The role of NPM1 alternative splicing in patients with chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease with heterogeneous clinical course. Recent studies revealed a link between NOTCH1 mutation and the overexpression of MYC and MYC-related genes involved in ribosome biogenesis and protein biosynthesis, such as nucleophosmin-1 (NPM1), in CLL cells. In the present study, we aim to evaluate the impact of the NOTCH1 mutation on the MYC and MYC induced NPM1 expression in CLL cells via quantification of their transcripts. METHODS: Using qRT-PCR, we analyzed the levels of MYC and three main NPM1 splice variants in 214 samples collected from CLL patients. We assessed the impact of each splice variant on CLL prognostic markers, including the IGHV, TP53, NOTCH1, SF3B1, and MYD88 mutational status, cytogenetic aberrations, and laboratory features. RESULTS: Significantly higher levels of NPM1.R1 transcripts in patients with unmutated compared to mutated IGHV status were found. The median time to first treatment (TTFT) in patients with a high level of NPM1.R1 was significantly shorter compared to the group with low NPM1.R1 levels (1.5 vs 33 months, p = 0.0002). Moreover, in Multivariate Cox Proportional Hazard Regression Model NPM1.R1 splice variant provided an independent prognostic value for TTFT. CONCLUSION: In conclusion, our study indicates the prognostic significance of the level of NPM1.R1 expression and suggests the importance of splicing alterations in the pathogenesis of CLL.

Pre-emptive analgesia with methylprednisolone and gabapentin in total knee arthroplasty in the elderly.

The aim of this study is to assess whether administration of gabapentin and methylprednisolone as "pre-emptive analgesia" in a group of patients above 65 years of age would be effective in complex pain management therapy following total knee arthroplasty (TKA). One hundred seventy patients above 65 years were qualified for the study, with exclusion of 10 patients due to clinical circumstances. One hundred sixty patients were randomly double-blinded into two groups: the study group (80 patients) and the control group (80 patients). The study group received as "pre-emptive" analgesia a single dose of 300 mg oral (PO) gabapentin and 125 mg intravenous (IV) methylprednisolone, while the control received a placebo. All patients received opioid and non-opioid analgesic agents perioperatively calculated for 1 kg of total body weight. We measured (1) pain intensity level at rest (numerical rating scale, NRS), (2) life parameters, (3) levels of inflammatory markers (leukocytosis, C reactive protein CRP), and (4) all complications. Following administration of gabapentin and methylprednisolone as "pre-emptive" analgesia, the NRS score at rest was calculated at 6, 12 (p < 0.000001), 18 (p < 0.00004) and 24 (p = 0.005569) h postoperatively. Methylprednisolone with gabapentin significantly decreased the dose of parenteral opioid preparations (p = 0.000006). The duration time of analgesia was significantly longer in study group (p < 0.000001), with CRP values lower on all postoperative days (1, 2 days-p < 0.00001, 3 days-p = 0.00538), and leukocytosis on day 2 (p < 0.0086) and 3 (p < 0.00042). No infectious complications were observed in the first postoperative days; in the control group, one patient manifested transient ischemic attack (TIA). The use of gabapentin and methylprednisolone as a single dose decreased the level of postoperative pain on the day of surgery, the dose of opioid analgesic preparations, and the level of inflammatory parameters without infectious processes.

The effect of pre-operative high doses of methylprednisolone on pain management and convalescence after total hip replacement in elderly: a double-blind randomized study.

PURPOSE: The aim of the study was to assess whether administration of a single dose of methylprednisolone in the group patients above 65 years of age will be effective in complex analgesic management after total hip arthroplasty (THA). METHODS: Seventy-seven patients above 65 years old were double-blind randomized into two: the study and controls groups. Pre-operatively, the study group received as a single dose of 125 mg intravenous methylprednisolone, while the others saline solution as placebo. Peri-operatively, all the patients were administered opioid and nonopioid analgesic agents. We measured the levels of inflammatory markers (leukocytosis, C-reactive protein-CRP), pain intensity level (visual analog scale-VAS; numerical rating scale-NRS), the life parameters, and noted complications. RESULTS: Following administration of methylprednisolone were significantly lower levels of CRP on all the four post-operative days; leukocytosis on the second day; the VAS/NRS score at rest after six, 12, and 18 hours post-operatively, diminished the dose of parenteral opioid preparations (oxycodone hydrochloride), the duration of analgesia by peripheral nerve block was significantly higher as compared with the placebo group (p < 0.000001). No infectious complications were noted; there was one patient who developed post-operative delirium. CONCLUSION: A single dose of methylprednisolone significantly reduces the level of post-operative pain at rest on the day of THA in the group patients above 65 years of age, decreases the dose of opioid analgesic agents, and significantly decreases the level of inflammatory markers, without infectious processes.

Cofilin-1 Maintains Prosurvival Signaling in Chronic Lymphocytic Leukemia Cells.

BACKGROUND/AIM: Despite numerous studies, the etiology of chronic lymphocytic leukemia (CLL) remains unknown. A hypothesis of autoantigen stimulation in leukemic clone selection might explain 'stereotypy' of B-cell receptors. In healthy cells, cofilin-1 (CFL1) has multiple functions. Its role was described in several malignancies. The aim of this study was characterization of the role of CFL1 in CLL. Materialas and Methods: Cells from peripheral blood of 180 patients and 42 healthy volunteers (HVs) were isolated. Gene expression was assessed with reverse transcription polymerase chain reaction (RT-qPCR); western blot was performed for determination of protein level and activity. After silencing of CFL1 gene, cell ability for migration and chemotaxis was investigated with Transwell method. Post-silencing, apoptosis and cell cycle was determined by flow cytometry. RESULTS: In RT-qPCR, we observed significantly higher expression of CFL1. Higher activity of protein in CLL cells when compared to HVs was detected. Knock-down of CFL1 led to decreased chemotaxis and migration of CLL cells versus cells from HVs. Apoptosis was increased amongst cells with silenced CFL1 and correlated with higher proportion of cells in the G2/M phase. CONCLUSION: Significantly higher expression of CFL1 mRNA in CLL and higher protein activity might indicate high utilization of CFL1 in malignant cells, maintaining their viability, as its inhibition affected viability, cell-cycle progression and motility of leukemia cells.

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells.

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Oral cavity health among cystic fibrosis patients: Literature overview.

Cystic fibrosis is a genetic disorder in which the mutation of the Cystis Fibrosis Transmembrane Conductance Regulator (CFTR) gene that codes the protein forming a chloride channel of epithelial cells results in its distorted functioning. The manifestations of the disorder are mainly observed in the respiratory and digestive system. Accumulation of sticky and thick mucus is the dominant clinical symptom; it leads to chronic infections and gradual tissue destruction. Although cystic fibrosis remains incurable, it is currently feasible to extend patients' life expectancy thanks to modern therapy possibilities. As cystic fibrosis is no longer the domain of pediatricians, health care to CF patients needs to be provided by doctors of various specializations. The multidisciplinary team of doctors should include a dentist aware of specific prevention and treatment needs of this group of patients. It results from the fact that in the course of cystic fibrosis it is possible to observe a variety of changes in the oral cavity environment. The study presents dental issues observed in CF patients and reported in literature. Particular attention was paid to dental caries, mineralization disorders of hard dental tissues, gingivitis and the change in the content and properties of saliva; moreover, prevention and treatment options regarding oral cavity health is this group of patients were taken into consideration.

Analysis of NPM1 splice variants reveals differential expression patterns of prognostic value in acute myeloid leukemia.

Mutations of the nucleophosmin-1 (NPM1) gene in cytogenetically normal (CN) acute myeloid leukemia (AML) identify a group of patients with more favorable prognosis. NPM1 encodes three main alternatively spliced isoforms R1(B23.1), R2(B23.2), and R3(B23.3). The expression of splice variants R1, R2 and R3 were higher in AML patients compared to normal cells of healthy volunteers (HVs), although RNA-seq analysis revealed enhanced R2 expression also in less differentiated cells of HVs as well as in AML cells. The variant R2, which lacks exons 11 and 12 coding for the nucleolar localization domain, might behave similar to the mutant form of NPM1 (NPM1mut). In accordance, in CN-AML high R2 expression was associated with favorable impact on outcome. Moreover, functional studies showed nucleolar localization of the eGFP-NPM1 wildtype and cytoplasmic localization of the eGFP-NPM1 mut protein. While the eGFP-NPM1 R2 splice variant localized predominantly in the nucleoplasm, we also could detect cytoplasmic expression for the R2 variant. These results support a unique biological consequence of R2 overexpression and in part explain our clinical observation, where that high R2 variant expression was associated with a better prognosis in CN-AML patients.

Long-term release of fluoride from fissure sealants-In vitro study.

The intensity of the cariostatic activity of fluoride ions can be attributed to their multidirectional influence on the caries process. They are an irreplaceable factor that helps sustain mineral balance of dental tissues, simultaneously demonstrating antibacterial properties. As a consequence, many manufacturers of fissure sealants include fluoride ions in their products. The aim of this in vitro study was to determine long-term fluoride release from four fissure sealants (Conseal F, Fissurit FX, Delton Fs+, Admira Seal). During a 14-week-long observation, all the materials showed a relatively constant level of F- release; however, it is crucial to mention that within the first 48h, the most significant increase in fluoride release was found for Fissurit and Delton sealants. Based on the overall assessment, the highest total amount of the released fluoride ions was observed for Delton, and the lowest level was reported for Admira Seal.

Assessment of Heat Hazard during the Polymerization of Selected Light-Sensitive Dental Materials.

Introduction. Polymerization of light-cured dental materials used for restoration of hard tooth tissue may lead to an increase in temperature that may have negative consequence for pulp vitality. Aim. The aim of this study was to determine maximum temperatures reached during the polymerization of selected dental materials, as well as the time that is needed for samples of sizes similar to those used in clinical practice to reach these temperatures. Materials and Methods. The study involved four composite restorative materials, one lining material and a dentine bonding agent. The polymerization was conducted with the use of a diode light-curing unit. The measurements of the external surface temperature of the samples were carried out using the Thermovision®550 thermal camera. Results. The examined materials significantly differed in terms of the maximum temperatures values they reached, as well as the time required for reaching the temperatures. A statistically significant positive correlation of the maximum temperature and the sample weight was observed. Conclusions. In clinical practice, it is crucial to bear in mind the risk of thermal damage involved in the application of light-cured materials. It can be reduced by using thin increments of composite materials.

The Effect of File Size and Type and Irrigation Solutions on the Accuracy of Electronic Apex Locators: An In Vitro Study on Canine Teeth.

Measurements of the root canal during endodontic treatment have a significant influence on the course of the therapeutic process as well as on its final result in both human and veterinary medicine. The apical constriction should be the termination point for the preparation and filling of the root canal. This research was conducted with the use of a Septodont kit consisting of a small chamber filled with the examined solution in which a healthy second incisor was placed. The step back method was applied for the root canal preparation and master apical file of 30 was used. The working length was 22 mm. The examination was conducted with the use of steel as well as nickel titanium hand instruments. Different irrigation solutions and two types of apex locators were used. Measurements of the working length of the root canal showed dependence on the size of the instrument. Examinations carried out in various environments showed that analogical measurements were obtained only for sodium hypochlorite solutions. In other environments the measured sections were shortened. Comparative examinations with the use of steel instruments demonstrated insignificant measurement differences. Compared to these results, the measurements in nickel titanium group were characterized by more considerable deviations.

Specific cytotoxic T-cell immune responses against autoantigens recognized by chronic lymphocytic leukaemia cells.

Mounting evidence suggests that autoreactivity and inflammatory processes are involved in the pathogenesis of chronic lymphocytic leukaemia (CLL). Cytoskeletal proteins, including non-muscle myosin heavy chain IIA (MYHIIA), vimentin (VIM) and cofilin-1 (CFL1), exposed on the surface of apoptotic cells have been identified as autoantigens that are recognized by the specific B-cell receptors of the CLL cells. In 212 CLL patients analysed with quantitative reverse transcriptase-polymerase chain reaction we found CFL1 overexpression and low expression of MYH9 in comparison with healthy volunteers. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 66·7%, VIM in 87·5% and CFL1 in 62·5% CLL patients in an Enzyme-Linked ImmunoSpot assay. Low frequencies of autoreactive peptide-specific T cells were detected against MYHIIA, VIM and CFL1 in CLL patients ex vivo; most of the detected cells had an effector-memory phenotype. Our findings support the existence of cytotoxic immune responses against three autoantigens that have been identified as targets of CLL clonotypic B-cell receptors. The presence of autoreactive CD8(+) T cells against MYHIIA, VIM and CFL1 in CLL patients indicates the involvement of antigen-specific autoreactive T cells in the pathogenesis of CLL.

Accumulation of CD5+CD19+ B lymphocytes expressing PD-1 and PD-1L in hypertrophied pharyngeal tonsils.

Programmed death-1 (PD-1) is one of the most important inhibitory co-receptors expressed predominantly on activated T and B lymphocytes whose expression could be sustained by permanent antigenic stimulation accompanying chronic or recurrent tonsillitis. The expression of PD-1 and PD-1L was analyzed using flow cytometry on hypertrophied tonsils collected from 57 children. We observed high expression of PD-1 and PD-1L on certain lymphocytes subpopulations of hypertrophied tonsils; among T cells, the expression of PD-1 on protein level was higher on CD4+ cells (70.3 %) than on CD8+ cells (35 %). Interestingly, a limited expression of PD-1 was observed on CD19+ B lymphocytes (6.5 %), while CD5+CD19+ B cells overexpressed PD-1 (52.5 %). Moreover, the expression of PD-1L was also higher on CD5+CD19+ B cells (16.5 %) than on CD19+ B cells (3.5 %) and on CD4+ T cells (20 %) than on CD8+ T cells (10 %). PD-1 and PD-1L expressions correlated only on CD5+CD19+ cells. In conclusion, high expression of PD-1 and PD-1L on T and B cells could represent hallmark of immune system adaptation to chronic antigenic exposition in patients with tonsillitis.

Expression of Programmed Death 1 Ligand in Different Compartments of Chronic Lymphocytic Leukemia.

BACKGROUND: The programmed death 1 (PD-1) receptor pathway is responsible for the negative regulation of both T and B lymphocytes upon activation of these cells. There is growing evidence that chronic lymphocytic leukemia (CLL) cells exploit the PD-1 ligand (PD-L1) to resist antitumor immune reactions and maintain their survival by shaping their own microenvironment. METHODS: We used a quantitative RT-PCR method to analyze PD-L1 gene expression in bone marrow and peripheral blood mononuclear cells, representing the proliferation and accumulation compartments of CLL. RESULTS: PD-L1 expression was found to be significantly higher in 112 CLL patients than in controls. Levels of PD-L1 expression in bone marrow and peripheral blood were comparable and showed a positive correlation. Furthermore, expression of PDL1 strongly correlated with expression of PD-1 receptor in mononuclear cells from the same compartment, and was not affected by incubation with immunomodulatory drug thalidomide. CONCLUSION: PD-L1 expression is shared between CLL cells localized in distinct disease compartments, demonstrating that PD-1/PD-L1 a universal target for therapy.

Cytotoxic activity of valproic acid on primary chronic lymphocytic leukemia cells.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in western civilization. The accumulation of CD5+CD19+ B lymphocytes in peripheral blood is due to a defect in the apoptotic pathway rather than excessive proliferation in the bone marrow and lymph nodes. Despite a number of treatments, CLL remains an incurable disease. Valproic acid (VPA) activity, as a histone deacetylase inhibitor, could restore the epigenetic changes underlying the pathogenesis of CLL and thus induce cell death. OBJECTIVES: In the present study we hypothesized that VPA could induce CLL primary cells death through activation of apoptosis. MATERIAL AND METHODS: Peripheral blood samples were obtained from 53 CLL patients. Peripheral blood mononuclear cells were isolated through density gradient centrifugation and were the subject of a 24-hour cell culture with 10 mM of VPA. The cytotoxic effect of VPA was evaluated with an XTT test and thereafter confirmed using Annexin V-FITC/PI staining and flow cytometry techniques. RESULTS: In this study, a median VPA cytotoxicity of 13.88% with a range of 0-54.65% was observed. Annexin V/PI staining confirmed that the demonstrated cytotoxicity was caused by increased apoptosis in the VPA treated cells as compared to control cells. Statistical analysis showed that VPA's effect on CLL cells depends on lactate dehydrogenase serum levels, but is independent of all other prognostic markers. CONCLUSIONS: The results of the present experiments found that VPA at a clinically applicable concentration significantly induces apoptosis independently of the disease stage and might be a valuable therapeutic agent for all CLL patients.

The function of a novel immunophenotype candidate molecule PD-1 in chronic lymphocytic leukemia.

Programmed death-1 (PD-1) is a negative receptor expressed on lymphocytes including malignant B cells in chronic lymphocytic leukemia (CLL). In this work, we found that patients with CLL had a higher expression of PD-1 transcript (PDCD1) than healthy volunteers (p < 0.0001). PDCD1 expression was comparable between CLL cells from accumulation (peripheral blood) and proliferation (bone marrow) disease compartments. In blood samples of patients with mutated IGHV genes PDCD1 expression was higher than with unmutated IGHV (p = 0.0299). We demonstrated that phosphorylation of SYK and LYN, key B-cell receptor signaling kinases, was independent of PD-1 expression in patients with CLL, while ZAP-70 phosphorylation in negative tyrosine residue 292 showed strong inverse correlation (r = - 0.8, p = 0.0019). No associations between five single nucleotide polymorphisms of PDCD1, their expressions and susceptibility to CLL were found. In conclusion, PD-1 might be an independent, universal marker of CLL cells and a part of their activated phenotype, and subsequently might modulate the function of ZAP-70.

Celiac disease and its impact on the oral health status - review of the literature.

The paper presents the most recent reports on celiac disease, especially its manifestations within the oral cavity. Particular attention should be paid to typical dental enamel defects with a various degree of advancement: discolorations, horizontal groves and pits, and even significant structural destruction causing the change of the dental crown. Symmetric location of defects within all dentition sections, and within the same anatomic groups of teeth (the most frequently: incisors and first permanent molars), is specific for celiac disease. The changes described above may be the only manifestation of celiac disease; therefore, in the case of their occurrence further studies towards gluten intolerance are recommended even when other symptoms are not present. In celiac patients, recurrent aphthae and other disorders of the oral mucosa such as ulceration, erythema, atrophic glossitis, as well as dryness and a burning sensation (particularly of the tongue) may be present, which may be caused by malnutrition. Delayed tooth eruption may also be a consequence of alimentary deficiency in celiac disease.

The first total laparoscopic pancreatoduodenectomy in Poland.

We present a case of a 55-year-old female patient with pancreatic head cancer who was treated with total laparoscopic pylorus-preserving pancreatoduodenectomy (TLPD) on 13.12.2013. The procedure as well as the postoperative course was uncomplicated. The patient was mobilized on the day of surgery; a liquid diet was introduced on day 1 and a full hospital diet on day 2 postoperatively. Drains were removed on the 3(rd) day after the procedure. Length of hospital stay was 6 days. The final pathology report confirmed the diagnosis of cancer. According to our knowledge this is the first report on total laparoscopic pancreatoduodenectomy in Poland performed by an entirely Polish team of surgeons. In our opinion, TLPD is feasible and similarly to other laparoscopic operations may improve postoperative recovery.

Total expression of HLA-G and TLR-9 in chronic lymphocytic leukemia patients.

Suppressed immune status facilitates immune escape mechanisms that allow chronic lymphocytic leukemia cells to proliferate and expand. The expression of HLA-G could effectively inhibit the immune response. In immune response inhibitory signals follow activation of immune system which might be occur during bacterial or viral infection in CLL patients. In the current study we characterized two components of immune system, inhibitory molecule HLA-G with its receptor - CD85j and Toll-like receptor 9. The material was obtained from 41 CLL patients and 41 HV with similar median age. In CLL patients expression of intracellular and surface HLA-G and soluble HLA-G levels were significantly higher than in HV. We found higher expression of CD85j compared to HV and the positive correlation between expression of HLA-G and CD85j. All the CLL cells expressed TLR-9, and the level of expression positively correlated with expression of HLA-G and CD85j. Patients with higher expression of intracellular expression of TLR-9 have significantly longer treatment-free survival than patients with low expression of TLR-9 (57 months vs. 8 months, respectively). Summarizing in CLL we characterized activatory and inhibitory components of immune system that might be connected functionally. Analysis of TLR-9 expression might have additional prognostic value for CLL patients.