high concentrations of folinic acid restored growth in the presence of methotrexate. Finally, we show that repression of DHFR in a mouse model of infection was sufficient to eliminate host mortality. Our work conclusively establishes DHFR as a valid antifungal target in C. albicans.

Nucleosides block AICAR-stimulated activation of AMPK in skeletal muscle and cancer cells.

AMP-activated kinase (AMPK) is a major regulator of energy metabolism and a promising target for development of new treatments for type 2 diabetes and cancer. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an adenosine analog, is a standard positive control for AMPK activation in cell-based assays. Some broadly used cell culture media, such as minimal essential medium α (MEMα), contain high concentrations of adenosine and other nucleosides. We determined whether such media alter AICAR action in skeletal muscle and cancer cells. In nucleoside-free media, AICAR stimulated AMPK activation, increased glucose uptake, and suppressed cell proliferation. Conversely, these effects were blunted or completely blocked in MEMα that contains nucleosides. Addition of adenosine or 2'-deoxyadenosine to nucleoside-free media also suppressed AICAR action. MEMα with nucleosides blocked AICAR-stimulated AMPK activation even in the presence of methotrexate, which normally markedly enhances AICAR action by reducing its intracellular clearance. Other common media components, such as vitamin B-12, vitamin C, and α-lipoic acid, had a minor modulatory effect on AICAR action. Our findings show that nucleoside-containing media, commonly used in AMPK research, block action of the most widely used pharmacological AMPK activator AICAR. Results of cell-based assays in which AICAR is used for AMPK activation therefore critically depend on media formulation. Furthermore, our findings highlight a role for extracellular nucleosides and nucleoside transporters in regulation of AMPK activation.

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression—significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4+ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

Methotrexate inhibits effects of platelet-derived growth factor and interleukin-1\u03b2 on rheumatoid arthritis fibroblast-like synoviocytes

BackgroundA key feature of joints in rheumatoid arthritis (RA) is the formation of hyperplastic destructive pannus tissue, which is orchestrated by activated fibroblast-like synoviocytes (FLS). We have demonstrated that the RA risk gene and tumor suppressor Limb bud and heart development (LBH) regulates cell cycle progression in FLS. Methotrexate (MTX) is the first-line treatment for RA, but its mechanisms of action remain incompletely understood. Here, we studied the effects of MTX on mitogen-induced FLS proliferation and expression of cell cycle regulators in vitro.MethodsPrimary FLS from patients with RA or osteoarthritis were stimulated with the mitogen platelet-derived growth factor (PDGF) and the cytokine interleukin-1β (IL-1β) in the presence or absence of MTX. Cells were then subjected to qPCR for gene expression and cell cycle analysis by flow cytometry.ResultsStimulation with PDGF and IL-1β increased the percentage of FLS in the G2/M phase and shifted the cell morphology to a dendritic shape. These effects were inhibited by MTX. Furthermore, PDGF + IL-1β reduced LBH mRNA expression. However, MTX treatment yielded significantly higher transcript levels of LBH, and of CDKN1A (p21) and TP53 (p53), compared to untreated samples upon mitogen stimulation. The expression of DNA methyltransferase-1 (DNMT1) was also higher in the presence of MTX and there was strong correlation between DNMT1 and LBH expression.ConclusionsTherapeutic concentrations of MTX abolish the effects of PDGF and IL-1β on tumor suppressor expression and inhibit mitogen-promoted FLS proliferation. These data demonstrate novel and important effects of MTX on pathogenic effector cells in the joint, which might involve epigenetic mechanisms.

Dihydrofolate reductase and membrane translocation: evolution of a classic experiment: Classic landmark papers, irrespective of their age, can teach students how best science is practiced and inspire new experiments.

Any PhD student or postdoc, who scrambles to write and submit a publication before getting scooped, can testify that science is a fast‐moving endeavor. Given our limited time and the ever‐increasing pace with which scientific studies are published, few students and postdocs—and PIs as a matter of fact—have time to keep up with the literature. “Reading” a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. Notwithstanding these constraints, I would argue that the scientific literature is a treasure trove of information and ideas that go beyond contemporary papers to include classical publications. However, it can be difficult to motivate students and postdocs to read old landmark publications that reported groundbreaking discoveries and opened up new avenues of research. Many think that these papers are of merely historic interest and have little to contribute to scientists expected to use cutting‐edge methods to produce high‐impact publications. > Reading a manuscript often means just skimming through the abstract, having a quick glimpse at the figures and searching the PDF file for keywords of immediate interest. However, classic papers still have a lot to offer. For once, they withstood the test of the time, as the reported results have shown to be correct and reproducible. This is unfortunately not the case for many high‐impact publications today, which, owing to the hype of selling it to the highest impact factor journal, just tell a cool story that in reality may be much less clear‐cut than reported. Furthermore, classic scientific publications often impress by a conceptual clarity and in many cases simplicity and thereby offer valuable lessons of how best science should be practiced. These are qualities I miss in many of today's papers that contain exceedingly large data sets …

Methotrexate Induces Apoptosis in Organ-Cultured Nasal Polyps Via the Fas Pathway.

Methotrexate (MTX) is very effective when used to treat chronic inflammatory diseases, and also induces apoptosis in nasal polyps (NPs). Increasing evidence suggests that Fas-Fas ligand (FasL) interactions activate multiple pathways involved in the regulation of immune and inflammatory cell functions. The aim of the present study was to identify pathways activated by Fas signaling when NPs were treated with MTX. Nasal polyps tissues were cultured using an air-liquid interface organ culture method. Cultures were maintained in the absence or presence of MTX (10 or 100 μM) for 24 hours. The authors used the reverse transcription-polymerase chain reaction method and Western blotting to identify pathways activated by Fas when NPs were treated with MTX. The Fas mRNA expression ratio was unchanged upon MTX treatment, but the FasL mRNA expression ratio was significantly higher in MTX-treated than nontreated polyps. In addition, the expression levels of the Fas and FasL proteins were significantly higher in polyps treated with both 10 and 100 μM MTX compared with nontreated polyps. Methotrexate induces apoptosis in NPs via the Fas pathway. Future studies should explore the topical use of MTX for NP control. Methotrexate may be a useful alternative steroid-sparing agent for the treatment of NPs.

Methotrexate Reduced TNF Bioactivity in Rheumatoid Arthritis Patients Treated with Infliximab.

Objectives. To evaluate methotrexate effect on tumor necrosis factor (TNF) alpha bioactivity during infliximab (IFX) therapy in rheumatoid arthritis (RA) patients and to correlate TNF bioactivity with antibody towards IFX (ATI) development and RA clinical response. Materials and Methods. Thirty-nine active women RA patients despite conventional synthetic disease modifying antirheumatic drugs (csDMARDs) requiring IFX therapy were enrolled, and clinical data and blood samples were recorded at baseline (W0) and at 6 weeks (W6), W22, and W54 of IFX treatment. TNF bioactivity as well as IFX trough and ATI concentrations were assessed on blood samples. Results. TNF bioactivity decreased from W0 to W54 with a large range from W22 at the time of ATI detection. From W22, TNF bioactivity was lower in presence of methotrexate as csDMARD compared to other csDMARDs. IFX trough concentration increased from W0 to W54 with a large range from W22, similarly to TNF bioactivity. Methotrexate therapy prevented ATI presence at W22 and reduced TNF bioactivity compared to other csDMARDs (p = 0.002). Conclusion. This suggests that methotrexate plays a key role in TNF bioactivity and against ATI development.

Apoptotic cell infusion treats ongoing collagen-induced arthritis, even in the presence of methotrexate, and is synergic with anti-TNF therapy.

BackgroundApoptotic cell-based therapies have been proposed to treat chronic inflammatory diseases. The aim of this study was to investigate the effect of intravenous (i.v.) apoptotic cell infusion in ongoing collagen-induced arthritis (CIA) and the interaction of this therapy with other treatments used in rheumatoid arthritis (RA), including methotrexate (MTX) or anti-TNF therapy.MethodsThe effects of i.v. apoptotic cell infusion were evaluated in a CIA mouse model in DBA/1 mice immunized with bovine type II collagen. The number and functions of antigen-presenting cells (APC), regulatory CD4+ T cells (Treg), and circulating anti-collagen auto-antibodies were analyzed in CIA mice.ResultsTreatment of arthritic mice with i.v. apoptotic cell infusion significantly reduced the arthritis clinical score. This therapeutic approach modified T cell responses against the collagen auto-antigen with selective induction of collagen-specific Treg. In addition, we observed that APC from apoptotic-cell-treated animals were resistant to toll-like receptor ligand activation and favored ex vivo Treg induction, indicating APC reprogramming. Apoptotic cell injection-induced arthritis modulation was dependent on transforming growth factor (TGF)-β, as neutralizing anti-TGF-β antibody prevented the effects of apoptotic cells. Methotrexate did not interfere, while anti-TNF therapy was synergic with apoptotic-cell-based therapy.ConclusionOverall, our data demonstrate that apoptotic-cell-based therapy is efficient in treating ongoing CIA, compatible with current RA treatments, and needs to be evaluated in humans in the treatment of RA.

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent.

373. Expression of Human Iduronidase from Sleeping Beauty Engineered Human B Lymphocytes as a Cellular Therapy for Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder cause by absence of the glycosidase alpha-L-idurondase (IDUA), resulting in the systemic accumulation of glycosaminoglycan storage materials heparan sulfate and dermatan sulfate. Affected individuals exhibit a range of manifestations including hepatosplenomegaly, skeletal dysplasias, and cardiopulmonary obstruction. The severe form of the disease (Hurler syndrome) is associated with progressive neurologic dysfunction and death by age 10. Current treatments for MPS I include enzyme replacement (ERT), and hematopoietic stem cell transplantation (HSCT). However, these treatments are expensive, insufficiently effective, and allogeneic HSCT is associated with considerable morbidity and mortality.As an alternate approach to MPS I, Immusoft Corporation is developing as a cellular therapeutic primary B cells that have been genetically engineered for long-term expression and secretion of IDUA. At the core of the technology is the generation of genetically engineered autologous human B cells, which are isolated and expanded from patient blood. Mature B cells (specifically long-lived plasma cells) are the ideal cell type to produce protein since they live for long periods of time, occupy diverse tissues throughout the body and are capable of secreting very high levels of protein. In collaboration with Discovery Genomics, Inc., the University of Minnesota and the Fred Hutchinson Cancer Research Center, Immusoft has developed a treatment for Mucopolysaccharidosis type I (MPS I). In initial tests, Sleeping Beauty (SB) transposons expressing the human iduronidase gene were introduced into cultured B lymphoblastoid cells with and without a source of SB transposase, demonstrating long-term, high level expression of IDUA activity in vitro. SB transposons expressing both IDUA and a methotrexate-resistant human dihydrofolate reductase allowed for a >10-fold expansion of IDUA-expressing cells in the presence of methotrexate, as determined by intracellular staining for human IDUA and flow cytometry. Overall IDUA expression was also increased >10-fold in the methotrexate-selected cell population. IDUA encoding SB transposons were also nucleofected along with a source of SB transposase into primary human memory B cells, demonstrating expression of IDUA at levels more than 100 times greater than that observed in cultures of untreated primary B cells. These data support the development of a clinical trial to test SB-engineered B cells in the treatment of MPS I, with implications for improved treatment of other lysosomal storage disorders as well.