Whole blood samples for faster real-time PCR analysis of thrombophilic mutations in SARS-CoV-2 virus positive patients.

High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.

No Inhibitory Effect of Heparinized Blood on Real-Time PCR Analysis of Thrombophilic Mutations.

We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalatetreated samples: 18.5 and 18.9, respectively. Significantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.

C-reactive protein, chemerin, fetuin-A and osteopontin as predictors of cardiovascular risks in persons with psoriasis vulgaris.

The study aimed to contribute to understanding the role of CRP, chemerin, fetuin-A and osteopontin and to assess their suitability as biomarkers of early stages of cardiovascular diseases in psoriasis vulgaris. Serum levels measured in 28 patients and 22 controls. Patients: increased levels of CRP (p<0.001), chemerin (p<0.05), osteopontin (p<0.05) and decreased levels of fetuin-A (p<0.05), significant relationships between CRP and fetuin-A (rho=0.530, p<0.01), CRP and chemerin (rho=0.543, p<0.01), CRP and age (rho=0.590, p<0.001), osteopontin and fetuin-A (r=-0.415, p<0.05), chemerin and PASI score (rho=-0.424, p<0.05). We confirmed specific roles of the biomarkers in psoriasis. CRP, fetuin-A and osteopontin could be considered appropriate markers for the detection of early stages of cardiovascular diseases.

Variation of selected genotoxic and epigenetic markers due to therapeutic exposure to PAHs and ultraviolet radiation.

BACKGROUND: Goeckerman therapy (GT) of psoriasis involves dermal application of crude coal tar containing polycyclic aromatic hydrocarbons (PAHs) and exposure to ultraviolet radiation (UVR). Little is known about GT influence on DNA epigenetics. OBJECTIVE: The study aim was to discover epigenetic mechanisms altered by the exposure related to the GT of psoriasis. METHODS: Observed group of patients with plaque psoriasis (n = 23) was treated by GT with 3 % CCT. Before and after GT, we analyzed the levels of benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts (BPDE-DNA), p53 protein in serum, 5-methylcytosine (5-mC, global DNA methylation), and methylation in selected CpG sites of p53 gene. RESULTS: We found a significant increase in the levels of BPDE-DNA (p < 0.01) and serum levels of p53 protein (p < 0.01) after GT, and an insignificant decrease in the percentage of 5-mC in peripheral blood DNA. Methylation of p53 CpG sites was affected neither by psoriasis nor by GT. The study confirmed good effectiveness of GT (significantly reduced psoriasis area and severity index; p < 0.001). CONCLUSION: Our findings indicate that there is a significantly increased genotoxic hazard related to the exposure of PAHs and UV radiation after GT of psoriasis. However, global DNA methylation and p53 gene methylation evade the effect of GT, as they remained unchanged (Tab. 4, Fig. 3, Ref. 50).

Incidence of venous thromboembolism in patients with colorectal cancer according to oncogenic status.

BACKGROUND: There are conflicting data regarding the role of KRAS mutation on the risk of venous thromboembolism (VTE) in colorectal cancer (CRC) patients. Moreover, the role of other biomarkers such as NRAS or BRAF has not been studied. PURPOSE: To analyze the incidence of VTE in a cohort of patients with CRC based on KRAS, NRAS, and BRAF status. METHODS: We performed a retrospective review of patients with unresectable locally advanced and metastatic CRC (mCRC) and known KRAS/NRAS/BRAF status, attended in the Medical Oncology Department of the Hospital General Universitario Gregorio Marañón (Madrid, Spain). The primary outcome was VTE defined as any venous thromboembolic event that occurred either 6 months before or at any time after the diagnosis of CRC. The biomarker status (KRAS, NRAS, and BRAF) and other predictors of thrombosis were collected. RESULTS: One hundred and ninety-four patients were identified and included in the analysis. Forty-one patients (21.1%) experienced VTE. The incidence was 19.1% in RAS-mutated patients, 28.6% in BRAF-mutated patients and 21% in triple wild-type patients (p = NS). In multivariate analysis, ECOG ≥ 2 was the only independent predictor of VTE (OR 8.73; CI 95% 1.32-57.82; p = 0.025). CONCLUSIONS: In our study, biomarkers have not been associated with an increased risk of VTE in CRC patients. A high incidence of VTE in BRAF-mutated patients has been observed and should be explored in further studies.

Serum Levels of Aryl Hydrocarbon Receptor, Cytochromes P450 1A1 and 1B1 in Patients with Exacerbated Psoriasis Vulgaris.

The aryl hydrocarbon receptor (AhR) is highly expressed in psoriasis skin lesions. The aim of this study was to investigate serum concentrations of AhR, cytochromes P450 (CYP) 1A1 and 1B1 in patients with exacerbated psoriasis vulgaris treated with combined therapy of ultraviolet radiation (UVR) and crude coal tar. The analyses were performed by using enzyme-linked immunosorbent assays. Before the treatment, the patients had significantly higher serum levels of AhR and CYP1A1 than healthy controls. AhR median noticeably decreased after the therapy; nevertheless, it remained significantly higher compared to the controls. CYP1A1 levels measured before and after the therapy did not differ significantly. Serum CYP1A1 positively correlated with AhR values before and after the treatment. The serum values of CYP1B1 were very low and we did not see any differences between the study group and the control group. The study demonstrated that serum levels of AhR and CYP1A1 could indicate their immunopathological and metabolic roles in exacerbated psoriasis.

Roles of miR-31 and endothelin-1 in psoriasis vulgaris: pathophysiological functions and potential biomarkers.

Psoriatic lesions are characterized by hyperproliferation, aberrant differentiation of keratinocytes resistant to apoptosis and inflammation. miR-31 plays pro-proliferative, pro-differentiative and pro-inflammatory roles and modulates apoptosis in psoriatic keratinocytes. Endothelin-1 (ET-1) is produced by psoriatic keratinocytes and suppresses apoptosis. Inflammation increases the production of ET-1, which in turn leads to the chronic stimulation of keratinocyte proliferation. The aim of this study was to identify the putative link between two potential biomarkers (miR-31 and ET-1) in patients with psoriasis. The study design included experimental group (29 patients with psoriasis), and the control group (22 blood donors). The PASI score evaluated the state of the disease (median: 18.6; interquartile range 14.5-20.9). Both, the serum level of ET-1 and the whole blood level of miR-31 were significantly increased (p<0.001 and p<0.05, respectively) in patients compared to the controls. However, a significant negative relationship between ET-1 and miR-31 was observed (Spearman's rho=-037, p=0.05). It is possible that a negative feedback loop will be present between miR-31 and ET-1. Our results indicate that miR-31 and ET-1, potential biomarkers of the disease, play significant roles in the pathophysiology of psoriasis.

Preparing Triple-Compound Heterozygous Control Material for Molecular Diagnostics of TPMT Allelic Variants.

The aim of the study is to present a novel approach for preparing triple-compound heterozygous reference material (TCH-RM) for thiopurine S-methyltransferase (TPMT) genotyping by using the gene synthesis technology. The polynucleotide chain we prepared consisted of three wild-type and three mutant segments corresponding to the TPMT 238G>C, 460G>A, and 719A>G polymorphic sites. TCH-RM characteristics were assessed via four methods: reverse hybridization, real-time PCR with hydrolysis probes, real-time PCR followed by subsequent melting temperature analysis, and DNA sequencing. Consequently, we investigated the TPMT genotype of 371 patients suffering from autoimmune diseases requiring immunosuppressive therapy with thiopurine drugs, mostly inflammatory bowel disease. All methods confirmed the triple heterozygous character and commutability of TCH-RM. In evaluating its stability we obtained very comparable data before and after six months of storage at -80 °C. The determined genotypes were as follows: 352 wild-type subjects (94.8%), 17 TPMT*3A heterozygotes (460G>A and 719A>G, 4.6%), one patient heterozygous for the TPMT*2 allele (238G>C, 0.3%), and one TPMT*3C heterozygote (719A>G, 0.3%). The frequencies of TPMT*1, *3A, *3C, and *2 in the patients were 97.5%, 2.3%, 0.1%, and 0.1 %, respectively. Assembling segments of synthetic DNA into long polynucleotide chains is a universal way of obtaining compound heterozygous material for performing any simultaneous analysis of polymorphic sites in the human genome. The batches are manufactured with a perfect concentration match of wildtype and mutant fragments, and can be made in large quantities for most diagnostic techniques.

Dopamine 2 and somatostatin 1-5 receptors coexpression in clinically non-functioning pituitary adenomas.

This study investigated quantitated expression of dopamine 2 receptor (D2R) and somatostatin receptors of the five types (SSTR1-SSTR5) in a large series of clinically non-functioning pituitary adenomas (CNFAs). Co-expression of these receptors in individual adenomas was studied as well as correlation between receptor types. Adenoma tissue from 198 patients who underwent surgery for CNFAs was analyzed by immunohistochemistry and quantitative real-time PCR. D2R and SSTR1-3 mRNA was expressed in all 198 adenomas. SSTR4 and SSTR5 were detectable in 85 % and 61 % of adenomas, respectively. Expression of D2R was significantly higher than that of the somatostatin receptors. The median relative expressions were as follows from highest D2R >> SSTR3 > SSTR2 > SSTR1 > SSTR5 > SSTR4. High relative expression (ratio to beta-glucuronidase mRNA > 1) of D2R was found in 60 % of tumors, high expression of SSTR1 in 7.5 %, SSTR2 in 7 %, SSTR3 in 4 % and SSTR5 in 0.5 %. The quantity of D2R correlated positively with expression of SSTR2 and SSTR3, and negatively with SSTR1 and SSTR5. Among histological adenoma types, SSTR1 was significantly higher in null-cell adenomas and SSTR3 was lower in silent corticotroph adenomas. In conclusions, in CNFAs, high expression of somatostatin receptors is much less common than that of D2R, and co-expression of both these receptors is exceptional. D2R and SSTR3 seem to be the most promising targets for pharmacological treatment.

ATM and TGFB1 genes polymorphisms in prediction of late complications of chemoradiotherapy in patients with locally advanced cervical cancer.

The purpose of our study was to evaluate a possible correlation between genetic polymorphisms in ATM and TGFB1 genes and late toxicity of chemoradiotherapy for locally advanced cervical cancer. Fifty five patients with FIGO stage IIB and higher without a disease recurrence with a mean follow up of 6 years were included. Late toxicity was assessed by EORTC/RTOG late toxicity criteria. Univariate and multivariate logistic regression model was used for statistical analysis. Degree of association between polymorphisms and late toxicity of chemotherapy was assessed on the basis of phi-coefficient (φ) as well. We did not find any association between 5557G>A polymorphism in the ATM gene or single TGFB1 polymorphisms and late toxicity. TGFB1 compound homozygosity (-1552delAGG, -509C>T, L10P) was a significant predictive factor of grade III-IV and any grade of complications in both univariate and multivariate logistic regression analyses and statistical significance of association between polymorphisms and late toxicity of chemoradiotherapy was confirmed also by the evaluation of phi-coefficient (φ). We conclude that haplotypes instead of single nucleotide polymorphic sites in the genes may better characterize the individual radiosensitivity.

[Use of natriuretic peptides in diagnosis of left ventricular hypertrophy in obese hypertensive patients with metabolic syndrome]. / Vyuzití natriuretických peptidu v dia-gnostice hypertrofie levé komory srdecní u obézních hypertoniku s metabolickým syndromem.

INTRODUCTION: Presence of left ventricular (LV) hypertrophy significantly increases cardiovascular risk in patients suffering from hypertension. Diagnostics of LV hypertrophy in hypertensive patients is not easy and there is still no method of enabling a simple and sufficiently sensitive dia-gnosis across a large patient population. The golden standard in LV hypertrophy diagnostics is echocardiography, and there are adverse opinions regarding the use of natriuretic peptides BNP and NT proBNP (NP) to diagnose LV hypertrophy. PATIENTS AND METHODS: We examined through echocardiography 173 hypertensive patients with signs of metabolic syndrome and a moderate increase in blood pressure (130- 159/ 85- 99 mm Hg) with an average age of 54.8 ± 13.54 years, i.e. 119 men and 54 women, who were divided into 2 groups; 1 with BMI > 30 (group A with a severe obesity) and the other without obesity, BMI < 30 (group B). Both groups were examined for BNP and NT proBNP levels. RESULTS: We found a positive correlation between NP and LVMi, both for BNP (r = 0.169; p = 0.033) and for NT proBNP (r = 0.240; p = 0.002). NT proBNP statistically significantly predicts the given LV hypertrophy LK in people with BMI < 30 but not in obese people (BMI > 30). CONCLUSION: Obese patients suffer from a higher occurrence of left ventricular hypertrophy and paradoxically a lower NP value than patients with a metabolic syndrome (MS) who are not obese. Natriuretic peptides have a limited diagnostic value when assessing left ventricular hypertrophy. They are only of value in patients who are not obese and whose kidney function and systolic myocardial function have not been impaired.

Methylation in the p53 promoter in epithelial ovarian cancer.

OBJECTIVE: Ovarian cancer is a leading cause of death from gynecologic tumors, however, the molecular and especially epigenetic events underlying this transformation are poorly understood. Promoter methylation status of tumor suppressor genes may be associated with transcriptional silencing and tumor progression. It has been shown that methylation of CpG dinucleotides located in the promoter region of p53 is associated with low expression levels of this gene. The aim of this study was to investigate promoter methylation of p53 gene in ovarian cancer by comparison with normal ovarian tissue. METHODS: To search for promoter methylation of p53 gene we used methylation-specific PCR (MSP) to compare the methylation status of 66 tissue samples of ovarian cancer with 37 control samples. RESULTS: In our study methylation specific PCR revealed p53 promoter methylation in 34 of 66 (51.5 %) of specimens with ovarian cancer. CONCLUSION: These results indicate that methylation in p53 promoter region may play an important role in carcinogenesis of ovarian cancer and could potentially be used in screening of ovarian cancer, and may have implications for future chemotherapy based on epigenetic changes.

The purple toe syndrome in female with Factor V Leiden mutation successfully treated with enoxaparin.

Purple toe syndrome is a rare complication of warfarin therapy. It occurs usually after 3 to 8 weeks of therapy and it is caused by cholesterol emboli from atheromatous plaque. Sudden onset of pain in affected area, typically in toes and feet, is the main characteristic of the syndrome. We describe a case of a 65-year-old female with purple toe syndrome after 6 weeks of warfarin. Indication of warfarin was a proximal deep venous thrombosis, which developed after prolonged immobilization. Factor V (FV) Leiden and persistent high FVIII activity were found as additional eliciting factors for venous thromboembolism. After warfarin withdrawal and enoxaparin treatment, symptoms disappeared promptly but a slight discoloration of the toe persists.

Molecular predictive factors of outcome of radiotherapy in cervical cancer.

Radical radiotherapy with concurrent cisplatin-based chemotherapy is an established treatment for cervical cancer patients with stage FIGO IIB and higher. The tumor control can be achieved in 40-80% of patients, the treatment is associated with the risk of late postiradiation complications in 10 - 15% of cases. Detection of the factors predictive for tumor control and late morbidity is a possible direction how to individualize radiotherapy dose and technique. The aim of our review is to summarize results of studies inquiring various molecular markers predicting tumor response to radiotherapy and a risk of late complications. A lot of candidate molecules were evaluated in histochemical studies: membrane receptors (EGFR, HER-2), cell cycle regulators (p53, p21), proliferative markers (Ki-67), hypoxia and angiogenetic factors (HIF, VEGF), HPV status, and others (COX-2), with promising results in some of them (HPV, HIF-1α, Ku80, ATM polymorphism). Microarray studies identified decades of genes with different expression in radiosensitive/radioresistant cervical tumors and sets of genes are able to comletely separate responding and nonresponding tumors, but these sets differ across studies. Further well designed studies will be necessary to achieve results matured for use in clinical practice.

Steady-state of azathioprine during initiation treatment of pediatric inflammatory bowel disease.

BACKGROUND AND AIM: Azathioprine (AZA) has a slow onset of action in treatment of pediatric inflammatory bowel disease (IBD). It is anticipated, that this delay correlates to the kinetics of 6-thioguanine nucleotides (6-TGN) accumulation. The aim of this study was to evaluate the time to steady state of 6-TGN concentration in red blood cells. METHODS: The inclusion criteria were: a) age 0-19 years b) IBD diagnosis c) AZA treatment initiation. High performance liquid chromatography was used for the 6-TGN analysis. Concentrations of metabolites were studied in weeks 0, 1, 2, 5, and 8 after beginning of treatment. RESULTS: The inclusion criteria were matched to 18 patients with IBD. The median time to steady state of 6-TGN was 55.3 days. The mean 6-TGN concentration at the steady state achieved 326 (SD 154) pmol/8.108 erythrocytes. High erythrocyte TPMT activity corresponds to the low steady state 6-TGN concentration and vice versa. This correlation reached statistical significance (p<0.01) for the dose expressed in mg per square meter of body surface area. CONCLUSION: The time to steady state of 6-TGN erythrocyte concentration is significantly shorter than would expected according to clinical observation describe earlier.

Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis.

BACKGROUND: Matrix metalloproteinases are notable contributors to neuroinflammation and blood-brain barrier disruption in multiple sclerosis (MS). OBJECTIVE: The goal of this study was to determine the serum levels of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-2 (MMP-2), and their tissue inhibitors (TIMP-1) and (TIMP-2), and to investigate their possible relations to type, disability, and severity of MS. MATERIALS AND METHODS: Eighty-seven patients with definite MS according to the McDonald criteria and 50 healthy controls were enrolled in the study. Their clinical status was evaluated with the Expanded Disability Status Scale. Serum levels were analyzed by enzyme-linked immunoassay. RESULTS: A significant elevation in MMP-9 serum levels and in the MMP-9/TIMP-1 ratio was found in the whole MS group (P<0.001), in the relapsing-remitting MS (RRMS) (P<0.001), and secondary-progressive MS (SPMS) (P<0.001) groups when compared with the controls. A significant elevation in MMP-2 serum levels and in the MMP-2/TIMP-2 ratio was observed in the primary progressive (P<0.001) and the SPMS (P<0.002) groups when compared with the RRMS group, and this increase was also associated with the disability (P<0.001) and severity (P<0.05) of the disease. CONCLUSION: We confirmed that metalloproteinases are useful biological markers in MS, providing information about the clinical type, disability, and severity of the disease.

[K-ras mutation as a prognostic factor in colorectal carcinoma]. / K-ras mutace u kolorektálního karcinomu jako prognostický faktor vývoje onemocnení.

INTRODUCTION: Colorectal carcinoma presents a serious problem in the Czech Republic: its incidence is on the increase and--according to some statistics takes first place among developed countries worldwide. Therefore, it is advised to incorporate examinational and the rapeutic algorithms with new modalities that will lead to early diagnostics or to a change in existing therapeutic procedures. CHARACTERIZATION OF K-RAS MUTATION: K-ras mutation belongs to the family of protooncogenes where a gene not having undergone mutation expresses proteins that regulate mitosis. Mutation cancels the regulatory function of these proteins, thus leading to the develop ment of tumors, especially carcinoma of the lungs, pancreas, and colorectum. PROJECT OBJECTIVE: The main objective of the project is to prove K-ras mutation in tumors of the colorectum: to detect tumor cells with K-ras mutation in peripheral blood; to detect K-ras mutation in liver metastases: and to verify the hypothesis claiming that tumors with K-ras mutation have a worse prognosis and often lead to disemination, mainly to the liver. METHODOLOGY AND COLLECTION OF DATA: The whole project is tied to an IGA grant and runs according to the strict rules of the protocol applied at the Surgical Clinic of the Pardubice Hospital, with its diagnostic part--PCR analysis being completed at the Biochemical Diagnostic Institute (UKBD) of the Teaching Hospital in Hradec Králové. RESULTS: The project has been running since June, 2004 to December 2006. 76 patients meeting defined parameters have been included in the file to date. K-ras mutation has been detected in the tumor tissue of 25 patients (33%). K-ras mutation hasn't been detected in the blood. DISCUSSION: Genetically analysis of a specific tumor has not yet become a standard part of the examinational and therapeutic algorithm. If an assumption of a worse course of illness and metastasizing--especially to the liver has been proven, the examination of Kras mutation in patients suffering from colorectal carcinoma should lead to the adjustment of their treatment and postoperative dispensarization, or the administration of chemotherapy and radiotherapy at stages when these modalities are not normally applied.

[Achieving Bcl-2/IgH negativity in peripheral blood/bone marrow after therapy implies better prognosis for patients with follicular lymphoma]. / Dosazení Bcl-2/IgH negativity v periferní krvi/kostní dreni po lécbe je u nemocných s folikulárním lymfomem spojeno s lepsí prognózou onemocnení.

UNLABELLED: Bcl-2/IgH rearrangement is a characteristic molecular rearrangement in patients with follicular lymphoma (FL), yet its prognostic significance is still unclear. OBJECTIVE: Evaluation of the implications of achieving Bcl-2/IgH negativity for the prognosis of FL patients. Twenty seven patients (54%) were receiving only chemotherapy (CHT), 23 patients (46%) were receiving chemotherapy combined with monoclonal antibody anti/CD20, rituximab (R-CHT). RESULTS: Molecular genetic remission was achieved in 7 out of 11 patients (64%) after R-CHT, and only in 2 out of 14 patients (14%) after CHT- this difference was statistically significant (p = 0.037). 4 weekly doses of rituximab were administered in a sequence to 17 out of 27 patients who had received only chemotherapy and failed to achieve complete remission. 12 out of 17 patients (71%) on this therapy were Bcl-2/IgH positive prior to treatment. 7 out of 12 (58 %) patients were no longer Bcl-2/IgH positive in a check performed after one month; the remaining 2 out of 5 patients had a negative Bcl-2/IgH record for the interval of 3 months (1 patient) or 6 (1 patient) months, respectively. The following factors were associated with the achievement of Bcl-2/IgH negativity at any point during the treatment: age < 65 years (p = 0.02) and performance status 0 + 1 according to WHO at baseline (p = 0.02). Patients who were Bcl-2/IgH negative after treatment had a lower recurrence/progression risk rate than the Bcl-2/IgH positive group of patients, i.e. 27% vs. 75% (p = 0.03), and a higher chance for progression-free survival, i.e. 81% vs. 38% (p = 0.004), event-free survival, i.e. 74% vs. 38% (p = 0.01), and overall survival, i.e. 87% vs. 74% (p = 0.05) at 2 years. CONCLUSION: In our experience, achieving Bcl-2/IgH negativity after follicular lymphoma therapy implies a better prognosis.

[The influence of long-term therapy with the insulin pump (CSII) in patients with type 1 diabetes mellitus on metabolic compensation and on the incidence of hypoglycaemia. Comparison with intensified conventional insulin therapy (MDI)]. / Vliv dlouhodobé terapié inzulinovou pumpou (CSII) u pacientu s diabetes mellitus 1. typu na metabolickou kompenzaci a výskyt hypoglykemií. Porovnání s intenzifikovanou konvencní inzulinovou terapií (MDI).

The principal objective of this paper is to verify, in clinical practice, the long-term affect (7 to 8 year follow up) and safety of insulin pump treatment in type 1 diabetes mellitus patients and to compare the results for diabetes compensation in patients treated with the insulin pump with a control group of patients treated with intensified insulin therapy using the MDI (multiple daily injection) method. PATIENT SAMPLE AND METHOD: We followed up 35 patients treated with the insulin pump and 35 patients in the control group. We evaluated the monitored parameters for both patient groups at the beginning and at the end of the follow up period. With respect to glycated haemoglobin, we evaluated the results on a yearly basis, and also on year by year changes. We assessed the incidence of hypoglycaemia in both groups on a yearly basis. The following aspects were considered in order to determine the level of statistical significance of the different parameters: (1) we compared the initial state with that seen at the end of the follow up, (2) we analysed the year by year changes in glycated haemoglobin, (3) we compared the patients treated with the insulin pump with those in the control group. Diabetes compensation was evaluated on the basis of measurement of glycated haemoglobin and calculation of glycaemia. Comparison of the incidence of hypoglycaemia was done for all hypoglycemic events and then separately for severe hypoglycaemia. Also evaluated were changes in weight and in insulin dose in 24 hours. RESULTS: The group of patients treated with the insulin pump recorded a dramatic decrease in glycated haemoglobin in the course of the follow up, p < 0.001, and also in average glycaemia, p < 0.001. In the control group only a transitory significant decrease of HbA1c, p < 0.05, was recorded in the first and second year of follow up, later the result was insignificant, i.e. p > 0.05, as compared with the initial state. In this group of patients, no significant improvement in average glycaemia, p > 0.05, was recorded when compared with the initial state. Comparison ofthe two groups of patients showed that HbA(1c), p < 0.001, and average glycaemia, p < 0.001, were worse with statistical significance in patients treated with the insulin pump at the beginning of the follow up. At the end of the follow up period, there was no significant difference between the two groups in terms ofglycated haemoglobin, p > 0.05, but a statistically significant difference was recorded in average glycaemia, p < 0.001, in favour of the group of patients treated with the insulin pump. The use of the insulin pump resulted in a statistically significant decrease in the incidence of severe hypoglycaemic events as compared with the control group, p = 0.010. This decrease was reflected in the measured parameters from the third year of the study to the end of follow up. However, at the beginning of the study and in the first and second years of follow up there was no statistically significant difference between the two groups in terms of incidence of severe hypoglycaemic episodes, p > 0.05. No statistically significant difference between the two groups was recorded in the incidence of all hypoglycaemic episodes from the beginning of the follow up to its end, p > 0.05. In both groups of patients, a statistically significant gain in weight was seen from the beginning of the study to the end of the follow up period, however, its statistical significance was lower (p < 0.05) in the group of patients treated with insulin pump than in the control group (p < 0.001). We proved a statistically significant decrease in daily insulin dose (p < 0.001 )was required in the group of patients treated with insulin pump, whilst no statistically significant change in the dose was recorded in the control group (p > 0.05). CONCLUSION: In the course of the follow up, we proved that treatment with the insulin pump in type 1 diabetes is more beneficial to patients than MDI treatment. This was reflected by both better compensation of diabetes and a lower incidence of severe hypoglycaemic episodes.