Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA. The active DNA demethylation process may be linked with aberrant methylation and can be involved in leukemogenesis. The levels of 5-methylcytosine oxidation products were analyzed in minimally invasive material: the cellular DNA from peripheral blood cells and urine of patients with AML and MDS along with the control group, using isotope-dilution two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. The receiver operating characteristic curve analysis was used for the assessment of the ability to discriminate patients’ groups from the control group, and AML from MDS. The most diagnostically useful for discriminating AML patients from the control group was the urinary excretion of 5-hydroxymethylcytosine (AUC = 0.918, sensitivity: 85%, and specificity: 97%), and 5-(hydroxymethyl)-2′-deoxyuridine (0.873, 74%, and 92%), while for MDS patients 5-(hydroxymethyl)-2′-deoxycytidine in DNA (0.905, 82%, and 98%) and urinary 5-hydroxymethylcytosine (0.746, 66%, and 92%). Multi-factor models of classification trees allowed the correct classification of patients with AML and MDS in 95.7% and 94.7% of cases. The highest prognostic value of the analyzed parameters in predicting the transformation of MDS into AML was observed for 5-carboxy-2′-deoxycytidine (0.823, 80%, and 97%) and 5-(hydroxymethyl)-2′-deoxyuridine (0.872, 100%, and 75%) in DNA. The presented research proves that the intermediates of the active DNA demethylation pathway determined in the completely non-invasive (urine) or minimally invasive (blood) material can be useful in supporting the diagnostic process of patients with MDS and AML. The possibility of an early identification of a group of MDS patients with an increased risk of transformation into AML is of particular importance.

H. pylori gastritis is strongly associated with the upregulation of the expression of several matrix metalloproteinases (MMPs) in the gastric mucosa. However, the role of MMP-2 and MMP-9, and their inhibitors (tissue inhibitors of metalloproteinases -TIMPs) produced by immune cells in infected children have not been clearly defined. Moreover, the effects of H. pylori eradication therapy on MMPs and TIMPs production has not been evaluated. A total of 84 children were studied: 24—with newly diagnosed H. pylori gastritis, 25—after H. pylori eradication therapy (17 of them after successful therapy), 24—with H. pylori-negative gastritis, and 11—controls. Plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 by ELISA; MMPs and TIMPs expression in lymphocytes; neutrophils and monocytes in peripheral blood by multiparameter flow cytometry; and mucosal mRNA expression levels of MMPs and TIMP-1 in gastric biopsies by RT-PCR were evaluated. Children with H. pylori-related gastritis showed the following: (1) increased MMP-2 and TIMP-2 plasma levels, (2) increased intracellular expression of MMP-2 in the circulating lymphocytes and neutrophils, (3) low frequencies of circulating TIMP-1+ and TIMP-2+ leukocytes, and (4) high expression of mRNA for MMP-9 along with low expression of mRNA for MMP-2 in the gastric mucosa. Unsuccessful H. pylori eradication was associated with the following: (1) high plasma levels of MMP-9 and TIMP-1, (2) increased pool of TIMP-1+ lymphocytes as well as high expression of MMP-9 in circulating lymphocytes, and (3) high expression of mRNA for MMP-9 in the gastric mucosa. Our data suggest that MMPs are important contributors to stomach remodelling in children with H. pylori-related gastritis. Unsuccessful H. pylori eradication is associated with increased MMP-9 in plasma, circulating lymphocytes, and gastric mucosa.

Biological therapies for Crohn’s disease (CD), including infliximab, adalimumab, and vedolizumab, show variable efficacy. While some predictive biomarkers exist, data on regulatory immune cells are limited. This study examined whether baseline levels of circulating T and B cell subsets can predict response to these treatments. We recruited 43 adults with conventional treatment-resistant active CD (CDAI > 330) and 16 healthy controls. Blood samples were analysed by flow cytometry at baseline (week 0) and after induction therapy (week 12 or 14, depending on the received drug) to measure T and B cell subsets and correlate them with disease activity. CD patients at baseline showed a significantly reduced frequency of memory B cells, CD5⁺CD1d⁺ B cells, plasmablasts, and transitional B cells. Additionally, significant negative correlations were identified between transitional B cells and calprotectin/platelets, and between CD5⁺CD1d⁺ B cells and calprotectin. All CD patients responded clinically to biologic therapy. In those treated with infliximab or adalimumab, mature naïve B cells decreased, with a trend toward increased CD24^hiCD27⁺ B cells. Adalimumab responders showed a trend toward higher CD161 expression on Tregs, while vedolizumab-treated patients had a slight increase in plasmablasts. Biologic therapies in CD revealed treatment-specific immune correlations: infliximab/adalimumab responses involved B and T cell changes linked to inflammation, while VDZ response correlated with CD4⁺ and CD5⁺CD1d⁺ B cells. Our study suggests that infliximab/adalimumab induction therapy in CD expands circulating CD24^hiCD27⁺ B cells and reduces mature naïve B cells, while vedolizumab increases plasmablasts. These B-cell changes may reflect distinct mechanisms and serve as potential response biomarkers.

Background: DNA methylation changes are recognized as a key hallmark of biological aging. We analyzed the products of active DNA demethylation pathways in DNA isolated from leukocytes and the urine of healthy volunteers aged between 1.5 and 71 years. Results: A statistically significant inverse correlation between age and the analyzed modifications in urine was found for 5-hydroxymethyluracil, 5-hydroxymethylcytosine, 5-(hydroxymethyl)-2′-deoxycytidine, 5-methyl-2′-deoxycytidine, 5-carboxycytosine and 5-formylcytosine. The highest correlation coefficient was observed for 5-hydroxymethyluracil ( r = − 0.66). Interestingly, statistically significant inverse correlations were observed for 5-(hydroxymethyl)-2′-deoxycytidine and 5-methyl-2′-deoxycytidine in DNA. Conclusions: Changes in epigenetic modifications with age are more pronounced in urine than in cellular DNA. However, DNA modifications are limited to a single tissue (leukocytes), whereas modifications detected in urine reflect processes occurring at the level of the entire organism.