[The assessment of risk factors for glucose metabolism disorders]. / The assessment of risk factors for glucose metabolism disorders.

AIM: The aim of the study was to assess risk factors for glucose metabolism disorders. MATERIALS AND METHODS: The study involved 197 people, 92 women and 105 men aged 63.21±8.74 years. In order to assess glucose metabolism disorders, patients were divided into three groups. The first group (DM) consisted of 60 people (16 women and 44 men aged 61.92±8.46 years). These were people with type 2 diabetes. Second group (IFG IGT) consisted of 67 people (35 women and 32 men aged 65±8.5 years). These were people who were diagnosed with impaired fasting glucose or impaired glucose tolerance. The third group, the control one (K) consisted of 70 people (41 women and 29 men aged 62.6±9.06 years). They were healthy individuals. Age, BMI, blood pressure, heart rate, pulse pressure, lipids in the blood serum, serum creatinine, glomerular filtration rate (GFR), the number of pack-years were assessed. Each patient responded to diabetes risk questionnaire. RESULTS: Univariate analyzes showed statistically significant differences between groups due to: systolic blood pressure, heart rate, pulse pressure, total cholesterol, LDL cholesterol, HDL cholesterol, the number of points obtained in diabetes risk questionnaire, serum creatinine and GFR. Multivariate analyzes demonstrated that in IFG IGT group independent risk factors that increased the risk of prediabetes were elevated levels of total cholesterol (OR 1,945, 95%CI [1,195-3,166], p=0,007) and the result of the diabetes risk questionnaire (OR 1,191, 95%CI [1,061-1,337], p=0,003). In diabetes mellitus (DM) group independent risk factors that increased the risk of diabetes were pulse pressure (OR 1,123, 95%CI [1,063-1,188], p<0,001) and the result of the diabetes risk questionnaire. Independent risk factor that lowered the risk of diabetes (OR 0,068, 95%CI [0,018-0,251], p<0,001) and prediabetes (OR 0,324, 95%CI [0,109-0,969], p=0,044) was higher serum HDL cholesterol. CONCLUSIONS: Elevated level of total cholesterol and the result of the diabetes risk questionnaire are independent risk factors that increase the risk of prediabetes. Puls pressure and the result of the diabetes risk questionnaire are independent risk factors that increase the risk of diabetes. Independent risk factor that lowers the risk of glucose metabolism disorders is higher serum HDL cholesterol.

[The evaluation of color vision and its diagnostic value in predicting the risk of diabetic retinopathy in patients with glucose metabolism disorders]. / Ocena widzenia barw i jej wartosc diagnostyczna w przewidywaniu ryzyka wystapienia retinopatii cukrzycowej u chorych z zaburzeniami gospodarki weglowodanowej.

AIM: The aim of the study was to evaluate color vision and its diagnostic value in predicting the risk of diabetic retinopathy in patients with glucose metabolism disorders. MATERIALS AND METHODS: The study involved 197 people, 92 women and 105 men aged 63.21 ± 8.74 years. In order to assess glucose metabolism disorders, patients were divided into three groups. The first group (DM) consisted of 60 people (16 women and 44 men aged 61.92 ± 8.46 years). These were people with type 2 diabetes. Second group (IFG IGT) consisted of 67 people (35 women and 32 men aged 65 ± 8.5 years). These were people who were diagnosed with impaired fasting glucose or impaired glucose tolerance. The third group, the control one (K) consisted of 70 people (41 women and 29 men aged 62.6 ± 9.06 years). They were healthy individuals. In order to assess diabetic retinopathy study population was divided into two groups. The first group (BZ) consisted of 177 patients (84 women and 93 men aged 62.9 ± 8.78 years) without diabetic retinopathy. The second group (NPDR) consisted of 20 patients (8 women and 12 men aged 65.95 ± 8.17 years) with diabetic retinopathy. Glucose metabolism disorders were diagnosed with glucose tolerance test (OGTT). Evaluation of retinopathy was based on eye examination. All patients underwent binocular Farnsworth-Munsell 100 Hue color vision test (test result is a Total Error Score - TES). RESULTS: In the healthy control group (K) there were less patients with diabetic retinopathy (p = 0,0101), and less patients with abnormal color vision test (p = 0,0001) than in other groups. Majority of patients in K group had generalized abnormalities of color vision while other groups demonstrated tritanomalia (p = 0,0018). It was discovered that sTES value adequately distinguishes group K from group IFG, IGT, DM (AUC = 0,673), group K from group DM (AUC = 0,701), and group K from group IFG IGT (AUC = 0,648) sTES does not differentiate groups IGT, IFG and DM (AUC = 0,563). It was shown that in IGT, IFG group sTES was an independent risk factor that increased the risk of prediabetes almost six times (OR 5,924, 95% CI [1,169- 30,036], p = 0,032). In DM group sTES was an independent risk factor, which increased the risk of diabetes more than thirteen times (OR 13,408, 95% CI [2,324-77,345], p = 0,004). Higher values of sTES occurred in patients with retinopathy (NPDR) (p <0,001) and multivariate analyzes demonstrated that sTES ≥1.04 was an independent risk factor that increased the risk of retinopathy over twenty times (OR 21,380, 95% CI [1,101 -89,241], p <0,001). CONCLUSIONS: sTES is an independent risk factor for glucose metabolism disorders that increases the risk of prediabetes almost six times and the risk of diabetes more than thirteen times. sTES ≥1,04 is an independent risk factor for diabetic retinopathy. sTES ≥1,04 increases the risk of diabetic retinopathy over twenty times. The color vision 100 Hue test can be useful in detecting glucose metabolism disorders even before the ophthalmoscopic manifestation of retinopathy. Early detection rises the possibility to prevent or delay the development of diabetes through lifestyle changes and implementation of healthy behaviors.

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.

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.

[Does acetylsalicylic acid and vitamin K antagonists are risk factors of macular degeneration related with age?]. / Czy kwas acetylosalicylowy i antagonisci witaminy K sa czynnikami ryzyka wystapienia zwyrodnienia plamki zóltej zwiazanego z wiekiem?

AIM: The aim of this study was to evaluate the effects of chronic use of acetylsalicylic acid (ASA) and vitamin K antagonists (VKA) on the incidence of age related macular degeneration (AMD). MATERIALS AND METHODS: The study included 292 individuals (187 women, 105 men, aged 45-94 (mean 73.2 ± 10.2 years). All individuals completed a survey and underwent a full eye examination. Patients were divided into four groups according to the presence or absence of degenerative changes at the bottom of an eye: group D - 80 patients with drusen (23 men and 57 women), 27,4% of studied population, group GA - 25 patients with geographic atrophy (10 men, 15 women), 8,56% of studied population, group CNV - 52 patients with neovascular form of AMD (28 men, 24 women), 17,81% of studied population, group Z - 135 healthy people (44 men, 91 women), 46,23% of studied population. Among study group 79 patients (27,1%) used aspirin, 32 people (11%) used vitamin K antagonists (acenocoumarol or warfarin), 181 people (61,9%) didn't use any drug. RESULTS: Patients from D and CNV group took ASA and VKA more often than patients from GA and Z group. The percentages were: in a group Z - 30,37%, in group D - 46,25%, in group GA - 32%, in group CNV - 48,08% (p=0,0407). There was no relationship between belonging to a group and use of ASA (p=0,3169). A statistically significant relationship between belonging to a group and use of VKA was discovered. The number of people using VKA in group D and CNV was statistically significantly higher than in the healthy control group and percentage were as follows: in group Z - 8,15%, in group D - 17,5%, in group GA 0%, in group CNV - 13,46% (p=0,0159). Patient groups differed statistically significantly due to age (p=0,0043), sex (p=0,0197), family history of macular diseases (p <0,0001), smoking (p=0,011), prevalence of hypercholesterolemia (p=0,0437), ischemic heart disease (p= 0,0173). The consumption of fish at least once a week and eating fruits and vegetables more often than once a day was associated with a reduced incidence of AMD p=0,0009, p=0,0003. Patients without AMD assessed their quality of life at a higher level than people with AMD (p<0,0001). ASA and VKA intake was found not to be an independent risk factor for AMD. Positive family history was an independent risk factor for AMD in all groups. Also age ≥75, fish consumption > 1 week, male gender were independent risk factors for AMD in specified groups. CONCLUSIONS: Patients with drusen and exudative form of AMD took ASA and VKA more often than healthy people and patients with geographic atrophy. ASA or VKA intake was found not to be an independent risk factor for AMD. Positive family history was an independent risk factor for AMD in all groups. In selected groups: age ≥75, male gender and reduced consumption of fish was found to be an independent risk factor for AMD. Number of people taking ASA and VKA is increasing and further studies are needed to assess their impact on the organ of vision.

T-Cell Engagers-The Structure and Functional Principle and Application in Hematological Malignancies.

Recent advancements in cancer immunotherapy have made directing the cellular immune response onto cancer cells a promising strategy for the treatment of hematological malignancies. The introduction of monoclonal antibody-based (mAbs) targeted therapy has significantly improved the prognosis for hematological patients. Facing the issues of mAb-based therapies, a novel bispecific antibody (BsAb) format was developed. T-cell engagers (TCEs) are BsAbs, which simultaneously target tumor-associated antigens on tumor cells and CD3 molecules present on T-cells. This mechanism allows for the direct activation of T-cells and their anti-tumor features, ultimately resulting in the lysis of tumor cells. In 2014, the FDA approved blinatumomab, a TCE directed to CD3 and CD19 for treatment of acute lymphoblastic leukemia. Since then, numerous TCEs have been developed, allowing for treating different hematological malignancies such as acute myeloid leukemia, multiple myeloma, and non-Hodgkin lymphoma and Hodgkin lymphoma. As of November 2023, seven clinically approved TCE therapies are on the market. TCE-based therapies still have their limitations; however, improving the properties of TCEs, as well as combining TCE-based therapies with other forms of treatment, give hope to find the cures for currently terminal diseases. In this paper, we summarized the technical basis of the TCE technology, its application in hematology, and its current issues and prospects.

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.

Aberrant Expression of TLR2, TLR7, TLR9, Splicing Variants of TLR4 and MYD88 in Chronic Lymphocytic Leukemia Patients.

Functional toll-like receptors (TLRs) could modulate anti-tumor effects by activating inflammatory cytokines and the cytotoxic T-cells response. However, excessive TLR expression could promote tumor progression, since TLR-induced inflammation might stimulate cancer cells expansion into the microenvironment. Myd88 is involved in activation NF-κB through TLRs downstream signaling, hence in the current study we provided, for the first time, a complex characterization of expression of TLR2, TLR4, TLR7, TLR9, and MYD88 as well as their splicing forms in two distinct compartments of the microenvironment of chronic lymphocytic leukemia (CLL): peripheral blood and bone marrow. We found correlations between MYD88 and TLRs expressions in both compartments, indicating their relevant cooperation in CLL. The MYD88 expression was higher in CLL patients compared to healthy volunteers (HVs) (0.1780 vs. 0.128, p < 0.0001). The TLRs expression was aberrant in CLL compared to HVs. Analysis of survival curves revealed a shorter time to first treatment in the group of patients with low level of TLR4(3) expression compared to high level of TLR4(3) expression in bone marrow (13 months vs. 48 months, p = 0.0207). We suggest that TLRs expression is differentially regulated in CLL but is similarly shared between two distinct compartments of the microenvironment.

The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia.

PURPOSE: NOTCH1 mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1 mut allelic burden. This study compared two methods of the NOTCH1 mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients. PATIENTS AND METHODS: This study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann-Whitney, Fisher's exact test, Kruskal-Wallis, Kaplan-Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data. RESULTS: We proved that ddPCR increased the detectability of the NOTCH1 mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1 mut group of patients compared to the NOTCH1 wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHV mut NOTCH1 wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1 mut determined by ddPCR constituted as a dependent prognostic factor for TTFT. CONCLUSION: The selection of the precise method of NOTCH1 mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1 mut irrespective of the quantity of allelic burden.

The Application of CAR-T Cells in Haematological Malignancies.

Chimeric antigen receptor (CAR)-T cells (CART) remain one of the most advanced and promising forms of adoptive T-cell immunotherapy. CART represent autologous, genetically engineered T lymphocytes expressing CAR, i.e. fusion proteins that combine components and features of T cells as well as antibodies providing their more effective and direct anti-tumour effect. The technology of CART construction is highly advanced in vitro and every element of their structure influence their mechanism of action in vivo. Patients with haematological malignancies are faced with the possibility of disease relapse after the implementation of conventional chemo-immunotherapy. Since the most preferable result of therapy is a partial or complete remission, cancer treatment regimens are constantly being improved and customized to individual patients. This individualization could be ensured by CART therapy. This paper characterized CART strategy in details in terms of their structure, generations, mechanism of action and published the results of clinical trials in haematological malignancies including acute lymphoblastic leukaemia, diffuse large B-cell lymphoma, chronic lymphocytic leukaemia and multiple myeloma.

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.

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.

Thalidomide regulation of NF-κB proteins limits Tregs activity in chronic lymphocytic leukemia.

BACKGROUND: Thalidomide may represent a novel therapeutic strategy in the treatment of chronic lymphocytic leukemia (CLL). Since the activation of nuclear factor kappa B (NF-κB) causes not only malignant transformation and tumor progression, but also allows tumor cells to evade immune surveillance, NF-κB signaling components might constitute a potential target for future therapy in CLL. OBJECTIVES: The current study is an attempt to characterize proteins regulated by thalidomide. Thalidomide's influence on NF-κB proteins and on regulatory T cells (Treg) in CLL was investigated. MATERIAL AND METHODS: A total of 15 patients with CLL were treated with a combined thalidomide/fludarabine regimen. Peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation. To evaluate glucocorticoid induced tumour-necrosis-factor-receptor-related protein (GITR) expression in regulatory T cells, cells incubated with anti-CD3, ani-CD4 and anti-CD25 were permeabilized and then stained with anti-FOXP3 and analyzed using flow cytometry. Human TNF enzyme-linked immunosorbent assay (ELISA) was used to determine the tumor necrosis factor (TNF) levels in the serum. To evaluate NF-κB activity, chemiluminescent oligonucleotide-based ELISA was performed. RESULTS: It was found that thalidomide regulates NF-κB activity differentially, and the activity of certain NF-κB components correlated with TNF levels and T regulatory cell (CD4 + CD25 high GITR + ). CONCLUSIONS: These results might indicate that thalidomide not only regulates TNF but also directly interferes with NF-κB components.