Search Results (4,729)

Multiple sclerosis is an immune-driven neurological disease that affects myelinated axons in the central nervous system. However, the trigger of the (dysregulated) immune reactions is not known. According to Wilkin’s primary lesion theory, myelin-reactive T cells present in the immune repertoire hyper-react to myelin antigens that are released from idiopathic lesions within the central nervous system. However, neither the cause of the primary lesion nor the cause of the immune hyper-reactivity is known. We investigated whether these unknown activation signals may be relayed by common herpesviruses. In this concept paper, we propose the novel paradigm that the trigger of autoimmunity in MS comprises a conspiracy of three common herpesviruses: human herpesvirus-6A as a potential trigger of primary lesions due to its proven capacity to cause oligodendrogliopathy, cytomegalovirus as a trigger for the formation of effector memory cytotoxic T cells with proven capacity to induce multiple sclerosis pathology in a non-human primate MS model and Epstein−Barr Virus due to its capacity to render B cells capable to effectively present a critical myelin antigen to these effector memory cytotoxic T cells. Full article

Background/Objectives: Gallic acid (GA) is a naturally occurring polyphenol with reported antioxidant and anticancer properties. This study investigated whether GA enhances carboplatin (CARB)-associated anticancer activity in HeLa cervical cancer cells through mechanisms related to oxidative stress, mitochondrial dysfunction, apoptosis, and cell cycle dysregulation, while comparatively evaluating cytotoxicity in HaCaT cells. Methods: The effects of GA and CARB, individually and in combination, were evaluated using cell viability assays, apoptosis and cell cycle analyses, intracellular reactive oxygen species (ROS) measurements, N-acetylcysteine (NAC)-mediated rescue experiments, mitochondrial membrane potential assessment, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry, and three-dimensional (3D) tumor spheroid models. Bioinformatic analyses were performed to explore pathways associated with the observed molecular responses. Results: The GA + CARB combination demonstrated enhanced cytotoxicity and apoptotic activity in HeLa cells compared with either monotherapy, while exhibiting comparatively lower toxicity in HaCaT cells. Combination treatment increased intracellular ROS levels, whereas NAC pretreatment partially reversed ROS accumulation and cytotoxicity, supporting a contributory role of oxidative stress in treatment-associated responses. The combination also induced mitochondrial membrane depolarization, increased G2/M arrest and SubG1 accumulation, and modulated apoptosis- and cell cycle-related gene expression. In 3D spheroid models, GA + CARB reduced spheroid growth and viability and disrupted spheroid integrity more effectively than single-agent treatments. Bioinformatic analyses identified interconnected pathways associated with oxidative stress, apoptosis, and cell cycle regulation. Conclusions: GA may enhance CARB-associated anticancer activity through mechanisms linked to oxidative stress, mitochondrial dysfunction, apoptosis, and cell cycle dysregulation. The incorporation of ROS/NAC rescue experiments and 3D spheroid validation further supports the biological relevance of the observed effects. Nevertheless, these findings remain preliminary and require confirmation in advanced in vivo and translational cervical cancer models. Full article

Background/Objective: Ultraviolet A and B (UVAB) radiation is a major environmental factor that induces DNA damage and upregulates programmed death-ligand 1 (PD-L1) expression in skin cells, thereby contributing to immune evasion and impaired tissue repair. This study evaluated the protective effects of two purified compounds, Cinnamtannin B1 (CTB-1) and Cinnamtannin D1 (CTD-1), as well as cinnamon extract, in UVAB-irradiated human keratinocyte HaCaT cells. Methods: HaCaT cells were exposed to low (20 kJ/m² UVA, 1.3 kJ/m² UVB), medium (30 kJ/m² UVA, 2 kJ/m² UVB), and high (40 kJ/m² UVA, 2.7 kJ/m² UVB) UVAB doses of UVAB radiation. Dose-dependent effects of CTB-1 and CTD-1 (0, 5, 10, 25, and 50 µg/ mL) and cinnamon extract (0, 5, 10, 50, and 100 µg/mL), as well as time-dependent effects (12, 24, and 72 h), were evaluated by measuring PD-L1 expression, cell viability, and DNA damage. Results: CTD-1 was the most effective compound, significantly reducing UVAB-induced PD-L1 expression and DNA double-strand breaks without compromising cell viability. CTB-1 also demonstrated protective effects at specific doses and time points; however, higher concentrations reduced cell viability. Cinnamon extract was protective at low concentrations but cytotoxic at higher doses. Conclusions: CTD-1, CTB-1, and cinnamon extract attenuated UVAB-induced cellular damage in HaCaT cells, with CTD-1 demonstrating the most favorable protective profile. These findings support the potential of cinnamon-derived compounds as therapeutic candidates for preventing UVAB-induced skin damage and immune dysregulation. Full article

Background: The tumor immune microenvironment, particularly the role of cytotoxic CD8+ T lymphocytes, is crucial in cancer progression but remains poorly understood in pituitary neuroendocrine tumors (PitNETs). The significance of CD8+ cell infiltration varies across PitNET subtypes, suggesting a complex interplay with tumor cell lineage. This study aimed to characterize the distribution of CD8+ tumor-infiltrating lymphocytes across different PitNET subtypes defined by the current WHO classification and to explore their association with clinicopathological features. Methods: We conducted a retrospective study on 40 surgically resected PitNETs. All cases were classified based on immunohistochemical expression of pituitary hormones and lineage-specific transcription factors (PIT-1, TPIT, SF-1). CD8+ lymphocyte density was quantified using immunohistochemistry and calculated as cells/mm². Exploratory statistical analysis was performed based on non-parametric tests to compare CD8+ cell density across tumor subtypes and with parameters like tumor size, invasiveness (Knosp grade), and proliferation index (Ki-67). Findings are to be treated as observational trends. Results: The highest density of CD8+ lymphocytes was observed in plurihormonal PIT-1-positive tumors [17.61 cells/mm² (IQR: 17.61–60.36)], followed by somatotroph [13.2 (6.6–15.72)] and mammosomatotroph [13.83 (0–21.38)] tumors. A difference in CD8+ density was found between PIT-1-positive and PIT-1-negative tumors (n1 = 34, n2 = 6, U = 49.5, p_exact = 0.050, r = 0.33); the medium effect size indicates a possible lineage-related trend. Another difference was observed between SF-1-positive and SF-1-negative tumors (p = 0.025), with SF-1 lineage tumors showing the lowest infiltration. No correlations were found between CD8+ density and tumor size, Knosp grade, or Ki-67 index. Conclusions: The distribution of intratumoral CD8+ T lymphocytes in PitNETs is highly heterogeneous and appears to be strongly dictated by the transcription factor-defined tumor lineage rather than by traditional clinicopathological markers of aggressiveness. PIT-1 lineage tumors harbor a more active immune microenvironment, while SF-1 lineage tumors are relatively ‘immune-poor’. These findings highlight the immunological diversity of PitNETs and support further investigation of the tumor immune landscape. Collaborative multi-institutional studies are required to validate these trends. Full article

Background: This study was conducted to identify genetic risk variants associated with nicotine addiction in the CHRNA5, HTR2A, DRD4, and CYP2A6 genes among electronic cigarette users who also smoke combustible cigarettes (dual users), and to assess potential genotoxic and cytotoxic damage in the oral mucosal cells of the study population. Methods: We included dual e-cig users (ECIG, n = 70), combustible cigarette smokers (CCU, n = 24), and non-smokers and non-e-cig users (NS, n = 110). Genetic variants in CHRNA5, HTR2A, DRD4, and CYP2A6 were genotyped. Micronucleus analysis was performed on oral mucosal cells to detect cellular abnormalities. Results: The ECIG group demonstrated greater nicotine addiction on the Fagerström Test for Nicotine Dependence (FTND, 5.5 vs. 1, p = 0.023). Salivary cotinine levels were significantly higher in the ECIG group compared to the CCU group (39 vs. 12 ng/mL, p < 0.001). The carriers of the A allele (rs16969968/CHRNA5) had higher FTND scores, carriers of the C allele (rs1800955/DRD4) used electronic cigarettes more frequently each day, and carriers of the T allele (rs4105144/CYP2A6) started using nicotine products at a younger age. The number of micronuclei and cellular abnormalities in the oral mucosa was higher in the ECIG and CCU groups compared to the NS group. Conclusions: Salivary cotinine levels and FTND are higher in dual e-cigarette users than in combustible cigarette users. Dual users exhibit risk alleles in the CHRNA5, DRD4, and CYP2A6 genes, which are associated with traits linked to increased nicotine addiction. Dual e-cigarette use poses comparable genotoxic risks to combustible smoking. Full article

Background/Objectives: Resistant pathogenic bacteria and fungi are a growing problem worldwide; therefore, the discovery of new active ingredients is an important challenge for which the functionalization of natural terpenes with biologically active heterocycles can provide a basis. To reach this goal, a series of 1,4-disubstituted-1,2,3-triazole conjugates was designed and synthesized starting from commercially available α-santonin. Methods: The key azido derivative intermediate was prepared according to literature procedures via Michael addition between dehydrosantonin and the TMSN₃/AcOH/Et₃N system at its highly reactive α-methylene-γ-lactone motif. Subsequently, the obtained azide was applied to regioselective Huisgen 1,3-dipolar cycloaddition reaction with a wide range of terminal alkynes bearing N-, S- and O-heterocycles. These include pyridine, pyrimidine, purine, quinoline, indol, or coumarin to afford the sesquiterpene–heterocycle chimaeras. All triazole conjugates were screened for in vitro antiproliferative activity by MTT assay against HeLa, MDA-MB231, SiHa, MCF-7 and A2780 human cancer cell lines compared with fibroblast cells (NIH/3T3) to check their cytotoxicity and antimicrobial effects on two Gram-positive (B. subtilis, S. aureus) pathogenic bacteria, two Gram-negative (E. coli and P. aeruginosa) pathogenic bacteria, and two yeasts (C. krusei and C. albicans). Results: The results indicated that most of the examined compounds expressed weak activity against human cell lines, while some of them showed moderate activity against S. aureus (up to 99% inhibition at 100 µg/mL conc.), C. krusei (up to 51% inhibition at 10 µg/mL conc.) and C. albicans (up to 52% inhibition at 10 µg/mL conc.). Conclusions: Further structural modification of the best, selective antibacterial and antifungal compounds may open the possibility to the development of effective natural sesquiterpene-based selective antimicrobial agents. Full article

This study assessed the potential of Oleaster leaves as a valuable cosmetic ingredient by comparing aqueous and hydroalcoholic ultrasound-assisted extracts of Oleaster leaves to those of the Olive tree. The hydroalcoholic Oleaster leaf extract showed a higher content in phenolic and flavonoid compounds, with an oleuropein and verbascoside content of 58 mg/g of dry leaves and 3.2 mg/g of dry leaves, respectively. To refine the comparison of their molecular composition, the extracts were analyzed using UHPLC-HRMS/MS. About twenty compounds, including secoiridoids, flavonoids and triterpenic acid derivatives, were annotated in both extracts, demonstrating their similarity. DPPH, CUPRAC and enzymatic SOD assays showed a good antioxidant activity with high inhibition (60–90%) for both Oleaster and Olive tree leaf extracts. The hydroalcoholic extracts at 62.5 µg/mL also exhibited 60–80% of protection against reactive oxygen species (ROS) in H₂O₂-stressed HaCaT cells, confirming this antioxidant capacity without demonstrating a severe cytotoxicity, which remained below 40%. The anti-inflammatory potential of the extracts was also demonstrated using COX-2 inhibition, which was around 70%, and by measuring the concentration of IL-8 in HaCaT cells under pro-inflammatory conditions, which decreased in the presence of extracts at a concentration of 50 pg/mL, similar to that observed for the positive control. Thus, the hydroalcoholic ultrasound extract of Oleaster leaves demonstrated its high potential to develop sustainable and active cosmetic ingredients. Full article

Daidzin is a soy-derived isoflavone with reported anti-obesity effects; however, the biological activity of microbial-derived daidzin remains poorly understood. In this study, we investigated the anti-adipogenic and anti-obesity potential of microbial-derived daidzin (hereafter referred to as Eco-3) in both in vitro and in vivo models. Eco-3 significantly suppressed adipocyte differentiation and lipid accumulation in 3T3-L1 preadipocytes and human adipose-derived stem cells (hASCs) without inducing cytotoxicity. Mechanistically, Eco-3 reduced the expression of key adipogenic regulators, including PPAR-γ and C/EBP-α, and modulated lipid metabolism-related proteins such as FAS and perilipin A. In addition, Eco-3 activated AMPK signaling while inhibiting the STAT-3 and STAT-5 pathway. In zebrafish models, Eco-3 significantly reduced lipid accumulation under both normal and diet-induced obesity conditions, as demonstrated by LipidGreen2 and Oil Red O staining. Collectively, these findings suggest that Eco-3 exerts anti-obesity effects through coordinated regulation of adipogenesis and lipid metabolism. Full article

Background: Oral candidiasis is an opportunistic infection caused by Candida albicans (C. albicans), highlighting the need to evaluate candidate substances that ensure both antifungal efficacy and mucosal safety. This study aimed to assess the potential of Sambucus williamsii var. coreana (S. williamsii var. coreana) extract as a naturally derived antifungal agent for topical oral application by investigating its antifungal activity against C. albicans and its cytotoxicity in human keratinocyte (HaCaT) cells. Methods: S. williamsii var. coreana was extracted with 70% ethanol, concentrated, and freeze-dried. The extract was prepared at concentrations of 1–40 mg/mL and applied under 6 h and 24 h exposure conditions. Antifungal activity against cultured C. albicans was evaluated using colony-forming unit (CFU) analysis, while cytotoxicity in HaCaT cells was assessed via the Water-soluble Tetrazolium Salt-1 assay after incubation at 37 °C in 5% CO₂ for 2 h. Statistical significance was analyzed using Student’s t-test, ANOVA, and Tukey’s HSD test (p < 0.05). Results: The S. williamsii var. coreana extract exhibited concentration- and time-dependent antifungal activity. A 99.99% inhibition of C. albicans was observed at 5 mg/mL. No detectable CFUs were observed at 30 mg/mL after 6 h and at 10 mg/mL after 24 h. HaCaT cell viability decreased in a concentration-dependent manner, with the half-maximal inhibitory concentration determined to be 10 mg/mL. Conclusions: The extract of S. williamsii var. coreana exhibited concentration- and time-dependent in vitro antifungal activity against C. albicans. However, the concentration associated with no detectable CFUs overlapped with the cytotoxic concentration range in HaCaT cells, indicating that further studies are required to define an appropriate concentration range for potential oral application. Full article

Background/Objectives: The toxic side effects and resistance-associated limitations of conventional chemotherapeutic agents necessitate the development of more effective and selective combination strategies incorporating naturally derived compounds. In this study, the cytotoxic, apoptotic, oxidative stress-associated, and immunomodulatory effects of thymoquinone (TQ), a bioactive compound derived from Nigella sativa, and docetaxel (Dos), a taxane-based chemotherapeutic agent, were investigated alone and in combination in OVCAR3 ovarian cancer cells using integrated two-dimensional (2D) and three-dimensional (3D) experimental models. Materials and Methods: Cell viability was evaluated following treatment with TQ (10–500 µM), Dos (1–500 nM), and the TQ + Dos combination, and synergistic interactions were assessed by IC₅₀⁻ and combination index-based analyses. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Cytokine levels were determined using ELISA, whereas apoptosis- and cell cycle-associated gene expression profiles were evaluated by RT-qPCR. Active caspase-3 expression was assessed by immunocytochemistry. Intracellular reactive oxygen species (ROS) accumulation was examined using DCFH-DA-based fluorescence imaging and antioxidant rescue experiments using N-acetyl-L-cysteine (NAC). In addition, the antitumor activity of the combination was further evaluated in OVCAR3-derived 3D tumor spheroid models using spheroid morphology, ATP-based viability, and live/dead fluorescence imaging analyses. Results: The TQ + Dos combination demonstrated enhanced cytotoxic and apoptotic activity in OVCAR3 cells compared with single-agent treatments and induced marked G2/M cell cycle arrest. Combination treatment increased pro-apoptotic gene expression and was associated with reduced expression of anti-apoptotic markers and modulated inflammatory cytokine profiles. Fluorescence-based analyses demonstrated marked intracellular ROS accumulation following TQ + Dos treatment, whereas NAC pretreatment partially attenuated oxidative stress and restored viability, suggesting partial involvement of ROS-associated mechanisms in treatment-induced cytotoxicity. Importantly, the combination maintained stronger cytotoxic and growth-inhibitory effects than either monotherapy in 3D ovarian cancer spheroids, where combination treatment induced pronounced spheroid shrinkage, viability loss, and structural disruption. Relatively lower toxicity observed in HaCaT cells suggested partial selectivity toward cancer cells. Conclusions: Collectively, these in vitro findings suggest that the TQ + Dos combination produces greater cytotoxic, apoptotic, and growth-inhibitory effects than either agent alone in ovarian cancer models and is associated with alterations in apoptosis-, cell cycle-, and oxidative stress-related responses. The observation of these effects in 3D spheroid models supports further investigation of this combination in more advanced preclinical systems. Full article

Background/Objectives: Tuberculosis, caused by Mycobacterium tuberculosis, remains a major global health challenge requiring novel prevention strategies. This study aims to developed an immunoinformatics-guided framework coupled with experimental screening to prioritize for multi-epitope mRNA vaccine design. Methods: Eight immunologically relevant antigens were computationally analyzed to predict cytotoxic (CTL) epitopes and helper T lymphocyte (HTL) epitopes. Population coverage, immune simulation, molecular docking, and normal mode analysis (NMA) were performed in silico. To evaluate peptide immunoreactivity, human IFN-γELISPOT assays were conducted using the candidate peptides, though HLA restriction was not experimentally validated. Results: The workflow identified 14 candidate CTL and 8 HTL epitopes, yielding an estimated global population coverage of 82.6% (60.7% in China; 51.2% in Indonesia). Immune simulations predicted robust humoral and Th1-associated cellular responses, though sustained CD8⁺ memory responses appeared limited. Docking and NMA suggested favorable structural interactions with TLR3 and TLR4. Crucially, the IFN-γ ELISPOT assay validated eight reactive epitopes that partially coincided with computational predictions within the tested donor group. Conclusions: This study establishes an integrated computational–experimental workflow for T cell epitope prioritization. The identified reactive epitopes provide a preliminary immunological basis and candidate pool for the future design and evaluation of multi-epitope mRNA vaccine strategies against tuberculosis. Full article

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, exemplifies one of the most formidable paradigms of tumor immune evasion (TIME) in pediatric oncology. Despite significant advances in multimodal therapy and the clinical integration of immunotherapeutic strategies, high-risk NB (HR-NB) remains largely refractory to durable immune control. This failure reflects not an absence of immune engagement, but the presence of a highly evolved and developmentally wired immune escape architecture. In this review, we synthesize emerging insights from single-cell, multi-omics, and functional studies to define how developmental lineage, cellular plasticity, metabolic rewiring, epigenetic regulation, and therapy-induced adaptation converge to engineer immune blindness in NB. We discuss how NB’s neural crest origin establishes a baseline of low immunogenicity, which is subsequently reinforced through coordinated suppression of antigen presentation, dominance of immune checkpoint signaling, and profound dysfunction of cytotoxic T and natural killer cells within an immunosuppressive tumor microenvironment. Central to this process is tumor-intrinsic plasticity, whereby lineage instability and dedifferentiation, exacerbated by therapeutic pressure, embed immune silence as a stable tumor state. We highlight evidence positioning RD3 as a master upstream regulator linking cellular identity to immune visibility, governing antigen presentation, innate immune sensing, checkpoint expression, and cytotoxic lymphocyte engagement. Beyond tumor-intrinsic mechanisms, we examine the roles of immunosuppressive myeloid populations, tumor-derived exosomes, metabolic stress, hypoxia, and ferroptosis-associated pathways in reinforcing immune paralysis. Finally, we outline emerging therapeutic strategies aimed at dismantling this architecture, including combinatorial checkpoint blockade, metabolic and epigenetic reprogramming, exosome-targeted interventions, and next-generation immune engineering platforms. Together, this review reframes TIME in NB as a programmable, developmentally rooted process and provides a mechanistic roadmap for restoring immune competence and therapeutic susceptibility in HR disease. Full article

Lymph nodes are central hubs of immune regulation and coordination, serving as primary sites for antigen presentation, lymphocyte activation, and the orchestration of adaptive immune responses. The composition and activation state of lymph node-resident immune cells critically shape both local and systemic immunity. Comprehensive immunophenotyping of these populations is therefore essential for understanding immune organization and functional heterogeneity. Here, we present an optimized protocol for the characterization of mouse lymph node-associated immune populations using 14-color multiparametric flow cytometry. The method combines lymph node isolation based on anatomical landmarks with mechanical dissociation and enzymatic digestion to generate high-quality single-cell suspensions suitable for downstream analysis. Furthermore, the described flow cytometry panel and gating strategy enable reliable identification and quantification of major lymphoid subsets, including helper CD4⁺ and cytotoxic CD8⁺ T cells with their differentiation states, as well as natural killer (NK) cells across distinct maturation stages. Although optimized for assessing lymphocyte maturation after lipopolysaccharide (LPS) challenge, the protocol serves as a reproducible platform for broad immunophenotyping of T and NK cell subsets in mouse lymphoid tissues under experimental conditions. Full article

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by unfavorable clinical outcomes. Despite significant progress in deciphering the genetic and epigenetic landscapes of T-ALL, the underlying molecular mechanisms, particularly in non-early T-cell precursor (non-ETP) T-ALL, remain incompletely understood. In this study, functional assays were performed using three well-characterized non-ETP T-ALL cell lines. In vivo therapeutic efficacy was evaluated using non-ETP T-ALL xenograft models. Transcriptomic profiling was performed by RNA sequencing (RNA-seq) followed by bioinformatic analysis. Publicly available clinical datasets from T-ALL patients were mined to analyze survival outcomes. We found that activation of CD40 ligand (CD40LG) or CD28 accelerates cell-cycle progression and enhances the migratory capacity of non-ETP T-ALL cells, with CD40LG uniquely upregulating CXCR4 to mediate bone marrow tropism. Further RNA-seq and functional validation identified Rho GTPase signaling, specifically RhoA/Rac1/Rac2, as a pivotal downstream effector of CD40LG/CD28, leading to therapeutic resistance to PI3K inhibition. Pharmacological blocking RhoA or Rac1 using small-molecule compounds not only induces remarkable cytotoxicity but also sensitizes resistant cells to PI3K inhibitors, both in vitro and in vivo. Clinically, elevated expression of CD40LG, CD28, RHOA, or RAC2 correlates with poor prognosis in non-ETP T-ALL patients. These findings uncover a novel CD40LG/CD28-Rho GTPase axis as a key driver of pathogenesis and a potential therapeutic vulnerability in non-ETP T-ALL, providing a new target for precision intervention and a promising strategy to overcome therapeutic resistance. Full article

The ICAM-1-derived cIBR peptide selectively binds to the I-domain of LFA-1, a receptor highly expressed on leukemia T cells; thus, the MTX-cIBR conjugate can be used to target methotrexate (MTX) to leukemic T cells and reduce its off-target toxicity. However, the uptake, biological mechanism, and selectivity of MTX-cIBR compared with unconjugated MTX remain unclear. Therefore, this study is aimed at evaluating the uptake, cytotoxicity, selectivity, apoptosis, cell cycle effects, and DHFR-related activity of MTX-cIBR in leukemia T cells compared with unconjugated MTX. MTX-cIBR exhibited cytotoxic activity comparable to MTX in LFA-1-expressing MOLT-4 cells but showed lower toxicity toward LFA-1-negative K562 cells, indicating improved selectivity. MTX uptake occurred through RFC and mFBP transport systems, whereas MTX-cIBR no longer depended on these pathways, suggesting altered cellular uptake after conjugation with cIBR by utilizing the LFA-1 receptor. Both compounds predominantly induced apoptosis with minimal necrotic cell populations. MTX induced S-phase arrest at lower concentrations and G2/M induced arrest at higher concentrations, whereas MTX-cIBR consistently promoted S-phase accumulation. In addition, MTX and MTX-cIBR downregulated the expression of DHFR, FPGS, and TYMS in MOLT-4 cells. Computational analyses further demonstrated that MTX exhibited lower binding free energy (ΔG) and greater binding stability toward DHFR than MTX-cIBR. These findings suggest that MTX-cIBR retains selective cytotoxic activity toward LFA-1-expressing leukemia T cells through altered cellular uptake and exhibits different interaction characteristics with DHFR compared with unconjugated MTX. Full article