Intracellular Accumulation and Secretion of YKL-40 (CHI3L1) in the Course of DMSO-Induced HL-60 Cell Differentiation.

YKL-40 (CHI3L1) is a matrix glycoprotein stored in human neutrophil-specific granules and released upon activation. While it is implicated in inflammation, cancer progression, and cell differentiation, its exact physiological role remains unclear. This study investigated the intracellular expression and secretion of YKL-40 by untreated and DMSO-treated HL-60 cells in association with surface expression of CD11b and CD66b throughout the differentiation process (up to 120 h). Secreted YKL-40 protein and mRNA levels of YKL-40, CD66b, and CD11b were measured by ELISA and quantitative RT-PCR, respectively. The intracellular YKL-40 and surface CD11b and CD66b expression were assessed by flow cytometry. A significant increase in CD11b expression confirmed DMSO-induced differentiation of HL-60 cells. Upon DMSO stimulation, YKL-40 mRNA expression increased in a time-dependent manner, unlike CD66b. The lack of CD66b (a granulocyte maturation and activation marker) on the surface of HL-60 cells might suggest that DMSO treatment did not induce full maturation or activation. The intracellular YKL-40 protein expression was increasing up to 96 h of DMSO treatment and then declined. YKL-40 secretion into the culture medium was detectable only at later time points (96 and 120 h), which was correlated with a decreased proliferation of DMSO-treated HL-60 cells. These findings suggest sequential changes in YKL-40 production and secretion during DMSO-induced differentiation of HL-60 cells and might contribute to a better understanding of YKL-40's involvement in both physiological processes and disease development, including multiple sclerosis.

Biochemical and cardiovascular predictors of PIMS-TS risk in children after COVID-19 recovery: preliminary results of the LATE-COVID-Kids Study.

Introduction: We aimed to characterize biochemical and cardiovascular predictors of the paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) risk based on the data from the LATE-COVID-Kids study. Methods: 148 consecutive COVID-19 convalescents hospitalized for the clinical evaluation after the acute phase of COVID-19 were classified into two groups related to symptoms: 33 children finally diagnosed with PIMS-TS and 115 children without PIMS-TS (control group). Results: PIMS-TS children were significantly younger (6.79 ±4.57 vs. 9.10 ±4.94 years). After adjustment, in comparison to those without, PIMS-TS children had a higher level of antithrombin III (111 ±9.30 vs. 105 ±11.4), higher heart rate (HR)/min (100 (89.0-111) vs. 90 (79.7-100)) and sinus rhythm (p = 0.03) but lower PQ interval (p = 0.02) on admission to hospital. The lymphocytes (absolute count and percentage) were significantly higher in children with PIMS-TS, and the opposite results were obtained for IgA and neutrophils. Furthermore, children with PIMS-TS had a higher level of thyroid stimulating hormone (2.76 (2.16-4.18) vs. 2.36 (1.73-2.83)) and red cell distribution width (p < 0.005) compared to those without. Conclusions: It is the first data on the possible predictors of PIMS-TS risk in the Long-COVID period. These results need to be further validated to next create the PIMS SCORE algorithm, which might enable the effective prediction of children with the risk of PIMS-TS occurrence after COVID-19 recovery.

Prognostic significance of IKZF1 deletions and IKZF1plus profile in children with B-cell precursor acute lymphoblastic leukemia treated according to the ALL-IC BFM 2009 protocol.

The strongest predictors of outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are minimal residual disease (MRD) and specific molecular abnormalities. One unfavorable prognostic factor is the presence of IKZF1 gene aberrations, particularly when co-occurring with high MRD level at the end of induction treatment. The present study determines the predictive value of a recently-defined IKZF1-plus (IKZF1plus ) microdeletion profile in 373 children with BCP-ALL treated according to the ALL-intercontinental Berlin-Frankfurt-Munster protocol 2009 protocol. IKZF1-wild type (IKZF1wt ) patients demonstrated lower leukemic burden parameters than those carrying IKZF1 deletion (IKZF1del [n = 26, 7.0%]) or IKZF1plus pattern (n = 34, 9.1%): (i) median blast percentage at diagnosis (78.0% vs. 86.9% vs. 86.0%; p = 0.021); (ii) median MRD level at day 15 of induction protocol (0.3% vs. 2.1% vs. 0.8%; p = 0.011); (iii) poor steroid response (7.6% vs. 26.5% vs. 12.5%; p = 0.010). Minimal residual disease level at day 33 (MRD33) exceeding 10-4 was more frequently observed in both the IKZF1del and IKZF1plus subgroups than in IKZF1wt patients (n = 9 [36.0%] vs. n = 13 [41.9%] vs. n = 70 [24.0%], p = 0.051). IKZF1plus individuals showed a tendency for a lower MRD reduction between day 15 and 33 compared to IKZF1del patients (p = 0.124). IKZF1del and IKZF1plus patients showed decreased relapse-free survival (HR [95%CI] for IKZF1wt as reference = 2.72 [1.21-6.11] and 2.00 [0.87-4.49], respectively, p = 0.023). Both genetic markers including IKZF1del and IKZF1plus microdeletion profile provide additional predictive value of treatment outcome in childhood BCP-ALL and may contribute to more efficient patient stratification; the same is true in MRD guided protocols, which are based on flow cytometric measurements on day 15 of induction protocol.

A multistep approach to the genotype-phenotype analysis of Polish patients with tuberous sclerosis complex.

The aim of this study was to evaluate a cost-effective diagnostic strategy for identification of casual variants for tuberous sclerosis complex (TSC) in the Polish population and to correlate the genetic results with selected phenotypic features. Fifty-five patients, aged 3-44 years, with a clinical diagnosis of TSC were enrolled into the study. All patients received a three-step analysis: next generation sequencing screening (NGS), multiplex ligation-dependent probe amplification (MLPA) and deep sequencing. This multistep approach obtained positive results in 51/55 (93%) patients: of the 51 positives TSC1 variants were observed in 16 (31%) and TSC2 variants in 35 (69%); these included 13 novel variants and two patients with mosaicism. Four patients (7%) had no mutation identified (NMI). Among the TSC1 gene variants, there were five nonsense, four frameshift, three large deletions, two missense and two splicing variants. For the TSC2 gene, 11 were missense, eight splicing, six frameshift, four large deletions, two in-frame deletions and four nonsense variants. The patients with TSC2 changes had their clinical diagnosis of TSC at a younger age than those with TSC1 changes (one year vs three years, p = 0.041). The TSC2 group demonstrated a higher number of major symptoms per patient (p = 0.04). Subependymal giant cell astrocytoma with concomitance of other brain lesions was more common in patients with missense mutations in either gene (23% vs 0%, p = 0.02). Such a multistep molecular diagnostic strategy could increase the possibility of detecting causal variants for TSC and may allow detection of mosaicism at low levels. Missense pathogenic variants in TSC1 or TSC2 gene might be associated with a higher risk of brain lesions.

Nanoparticles for Directed Immunomodulation: Mannose-Functionalized Glycodendrimers Induce Interleukin-8 in Myeloid Cell Lines.

New therapeutic strategies for personalized medicine need to involve innovative pharmaceutical tools, for example, modular nanoparticles designed for direct immunomodulatory properties. We synthesized mannose-functionalized poly(propyleneimine) glycodendrimers with a novel architecture, where freely accessible mannose moieties are presented on poly(ethylene glycol)-based linkers embedded within an open-shell maltose coating. This design enhanced glycodendrimer bioactivity and led to complex functional effects in myeloid cells, with specific induction of interleukin-8 expression by mannose glycodendrimers detected in HL-60 and THP-1 cells. We concentrated on explaining the molecular mechanism of this phenomenon, which turned out to be different in both investigated cell lines: in HL-60 cells, transcriptional activation via AP-1 binding to the promoter predominated, while in THP-1 cells (which initially expressed less IL-8), induction was mediated mainly by mRNA stabilization. The success of directed immunomodulation, with synthetic design guided by assumptions about mannose-modified dendrimers as exogenous regulators of pro-inflammatory chemokine levels, opens new possibilities for designing bioactive nanoparticles.

COVID-19 and cardiovascular complications - preliminary results of the LATE-COVID study.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) may affect many organs and may be responsible for numerous complications including cardiovascular problems. METHODS: We analysed consecutive patients (n = 51) admitted to the cardiology department between 1st October 2020 and 31st January 2021 due to symptoms which might have reflected cardiovascular complications following COVID-19. We collected data concerning clinical characteristics, results of laboratory tests, echocardiography and 24-hour ambulatory ECG recording. RESULTS: The post-COVID-19 complications appeared 1-4 months after disease recovery. Severe cardiovascular complications were observed in 27.5% of hospitalized patients. In comparison to those with mild complications, patients with severe complications had significantly higher prevalence of diabetes (36 vs. 8%; p = 0.01), decrease in ejection fraction (36% vs. 0%, p < 0.001), higher resting heart rate at admission (85 vs. 72 bpm; p < 0.001), and higher levels of C-reactive protein (p = 0.02) and troponin T (17.9 vs. 4.2 pg/ml; p = 0.01). Dyspnoea and exercise intolerance were also more frequent in patients with severe complications. CONCLUSIONS: Diabetes, elevated level of CRP and troponin, heart rate variability parameters and worsening of left ventricular ejection fraction are related to the severity of cardiovascular complications following COVID-19 infection.

Six molecular patterns leading to hemophilia A phenotype in 18 females from Poland.

INTRODUCTION: Female hemophilia is an intriguing rare disorder and few larger reports on its genetic etiology are available. While historically the diagnosis was satisfactorily reached by factor VIII activity assays, the clinical and potentially therapeutic heterogeneity of female hemophilia calls for comprehensive molecular diagnosis in each case. Currently, the genetic investigations are not a part of routine, state-funded, diagnostics in Poland, and thus molecular epidemiological data are missing. AIM: We set out to perform a comprehensive genetic analysis of Polish females with hemophilia A. PATIENTS/METHODS: Eighteen females with hemophilia A (including 2 with severe and 5 with moderate hemophilia phenotype) consented for genetic diagnostics. To establish F8 mutations, we used next-generation sequencing of a panel of genes associated with hematological disorders, standard assays for recurrent intragenic F8 inversions and MLPA when deletions were suspected. When appropriate we also used karyotyping, genomic microarrays and X chromosome inactivation assays. RESULTS: While abnormally skewed X-chromosome inactivation combined with a F8 variant on the active allele was, as expected, the most common genetic etiology, a number of other genetic scenarios were unraveled. This included: misdiagnosis (molecular diagnosis of vWd), Turner syndrome, compound heterozygosity and androgen insensitivity syndrome (a phenotypical 46,XY female with a novel androgen receptor gene mutation). We report 3 novel F8 mutations. CONCLUSION: Every case of female hemophilia warrants full genomic diagnostics, as this may change the diagnosis or reveal broader morbidity than a coagulation disorder (Turner syndrome, androgen insensitivity, or cardiovascular morbidity that we described previously in a SHAM syndrome carrier).

CCR6 blockade on regulatory T cells ameliorates experimental model of multiple sclerosis.

Regulatory T cells (Tregs) play a significant role in limiting damage of tissue affected by autoimmune process, which has been demonstrated in various experimental models for multiple sclerosis (MS) (mostly experimental autoimmune encephalomyelitis - EAE), rheumatoid arthritis, and type 1 diabetes. In this study, we demonstrated that Tregs increasingly migrate to central nervous system (CNS) during subsequent phases of EAE (preclinical, initial attack, and remission). In contrast, in peripheral tissues (blood, lymph nodes, and spleen), a significant accumulation of Tregs is mostly present during EAE remission. Moreover, an increased expression of CCR6 on Tregs in the CNS, blood, lymph nodes, and spleen in all phases of EAE was observed. The highest expression of CCR6 on Tregs from the CNS, lymph nodes, and spleen was noted during the initial attack of EAE, whereas in the blood, the peak expression of CCR6 was detected during the preclinical phase. The presence of Tregs in the CNS during EAE was confirmed by immunohistochemistry. To analyze additional functional significance of CCR6 expression on Tregs for EAE pathology, we modulated the clinical course of this MS model using Tregs with blocked CCR6. EAE mice, which received CCR6-deficient Tregs showed significant amelioration of disease severity. This observation suggests that CCR6 on Tregs may be a potential target for future therapeutic interventions in MS.

3-Bromopyruvate induces expression of antioxidant genes.

An alkylating compound, 3-bromopyruvic acid (3-3-bromopyruvic acid (BP)) is a promising anti-cancer agent, potentially able to act on multidrug-resistant cells. Its action has been attributed mainly to inhibition of glycolysis. This compound induces also oxidative stress at a cellular level. The effects of 3-BP on gene expression have not been studied although they may determine the survival of cells exposed to 3-BP. The aim of this paper was to examine the effect 3-BP on gene expression pattern in breast MCF-7 cancer cells. Detection of the differences in gene expression was performed using microarrays and dysregulated genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Exposure of cells to 100 µM 3-BP for 6, 12 and 24 increased expression and diminished expression of 39 and 6 genes, respectively. Among the induced genes, 22 belong to general cellular stress response genes, maintenance genes involved in redox homeostasis, responding to oxidative stress (among them metallothioneins, low-molecular-weight thiol homeostasis enzymes and genes coding for NAD(P)H-dependent oxidoreductases operating on complex organic substrates, including aldo-keto reductases). These results demonstrate that transient oxidative stress in cells exposed to 3-BP is followed by antioxidant response.

Terminal Sugar Moiety Determines Immunomodulatory Properties of Poly(propyleneimine) Glycodendrimers.

Poly(propyleneimine) dendrimers fully surface-modified with disaccharide moieties (maltose, cellobiose, and lactose) designed to mimic natural lectin receptor ligands were tested for their bioactivity in two myeloid cell lines: THP-1 and HL-60. Depending on the sugar modification, we observed variable activation of NF-κB, AP-1, and NF-AT signaling pathways: lactose-coated dendrimers had the strongest impact on marker gene expression and most signaling events with the notable exception of NF-κB activation in THP-1 cells. The two cell lines showed an overall similar pattern of transcription factor and gene expression activation upon treatment with glycodendrimers, suggesting the involvement of galectin and C-type lectin receptor types. An important result of this action was the overexpression of CD40 and IL8 genes, potentially leading to an activated, proinflammatory phenotype in the monocyte/macrophage cell lineage. These pharmacodynamic characteristics of glycodendrimers need to be taken into account during their pharmaceutical applications both in drug delivery and direct immunomodulation.

Glycodendrimer Nanocarriers for Direct Delivery of Fludarabine Triphosphate to Leukemic Cells: Improved Pharmacokinetics and Pharmacodynamics of Fludarabine.

Fludarabine, a nucleoside analogue antimetabolite, has complicated pharmacokinetics requiring facilitated transmembrane transport and intracellular conversion to triphosphate nucleotide form (Ara-FATP), causing it to be susceptible to emergence of drug resistance. We are testing a promising strategy to improve its clinical efficacy by direct delivery of Ara-FATP utilizing a biocompatible glycodendrimer nanocarrier system. Here, we present results of a proof-of-concept experiment in several in vitro-cultured leukemic cell lines (CCRF, THP-1, U937) using noncovalent complexes of maltose-modified poly(propyleneimine) dendrimer and fludarabine triphosphate. We show that Ara-FATP has limited cytotoxic activity toward investigated cells relative to free nucleoside (Ara-FA), but complexation with the glycodendrimer (which does not otherwise influence cellular metabolism) drastically increases its toxicity. Moreover, we show that transport via hENT1 is a limiting step in Ara-FA toxicity, while complexation with dendrimer allows Ara-FATP to kill cells even in the presence of a hENT1 inhibitor. Thus, the use of glycodendrimers for drug delivery would allow us to circumvent naturally occurring drug resistance due to decreased transporter activity. Finally, we demonstrate that complex formation does not change the advantageous multifactorial intracellular pharmacodynamics of Ara-FATP, preserving its high capability to inhibit DNA and RNA synthesis and induce apoptosis via the intrinsic pathway. In comparison to other nucleoside analogue drugs, fludarabine is hereby demonstrated to be an optimal candidate for maltose glycodendrimer-mediated drug delivery in antileukemic therapy.

Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line.

PURPOSE: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. METHODS: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. RESULTS: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 µM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25-100 µM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. CONCLUSION: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).

The impact of multiple sclerosis relapse treatment on migration of effector T cells--Preliminary study.

UNLABELLED: Migration of inflammatory cells from the blood to the central nervous system (CNS) is crucial for development of multiple sclerosis (MS). Inhibition of this process would allow to control disease activity. The first step confirming this approach would be the analysis of the impact of effective MS relapse therapy on migration of effector T cells. The aim of the study was to analyze the influence of methylprednisolone (MP) on the migratory activity of effector CD4+ T cells from MS patients. Moreover, to study the potential mechanism of this process we studied expression of chemokine receptors on migrating cells. MATERIAL AND METHODS: Peripheral blood samples were obtained from relapsing-remitting MS (RR-MS) patients during relapse (n=23) and from control group (n=23). After isolation CD4+ T cells were incubated with various concentrations of MP. Then they were stimulated in chemotaxis assay with chemokines CCL3 or CXCL10 or were used to CCR1 and CXCR3 expression analysis. RESULTS: CXCL10- and CCL3-stimulated migration of CD4+ T cells was significantly increased in MS. MP was able to reduce in vitro migration of effector T cells induced by CXCL10, but not by CCL3. Inhibition by MP was dose-dependent. Expression of analyzed chemokine receptors was unaltered after MP incubation. CONCLUSIONS: MP reduced CD4+ T cells migration induced by CXCL10 without affecting CXCR3 expression. These observations demonstrate one of the potential mechanisms of MP action in MS, distinct from inducing cell apoptosis, and suggests the new targets for development of more effective MS treatments.

Central Nervous System and Peripheral Expression of CCL19, CCL21 and Their Receptor CCR7 in Experimental Model of Multiple Sclerosis.

It is well documented that inflammatory chemokines play a significant role in the development of multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE). Recently, the involvement of homeostatic (or lymphoid) chemokines in the pathogenesis of autoimmune diseases has become an object of intensive study. In this work, quantitative analysis of CCL19, CCL21 and CCR7 expression in the central nervous system (CNS), as well as in inflammatory mononuclear cells isolated from several organs during the first attack, remission and the second attack of chronic-relapsing EAE (ChREAE), was performed. Using real-time PCR, RNAse Protection Assay and immunohistochemistry, the expression of both chemokines, as well as of their common receptor CCR7, was analyzed in the brain, spleen, lymph nodes and peripheral blood mononuclear cells. Increased expression of CCL19 and CCL21 was observed mostly in mononuclear inflammatory cells isolated from the CNS during active ChREAE. At the same time the expression of CCR7 in blood mononuclear leukocytes was reduced. This observation extends our current knowledge about the possible role of chemokines CCL19, CCL21 and their receptor CCR7 in the pathogenesis of ChREAE and, by extension, MS.