MicroRNA-125b: association with disease activity and the treatment response of patients with early rheumatoid arthritis.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs that regulate gene expression by targeting mRNA. It was proved that some miRNAs are significantly deregulated in rheumatoid arthritis (RA). MicroRNA-125b negatively regulates expression of TNF-α, which plays a crucial role in RA pathogenesis. The aim of this study was to determine the treatment outcome of patients with early RA based on the expression of circulating and cellular miR-125b. METHODS: Total RNA was isolated from the plasma and peripheral blood mononuclear cells (PBMCs) of 58 patients with early RA before and three months after treatment initiation and of 54 age- and sex-matched healthy controls (HC). The expression of miR-125b was measured by TaqMan quantitative PCR. The treatment responders were defined as patients achieving remission or low disease activity (28-joint count disease activity score (DAS28) <3.2). Receiver operating characteristic (ROC) curve and stepwise backward multivariable logistic regression analyses of miR-125b expression were used to predict the disease outcome at three and six months after initiation of treatment. RESULTS: The expression of miR-125b in the PBMCs and plasma of treatment-naïve early RA patients was significantly lower than that of HC and increased significantly after three months of treatment, particularly in responders. However, only the cellular expression of miR-125b was inversely correlated with disease activity. MiR-125b expression in PBMCs was higher in responders than in non-responders after three months (p = 0.042). Using ROC analysis, the cellular expression of miR-125b, but not the disease activity at baseline, predicted the treatment response after three months of therapy (area under the curve 0.652 (95 % CI 0.510 to 0.793); p = 0.048). CONCLUSION: The expression of miR-125b in PBMCs of treatment-naïve patients may present a novel biomarker for monitoring the treatment outcome during the early phase of RA.

Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

A comparative study of pharmacokinetics, urinary excretion and tissue distribution of platinum in rats following a single-dose oral administration of two platinum(IV) complexes LA-12 (OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV) and satraplatin (OC-6-43)-bis(acetato)amminedichloro(cyclohexylamine)platinum(IV).

PURPOSE: This study compared the pharmacokinetics, tissue distribution, and urinary excretion of platinum in rats after single oral doses of LA-12 and satraplatin. METHODS: Both platinum derivatives were administered to male Wistar rats as suspensions in methylcellulose at four equimolar doses within the range of 37.5-300 mg LA-12/kg body weight. Blood sampling was performed until 72 h, and plasma and plasma ultrafiltrate were separated. Moreover, urine was collected until 72 h, and kidney and liver tissue samples were obtained at several times after administration. Platinum was measured by atomic absorption spectrometry. The pharmacokinetics of platinum was analyzed by population modelling and post hoc Bayesian estimation as well as using non-compartmental pharmacokinetic analysis of the mean concentration-time curves. RESULTS: Platinum was detected in all plasma and ultrafiltrate samples 15 min after oral administration of both compounds and peaked between 3-4 h and 1-3 h, respectively. Similar for LA-12 and satraplatin, the C (max) and AUC values of plasma and ultrafiltrate platinum increased less than in proportion to dose. The mean C (max) and AUC values of plasma platinum observed after administration of LA-12 were from 0.84 to 2.5 mg/l and from 20.2 to 75.9 mg h/l. For ultrafiltrate platinum, the corresponding ranges were 0.16-0.78 mg/l and 0.63-1.8 mg h/l, respectively. The AUC of plasma platinum was higher after satraplatin (P < 0.001). However, administration of LA-12 resulted in significantly higher AUC values of ultrafiltrate platinum after the doses of 150 mg and 300 mg/kg (P < 0.01), respectively, and the C (max) values were significantly higher starting from the dose of 75 mg/kg LA-12 and upward (P < 0.01). Cumulative 72-h urinary recovery of platinum dose was below 5% for both compounds, and it decreased with the dose of satraplatin (P < 0.01), while a numerical decrease was observed after administration of LA-12 that did not reach statistical significance (P = 0.41). The renal clearance of free platinum was similar regardless of the dose and compound administered. Platinum concentrations in the liver homogenate exceeded those in the kidney. Distribution of platinum to tissues was higher after LA-12 compared to satraplatin. The difference in kidney platinum increased with dose and was twofold after 350 mg/kg LA-12. Liver platinum was twofold higher after LA-12 across all four doses. CONCLUSIONS: In conclusion, this first comparative pharmacokinetic study with LA-12 and satraplatin shows that characteristics of platinum exposure evaluated in the plasma, plasma ultrafiltrate and kidney and liver tissues increase less than in proportion to dose following a single-dose administration of 37.5-300 mg/kg to Wistar rats. These findings together with the dose-related elevation in the pharmacokinetic characteristics V/F and CL/F of platinum and ultrafiltrate platinum as well as a drop in platinum urinary recovery are consistent with a dose-related decrease in the extent of oral bioavailability most likely due to saturable intestinal absorption.

The new platinum(IV) derivative LA-12 shows stronger inhibitory effect on Hsp90 function compared to cisplatin.

BACKGROUND: Cisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells. RESULTS: LA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12. CONCLUSIONS: To generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.

Bcr-Abl fusion sequences do not induce immune responses in mice when administered in mouse polyomavirus based virus-like particles.

Mouse polyomavirus-like particles (MPyV-VLPs) carrying inside a fragment of the Bcr-Abl hybrid protein containing the epitope of chronic myeloid leukemia fusion region were prepared. A sequence encoding 171 amino acids covering Bcr-Abl breakpoint was fused to the C-terminal part of VP3 minor protein connecting it to the VP1 capsomeres. Chimeric particles, the Bcr-Abl VLPs, were tested for their ability to induce Bcr-Abl specific immune response in mice after their intranasal (i.n.) or intraperitoneal (i.p.) administration without any other adjuvants. Bcr-Abl VLPs induced strong anti-VP1 immune response in both i.n. and i.p. immunized mice. As expected, neither IgG nor IgM anti-Bcr-Abl specific antibodies were detected in the sera of immunized animals. Surprisingly, no specific CTL (cytotoxic T-lymphocyte) activity was proved using two different methods (in vitro cytotoxicity assay with CFSE-labeled target cells and highly sensitive cytotoxicity assay using MHC class I Bcr-Abl specific pentamers). In addition, no proliferative response of T-cells of i.n. immunized mice after in vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells was observed. Taken together, Bcr-Abl breakpoint epitopes appeared to be weak immunogens and even MPyV-VLPs did not provide sufficient adjuvant ability to support induction of immune responses specific to Bcr-Abl fusion zone epitope.

Cellular and humoral immune responses to chimeric EGFP-pseudocapsids derived from the mouse polyomavirus after their intranasal administration.

Mouse polyomavirus (MPyV) VP1-pseudocapsids carrying enhanced green fluorescent protein (EGFP-VLPs) were used for intranasal immunization of mice. EGFP-VLPs induced strong anti-VP1 but not anti-EGFP antibody production. In vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells (BMDCs) induced remarkable T-cell proliferative response specific for both VP1 and EGFP antigen and IL-2 and IFN-gamma production. Surprisingly, no specific cytotoxic activities against VP1 and EGFP proteins were detected. After intranasal administration of EGFP-VLPs, as well as after polyomavirus infection, a moderate reduction of CD4(+)CD25(+)Foxp3(+) T cells was observed in spleens but not in lymph nodes and peripheral blood, suggesting that both MPyV virions and pseudocapsids are able to induce changes in distribution of regulatory T cells. Treatment of EGFP-VLPs pulsed BMDCs with inhibitors of endosomal acidification proved that presentation of peptides on MHCgp class II is dependent on acidic endosomal environment. Substantial decrease of CD4-specific T-cell proliferation in the presence of proteasome inhibitor suggests that MHCgp class II might load VPL-derived peptides processed by proteasomes. Thus, polyomavirus derived VLPs appear to be promising delivery and adjuvant vehicles for therapeutic proteins.