The thiopurine drugs-azathioprine and mercaptopurine-are purine antimetabolites used for the treatment of autoimmune hepatitis. These drugs undergo metabolism through genetically determined pathways, which influences their effectiveness and toxicity. There is scarce information regarding the clinical effects of measuring drug metabolites in these patients. The goal of the study is to test the clinical significance of measuring thiopurine metabolites in patients unsuccessfully treated with thiopurines. Clinical and laboratory data collected for patients who were treated for autoimmune hepatitis between 2015 and 2018, and did not achieve full remission under thiopurine therapy and had thiopurine metabolite levels measured due to lack of response and suspicious side effects were chosen. We compared clinical and laboratory data before and after the therapy change. The study included 25 tests of thiopurine metabolites in 21 patients. Six tests had therapeutic levels. Three tests showed high levels leading to lowering the drug dose. In 11 cases, levels of 6-thioguanine nucleotide were low; the dose was not changed in 3 of these, and the dose was increased in the remaining 8. Shunting was observed in 5 cases, 2 of which were mild and the dose was not changed. In the remaining 3, the dose was decreased, and allopurinol was added. Significant improvements in liver enzymes were observed following dose adjustments. We showed that, in cases of suboptimal response to thiopurine treatment, measuring thiopurine metabolites had an important role in optimizing therapy. In most patients, changing the dose led to a significant improvement with no need to switch to secondline therapies.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a bacterial second messenger that modulates various processes including biofilm formation, motility, and host-microbe symbiosis. Numerous studies have conducted comprehensive analysis of c-di-GMP. However, the mechanisms by which certain environmental signals such as iron control intracellular c-di-GMP levels are unclear. Here, we show that iron regulates c-di-GMP levels in Pseudomonas aeruginosa by modulating the interaction between an iron-sensing protein, IsmP, and a diguanylate cyclase, ImcA. Binding of iron to the CHASE4 domain of IsmP inhibits the IsmP-ImcA interaction, which leads to increased c-di-GMP synthesis by ImcA, thus promoting biofilm formation and reducing bacterial motility. Structural characterization of the apo-CHASE4 domain and its binding to iron allows us to pinpoint residues defining its specificity. In addition, the cryo-electron microscopy structure of ImcA in complex with a c-di-GMP analog (GMPCPP) suggests a unique conformation in which the compound binds to the catalytic pockets and to the membrane-proximal side located at the cytoplasm. Thus, our results indicate that a CHASE4 domain directly senses iron and modulates the crosstalk between c-di-GMP metabolic enzymes.

Spatially segregated, socio-economically deprived communities in Europe are at risk of being neglected in terms of health care. In Hungary, poor monitoring systems and poor knowledge on the health status of people in these segregated areas prevent the development of well-informed effective interventions for these vulnerable communities.
We used data available from National Health Insurance Fund Management to better describe health care performance in segregated communities and to develop more robust monitoring systems.
A cross-sectional study using 2020 health care data was conducted on each general medical practice (GMP) in Hungary providing care to both segregated and nonsegregated (complementary) adult patients. Segregated areas were mapped and ascertained by a governmental decree that defines them as within settlement clusters of adults with low level of education and income. Age, sex, and eligibility for exemption certificate standardized indicators for health care delivery, reimbursement, and premature mortality were computed for segregated and nonsegregated groups of adults and aggregated at the country level. The ratio of segregation and nonsegregation specific indicators (relative risk, RR) was computed with the corresponding 95% confidence intervals (95% CI).
Broad variations between GMPs were detected for each indicator. Segregated groups had a significantly higher rate of health care service use than complementary groups (RR = 1.22, 95% CI: 1.219;1.223) while suffering from significantly reduced health care reimbursement (RR = 0.940, 95% CI: 0.929;0.951). The risk of premature mortality was significantly higher among segregated patients (RR = 1.184, 95% CI: 1.087;1.289). Altogether, living in a segregated area led to an increase in visits to health care services by 18.1% with 6.6% less health spending.
Adults living in segregated areas use health care services more frequently than those living in nonsegregated areas; however, the amount of health care reimbursement they receive is significantly lower, suggesting lower quality of care. The health status of segregated adults is remarkably lower, as evidenced by their higher premature mortality rate. These findings demonstrate the need for intervention in this vulnerable group. Because our study reveals serious variation across GMPs, segregation-specific monitoring is necessary to support programs sensitive to local issues and establish necessary benchmarks.

When compartmentally mislocalized within cells, nucleic acids can be exceptionally immunostimulatory and can even trigger the immune-mediated elimination of cancer. Specifically, the accumulation of double-stranded DNA in the cytosol can efficiently promote antitumor immunity by activating the cGAMP synthase (cGAS) / stimulator of interferon genes (STING) cellular signaling pathway. Targeting this cytosolic DNA sensing pathway with interferon stimulatory DNA (ISD) is therefore an attractive immunotherapeutic strategy for the treatment of cancer. However, the therapeutic activity of ISD is limited by several drug delivery barriers, including susceptibility to deoxyribonuclease degradation, poor cellular uptake, and inefficient cytosolic delivery. Here, we describe the development of a nucleic acid immunotherapeutic, NanoISD, which overcomes critical delivery barriers that limit the activity of ISD and thereby promotes antitumor immunity through the pharmacological activation of cGAS at the forefront of the STING pathway. NanoISD is a nanoparticle formulation that has been engineered to confer deoxyribonuclease resistance, enhance cellular uptake, and promote endosomal escape of ISD into the cytosol, resulting in potent activation of the STING pathway via cGAS. NanoISD mediates the local production of proinflammatory cytokines via STING signaling. Accordingly, the intratumoral administration of NanoISD induces the infiltration of natural killer cells and T lymphocytes into murine tumors. The therapeutic efficacy of NanoISD is demonstrated in preclinical tumor models by attenuated tumor growth, prolonged survival, and an improved response to immune checkpoint blockade therapy.

In inflammatory bowel disease (IBD), conventional thiopurine users cease treatment in 60% of cases within 5 years, mostly because of adverse events or nonresponse. In this study, the authors aimed to investigate the role of 6-thioguanine nucleotide (TGN) measurements, geno/phenotyping of thiopurine S-methyltransferase (TPMT), and their mutual relationship with TG therapy in IBD.
An international retrospective, multicenter cohort study was performed at 4 centers in the Netherlands (Máxima Medical Centre) and the United Kingdom (Guy\'s and St. Thomas\' Hospital, Queen Elizabeth Hospital, and East Surrey Hospital).
Overall, 526 6-TGN measurements were performed in 316 patients with IBD. The median daily dosage of TG was 20 mg/d (range 10-40 mg/d), and the median duration of TG use was 21.1 months (SD, 28.0). In total, 129 patients (40.8%) had a known TPMT status. In the variant-type and wild-type TPMT genotype metabolism groups, median 6-TGN values were 1126 [interquartile range (IQR) 948-1562] and 467.5 pmol/8 × 10E8 red blood cells (RBCs) (IQR 334-593). A significant difference was observed between the 2 groups (P = 0.0001, t test). For TPMT phenotypes, in the slow, fast, and normal metabolism groups, the median 6-TGN values were 772.0 (IQR 459-1724), 296.0 (IQR 200-705), and 774.5 pmol/8 × 10E8 RBCs (IQR 500.5-981.5), with a significant difference observed between groups (P < 0.001, analysis of variance).
Our findings indicated that TPMT measurements at TG initiation can be useful but are not necessary for daily practice. TPMT genotypes and phenotypes are both associated with significant differences in 6-TGN levels between metabolic groups. However, the advantage of TG remains that RBC 6-TGN measurements are not crucial to monitor treatments in patients with IBD because these measurements did not correlate with laboratory result abnormalities. This presents as a major advantage in countries where patients cannot access these diagnostic tests.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.

It had not been reported that myocardial scar shown on gated myocardial perfusion SPECT (GMPS) might reduce after cardiac resynchronization therapy (CRT). In this study, we aim to investigate the clinical impact and characteristic of scar reduction (SR) after CRT.
Sixty-one heart failure patients following standard indication for CRT received twice GMPS as pre- and post-CRT evaluations. The patients with an absolute reduction of scar ≥ 10% after CRT were classified as the SR group while the rest were classified as the non-SR group. The SR group (N = 22, 36%) showed more improvement on LV function (∆LVEF: 18.1 ± 12.4 vs 9.4 ± 9.9 %, P = 0.007, ∆ESV: - 91.6 ± 52.6 vs - 38.1 ± 46.5 mL, P < 0.001) and dyssynchrony (ΔPSD: - 26.19 ± 18.42 vs - 5.8 ± 23.0°, P < 0.001, Δ BW: - 128.7 ± 82.8 vs - 25.2 ± 109.0°, P < 0.001) than non-SR group (N = 39, 64%). Multivariate logistic regression analysis showed baseline QRSd (95% CI 1.019-1.100, P = 0.006) and pre-CRT Reduced Wall Thickening (RWT) (95% CI 1.016-1.173, P = 0.028) were independent predictors for the development of SR.
More than one third of patients showed SR after CRT who had more post-CRT improvement on LV function and dyssynchrony than those without SR. Wider QRSd and higher RWT before CRT were related to the development of SR after CRT.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5\'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.

The blood-brain barrier (BBB) is the most restrictive and complicated barrier that keeps most biomolecules and drugs from the brain. An efficient brain delivery strategy is urgently needed for the treatment of brain diseases. Based on the studies of brain-targeting extracellular vesicles (EVs), the potential of using small apoptotic bodies (sABs) from brain metastatic cancer cells for brain-targeting drug delivery is explored. It is found that anti-TNF-α antisense oligonucleotide (ASO) combined with cationic konjac glucomannan (cKGM) can be successfully loaded into sABs via a transfection/apoptosis induction process and that the sABs generated by B16F10 cells have an extraordinarily high brain delivery efficiency. Further studies suggest that ASO-loaded sABs (sCABs) are transcytosed by b. End3 (brain microvascular endothelial cells, BMECs) to penetrate the BBB, which is mediated by CD44v6, and eventually taken up by microglial cells in the brain. In a Parkinson\'s disease (PD) mouse model, sCABs dramatically ameliorate PD symptoms via the anti-inflammatory effect of ASO. This study suggests that sABs from brain metastatic cancer cells are excellent carriers for brain-targeted delivery, as they have not only an extraordinary delivery efficiency but also a much higher scale-up production potential than other EVs.