Expansions of tumor-reactive Vdelta1 gamma-delta T cells in newly diagnosed patients with chronic myeloid leukemia.

Recent studies have underscored the importance of gamma-delta (γδ) T cells in mediating potent MHC-unrestricted cytotoxicity in numerous malignancies. Here, we analyzed Vδ1 and Vδ2 γδ T cell subsets in newly diagnosed chronic myeloid leukemia (CML) patients (n = 40) who had initiated tyrosine kinase inhibitor (TKI) therapy including imatinib (n = 22), nilotinib (n = 14) and dasatinib (n = 4). Patient peripheral blood samples were analyzed at diagnosis and monitored prospectively at 3, 6, 12 and 18 months post-TKI. γδ T cells isolated from healthy donors and CML patients were used against K562, LAMA-84 and KYO-1 cell lines and against primary CML cells in cytotoxicity assays. We found large expansions of Vδ1 and Vδ2 T cells in patients at diagnosis compared to age-matched healthy donors (n = 40) (p < 0.0001). The γδ T cell reconstitution in patients on imatinib and also on nilotinib showed significant reductions of Vδ1 T cell and Vδ2 T cell absolute counts at 3 months compared to diagnosis. Importantly, Vδ1 and Vδ2 T absolute cell counts remained at normal levels from 3 months throughout the follow-up. Next, we observed susceptibility to specific lysis of primary CML tumor cells by Vδ1 T cells from healthy donors. Furthermore, we determined inherent cytotoxic reactivity by autologous patients' Vδ1 T lymphocytes against primary CML tumor cells. Finally, the TCR clonality profiles showed in CML patients mostly polyclonal repertoires regardless of the TKI. Our results provide further evidence into γδ T cell antileukemia immunity in CML that might be beneficial for long-term disease control and treatment outcome.

Oxidative Stress in the Brain: Basic Concepts and Treatment Strategies in Stroke.

The production of free radicals is inevitably associated with metabolism and other enzymatic processes. Under physiological conditions, however, free radicals are effectively eliminated by numerous antioxidant mechanisms. Oxidative stress occurs due to an imbalance between the production and elimination of free radicals under pathological conditions. Oxidative stress is also associated with ageing. The brain is prone to oxidative damage because of its high metabolic activity and high vulnerability to ischemic damage. Oxidative stress, thus, plays a major role in the pathophysiology of both acute and chronic pathologies in the brain, such as stroke, traumatic brain injury or neurodegenerative diseases. The goal of this article is to summarize the basic concepts of oxidative stress and its significance in brain pathologies, as well as to discuss treatment strategies for dealing with oxidative stress in stroke.

Experience with cholesteatoma behind an intact tympanic membrane in children.

INTRODUCTION: To systematically investigate all surgeries for cholesteatoma behind an intact tympanic membrane at our department. To identify predictive factors that can help the surgeon to plan surgery, surgical techniques, and follow-up treatment. MATERIAL AND METHODS: This retrospective study evaluates 21 child patients, who were operated in the period 2007-2017 on for cholesteatoma behind an intact tympanic membrane. RESULTS: A total of 202 primary operations were performed for cholesteatoma. In 21 cases (10,4%) there was a cholesteatoma behind an intact tympanic membrane and in 11 (5,45%) cases of it there was the congenital cholesteatoma. The most frequently affected area was the anterior-superior quadrant. The preoperative hearing loss increased significantly with disease severity (I-IV by Potsic). CONCLUSIONS: The classification system according to Potsic is sufficient and fully corresponds to the surgeon's needs. It has been clearly shown that a higher CC stage is associated with worse postoperative hearing results.

Early Inflammatory Response in the Brain and Anesthesia Recovery Time Evaluation After Experimental Subarachnoid Hemorrhage.

The main objective was to evaluate, whether the subarachnoid hemorrhage (SAH)-associated early inflammatory response has focal or global character, i.e., whether areas distant to hematoma may be affected by an early inflammatory response. The second objective was to evaluate the association of anesthesia recovery time for basic reflexes/neurological functions with severity of SAH. SAH was induced in rats using an endovascular perforation model. Anesthesia recovery time was evaluated for pain reaction recovery time (spinal level), spontaneous ventilation recovery time (brain stem level), and consciousness recovery time (neocortical level). mRNA expressions of TNFα, IL-1ß, IL-6, ICAM-1, and VCAM-1 in areas adjacent and distant to hematoma were evaluated between 2 and 8 h after SAH. Serum levels of TNFα, IL-1ß, and IL-6 were assessed at 4 and 8 h after SAH. Anesthesia recovery time of all selected parameters was associated with severity of SAH. The consciousness recovery time test had the best predictive value, while the spontaneous ventilation recovery time test was able to bring information in the shortest time. The mRNA expressions of pro-inflammatory cytokines were significantly increased in severe SAH groups in both adjacent and distant areas. The inflammatory response in mild/moderate SAH groups was less strong, peaking at 4 h after SAH. Serum levels of pro-inflammatory cytokines were ambiguous. Anesthesia recovery time may be useful for bleeding severity prediction in the SAH model; however, further validation is needed. Severe subarachnoid hemorrhage is associated with the strong early inflammatory response, which has a global character, while mild subarachnoid hemorrhage is accompanied by a weaker inflammation.

Cholinergic Anti-inflammatory Pathway and Stroke.

BACKGROUND: Stroke is devastating cerebrovascular event which is responsible for 6.7 million deaths each year worldwide. Inflammation plays an important role in the pathophysiology of stroke. Targeting inflammation after stroke is highly actual topic for both experimental and clinical research. METHODS: Research articles related to cholinergic anti-inflammatory pathway (CHAIP) and stroke were reviewed. The first part of review describes the basic characteristics of inflammatory response after stroke, main components and function of CHAIP. The second part reviews studies focused on CHAIP as a therapeutic target for ischemic and hemorrhagic stroke. Both pharmacological stimulation of α7 nAChR and vagus nerve stimulation after stroke are reviewed. RESULTS: Cholinergic anti-inflammatory pathway (CHAIP) is a physiological mechanism by which central nervous system regulates immune response and controls inflammation. Vagus nerve, spleen and α7 nicotinic acetylcholine receptor (α7 nAChR) are the main components of CHAIP. CONCLUSION: Targeting cholinergic anti-inflammatory pathway is a promising way of immunomodulation which attenuates inflammation in a complex manner without causing immunosuppression.

Depression and Anxiety after Acute Myocardial Infarction Treated by Primary PCI.

AIMS: The main objective of the study was to find out prevalence of depression and anxiety symptoms in the population of patients with AMI with ST-segment elevation (STEMI), treated with primary PCI (pPCI). Secondary target indicators included the incidence of sleep disorders and loss of interest in sex. METHODS AND RESULTS: The project enrolled 79 consecutive patients with the first AMI, aged <80 years (median 61 years, 21.5% of women) with a follow-up period of 12 months. Symptoms of depression or anxiety were measured using the Beck Depression Inventory II tests (BDI-II, cut-off value ≥14) and Self-Rating Anxiety Scale (SAS, cut-off ≥ 45) within 24 hours of pPCI, before the discharge, and in 3, 6 and 12 months). Results with the value p<0.05 were considered as statistically significant. The BDI-II positivity was highest within 24 hours after pPCI (21.5%) with a significant decline prior to the discharge (9.2%), but with a gradual increase in 3, 6 and 12 months (10.4%; 15.4%; 13.8% respectively). The incidence of anxiety showed a relatively similar trend: 8.9% after pPCI, and 4.5%, 10.8% and 6.2% in further follow-up. CONCLUSIONS: Patients with STEMI treated by primary PCI have relatively low overall prevalence of symptoms of depression and anxiety. A significant decrease in mental stress was observed before discharge from the hospital, but in a period of one year after pPCI, prevalence of both symptoms was gradually increasing, which should be given medical attention.

Period3 VNTR polymorphism influences the time-of-day pain onset of acute myocardial infarction with ST elevation.

It is well established that the incidence and infarct size in acute myocardial infarction (AMI) is subject to circadian variations. At the molecular level, circadian clocks in distinct cells, including cardiomyocytes, generate 24-h cycles of biochemical processes. Possible imbalance or impairment in the cell clock mechanism may alter the cardiac metabolism and function and increase the susceptibility of cardiovascular diseases. One of the key components of the human clock system PERIOD3 (PER3) has been recently demonstrated to affect circadian expression of various genes in different tissues, including the heart. The variable number tandem repeat (VNTR) polymorphism (rs57875989) in gene Period3 (Per3) is related to multiple phenotypic parameters, including diurnal preference, sleep homeostasis, infection and cancer. The aim of our study was to investigate the effect of this polymorphism in AMI with ST elevation (STEMI). The study subjects (314 patients of Caucasian origin with STEMI, and 332 healthy controls) were genotyped for Per3 VNTR polymorphism using an allele-specific polymerase chain reaction. A gender difference in circadian rhythmicity of pain onset was observed with significant circadian pattern in men. Furthermore, the Per3(5/5) variant carriers were associated with higher levels of interleukin-6, B-type natriuretic peptide and lower vitamin A levels. By using cosinor analysis we observed different circadian distribution patterns of AMI onset at the level of genotype and allelic frequencies. Genotypes with at least one 4-repeat allele (Per3(4/5) and Per3(4/4)) (N = 264) showed remarkable circadian activity in comparison with Per3(5/5) (N = 50), especially in men. No significant differences in genotype and/or allele frequencies of Per3 VNTR polymorphism were observed when comparing STEMI cases and controls. Our results indicate that the Per3 VNTR may contribute to modulation of cardiac functions and interindividual differences in development and progression of myocardial infarction.

ADMA, SDMA and L-arginine/ADMA ratio but not DDAH genetic polymorphisms are reliable predictors of diabetic nephropathy progression as identified by competing risk analysis.

BACKGROUND/AIMS: Complex interplay of genetic and (patho)physiological factors influence availability of nitric oxide during the development and progression of diabetic complications. We assessed predictive value of commonly studied methylated asymmetric and symmetric dimethylarginines (ADMA and SDMA) and selected single nucleotide polymorphisms (SNPs) in dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 genes for the progression of diabetic nephropathy (DN). METHODS: A total of 341 type 1 and type 2 diabetes patients with variable degree of kidney disease were included at baseline. Plasma levels of ADMA, SDMA and L-arginine were measured and six tagging SNPs in DDAH1 and 2 were determined. Progression of DN was defined as a transition from any given stage to a more advanced stage of albuminuria. Competing risk analysis was applied. RESULTS: Plasma levels of ADMA and SDMA significantly correlated with GFR. No significant genotype-phenotype relationship was ascertained for ADMA and DDAH variants, but SNP rs805304 exhibited marginally significant association with DN. ADMA, SDMA and L-arginine/ADMA ratio standardised to GFR were identified as significant predictors of DN progression but not GFR decline using multivariate competing risk analysis. CONCLUSIONS: In our study we confirmed potentially significant role of ADMA and SDMA for the assessment of risk of DN progression in European diabetic population.

Altered dopamine D1 and D2 receptor mRNA expression in mesencephalon from mice exposed to repeated treatments with methamphetamine and cannabinoid CB1 agonist methanandamide.

OBJECTIVES: In our previous studies we found that both acute administration of CB1 receptor agonist methanandamide and repeated methanandamide pre-treatment prior to methamphetamine challenge dose elicited increase in the CB1 receptor mRNA expression in the mouse mesencephalon. As a reciprocal cross-talk is reported between the cannabinoid CB1 and dopamine receptors, that are highly co-localized on brain neurones, we targeted possible changes in relative expression of dopamine D1 and D2 receptor mRNA in mesencephalon in mice sensitized by repeated treatments to methamphetamine stimulatory effects and cross-sensitized to methamphetamine by cannabinoid CB1 receptor agonist methanandamide pre-treatment. METHODS: To confirm development of behavioural sensitization or cross-sensitization, respectively, we observed changes in locomotion using the open field test. Mice were treated repeatedly with either methamphetamine or methamphetamine after repeated pre-treatment with methanandamide. After each measurement of locomotion one third of animals were sacrificed and the brain was stored. RNA was isolated from the midbrain and used for reverse transcription and subsequent real-time PCR. RESULTS AND CONCLUSION: As in many of our earlier studies with the same dosage regimen we found in the behavioural part both development of sensitization to methamphetamine stimulatory effects after repeated treatment and cross-sensitization to them by pre-treatment with cannabinoid receptor CB1 agonist methanandamide. Real-time PCR analyses showed an increase in D1 receptor mRNA expression after the first dose of methamphetamine (that persisted also after the last dose of methamphetamine) and after the first dose of methanandamide (which also persisted after the methamphetamine challenge dose). In opposite a significant decrease in D2 receptor mRNA expression both after the first dose of methamphetamine and methanandamide (that persisted also after the methamphetamine challenge doses) was registered. Thus, our results suggest that both methamphetmine and methanandamide treatment can provoke changes in dopamine receptor density in mouse mesenpcephalon, the increase in D1 and decrease in D2 receptor subtypes.

Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis.

Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis.

Altered cannabinoid CB1 receptor mRNA expression in mesencephalon from mice exposed to repeated methamphetamine and methanandamide treatments.

OBJECTIVES: Since among others also our previous studies suggested an interaction between the endocannabinoid system and methamphetamine brain mechanisms we focused on possible changes in relative expression of cannabinoid CB1 receptor mRNA in mesencephalon from mice sensitized by repeated treatments to methamphetamine stimulatory effects and cross-sensitized by cannabinoid CB1 receptor agonist methanandamide pre-treatment. METHODS: The Open Field Test was used to measure changes in terms of behavioural sensitization or cross-sensitization to drug effects on locomotion in male mice treated repeatedly with either methamphetamine or methamphetamine after pre-treatment with methanandamide. After each measurement one third of animals were sacrificed and the brain was stored. RNA was isolated from the midbrain and used for reverse transcription and subsequent real-time PCR. RESULTS AND CONCLUSION: The evaluation of behavioural drug effects showed both development of sensitization to methamphetamine stimulatory effects after repeated treatment and cross-sensitization to them by pre-treatment with cannabinoid receptor CB1 agonist methanandamide. Real-time PCR analyses revealed an increase in CB1 receptor mRNA expression after the first dose of methanandamide followed by decrease after the combined treatment with methamphetamine challenge dose. Our findings suggest that particularly repeated pre-treatment with CB1 agonist methanandamide can elicit increase in the mRNA expression level at least in the mouse mesencephalon neurons associated with cross-sensitization to methamphetamine stimulatory effects.

Haplotype analysis of the endothelial nitric oxide synthase gene in asthma.

Nitric oxide (NO) is an important mediator of physiologic processes in the airways. Evidence exists that genetic factors affect NO formation and contribute to the pathophysiology of asthma. The aims of this study were to determine the endothelial NO synthase (eNOS) haplotypes in Czech asthmatics and control subjects and examine their relation to asthma. We analyzed a total of six polymorphisms. Two SNPs in the promoter (C-786T and C-691T), two variants in the introns (27-bp repeat in intron 4 and G11T in intron 23), and two others in the exons (C774T in exon 6 and G894T in exon 7) were genotyped in 610 subjects (asthma, n = 294; healthy controls, n = 316), and a case-control association study was conducted. No significant differences in allele or genotype frequencies for individual polymorphisms were observed between patients with asthma and controls after correction for multiple comparisons. Nevertheless, a G to T exchange in intron 23 was related with specific sensitization for feather (p = 0.008, p(corr) < 0.05). However, the common haplotype -786T/-691C/27-bp 5 repeat variant/774C/894G/11T was associated with lower risk of asthma (p = 0.001, p(corr) < 0.05, odds ratio = 0.58, 95% confidence interval = 0.46-0.73). These findings suggest that endothelial NOS variants may be one of the factors participating in protection or susceptibility to asthma in our population.

C766T low-density lipoprotein receptor-related protein 1 (LRP1) gene polymorphism and susceptibility to breast cancer.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor with an important role in regulating the activity of proteinases in extracellular matrix. Several studies have also described its role in intracellular signaling. Previous studies showed that the expression of LRP1 is related to invasiveness of cancer cells. However, recent data on LRP1 suggest that this receptor can also be involved in tumor establishment and progression. METHODS: We investigated an association between the C766T polymorphism of the third exon of the LRP1 gene and breast cancer in a sample of women of Caucasian origin. Allele and genotype frequencies of this polymorphism were assessed in 164 women with breast cancer and in 183 age-compatible women without a history of any cancer disease. RESULTS: An increase in LRP1 T allele frequency in subjects with breast cancer was observed compared with controls (0.21 versus 0.15, P = 0.01963). A significant excess of genotypes with the T allele (homozygotes plus heterozygotes) was also observed (odds ratio 1.743, 95% confidence interval 1.112-2.732). CONCLUSION: The T allele of the C766T polymorphism in the LRP1 gene is associated with an increased risk of breast cancer development in women of Caucasian origin.