Krzysztof Safranow

Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Into Peripheral Blood in Patients After Stroke

Background and Purpose— In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods— We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient’s stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results— In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+lin-CD45- small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4+ VSEL SCs circulating in the PB. Conclusions— We conclude that stroke triggers the mobilization of CXCR4+ VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study.

Molecular basis of human CD36 gene mutations.

CD36 is a transmembrane glycoprotein of the class B scavenger receptor family. The CD36 gene is located on chromosome 7 q11.2 and is encoded by 15 exons. Defective CD36 is a likely candidate gene for impaired fatty acid metabolism, glucose intolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, Alzheimer disease, and modification of the clinical course of malaria. Contradictory data concerning the effects of antiatherosclerotic drugs on CD36 expression indicate that further investigation of the role of CD36 in the development of atherosclerosis may be important for the prevention and treatment of this disease. This review summarizes current knowledge of CD36 gene structure, splicing, and mutations and the molecular, metabolic, and clinical consequences of these phenomena.

677C>T and 1298A>C MTHFR polymorphisms affect methotrexate treatment outcome in rheumatoid arthritis

INTRODUCTION Methotrexate (MTX), widely used in the treatment of rheumatoid arthritis (RA), inhibits dihydrofolate reductase and folate-dependent enzymes. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and has been shown to be polymorphic, affecting the enzyme activity. METHODS To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and MTX efficacy in the treatment of RA, a total of 174 RA patients, treated with MTX plus methylprednisone 4 mg and folic acid 5 mg were analyzed. RESULTS In univariate regression analysis model, the MTHFR 677T allele was associated with significantly higher frequency of remission, whereas in the case of the 1298C allele, a tendency for higher remission rate was observed. In multivariate regression analysis, the presence of both 677T and 1298C alleles was associated with an increased frequency of remission. CONCLUSION The results of our study suggest that the MTHFR 677T and 1298C alleles may be associated with an increased rate of RA remission in patients treated with MTX receiving high doses of folic acid supplementation.

Mutation analysis of MLH1 and MSH2 genes performed by denaturing high-performance liquid chromatography

Mutation analysis of large genes, such as MSH2 and MLH1, is time-consuming and expensive. We investigated the sensitivity and specificity of DHPLC analysis for the detection of mutations within both MSH2 and MLH1. Studies included a series of 46 patients affected by colorectal cancer from HNPCC families. We confirmed 19 changes previously identified by DNA sequencing and, in a blind study, an additional 16 rare alterations including four mutations not previously described. Generally, false negative results were not observed. Elution profiles were highly characteristic for a given change and in 98.5% cases allowed the distinction between novel alterations and previously identified mutations and polymorphisms. For the detection of changes in almost all amplicons, it was sufficient to use just one denaturing temperature. DHPLC was confirmed to be highly sensitive, specific and a cost-effective technique with particularly high potential for the detection of MSH2 and MLH1 gene mutations in the diagnostic setting.

The LVIS/LVIS Jr. stents in the treatment of wide-neck intracranial aneurysms: multicentre registry

Background Wide-necked intracranial aneurysms have been a challenge for endovascular techniques. With the advent of adjunctive devices such as balloons or stents, recanalisation rates have decreased secondary to better packing. Purpose The purpose of this registry was to evaluate the safety and effectiveness of the new Low-profiled Visualized Intraluminal Support LVIS and LVIS Jr. stents in the treatment of unruptured wide-neck intracranial aneurysms. Methods The LVIS or LVIS Jr. stent-assisted coil embolisation was performed in 78 patients harbouring 78 intracranial aneurysms. There were 59 aneurysms located in the anterior circulation and 19 in the posterior circulation. Clinical data and 6-month follow-up angiograms are presented. Results The LVIS and LVIS Jr. stents were successfully delivered to the target aneurysm; however, there were seven cases in which the LVIS/LVIS Jr. stents had suboptimal opening and apposition to the parent vessel wall. The overall technical success for all groups was 91% (71 of 78 stents). There was complete angiographic occlusion in 66 (85%) of 78 cases and residual neck remnants in 12 (15%) cases. All patients had 6-month angiographic follow-up, which demonstrated complete occlusion of the target aneurysm in 64 (82%) cases, residual neck remnants in 5 (6%) cases and there was aneurysm filling in 9 (12%) cases. Conclusions The LVIS/LVIS Jr. stent system is safe and effective for the treatment of wide-neck intracranial aneurysms, providing suitable support of the coil mass, which allows for a high level of occlusion with low rates of recanalisation and subsequent treatments.

Plasma concentrations of TNF-α and its soluble receptors sTNFR1 and sTNFR2 in patients with coronary artery disease

Tumour necrosis factor alpha (TNF-alpha) is implicated in post-ischemic myocardial dysfunction. Two distinct TNF-alpha receptors are shed from cell membranes and circulate in plasma as soluble sTNFR1 and sTNFR2 proteins. The aim of the study was to establish factors associated with plasma concentrations of TNF-alpha and its receptors in patients with coronary artery disease (CAD). Since adenosine inhibits the expression of TNF-alpha, two functional polymorphisms in genes encoding enzymes participating in adenosine metabolism, i.e. AMP deaminase-1 (AMPD1, C34T) and adenosine deaminase (ADA, G22A), were analyzed. Plasma concentrations of TNF-alpha, sTNFR1, and sTNFR2 were measured using ELISA in 167 patients with CAD. Common factors significantly associated with higher TNF-alpha, sTNFR1, and sTNFR2 were lower glomerular filtration rate (GFR), older age, higher BNP, lower blood haemoglobin, and the presence of asthma or chronic obstructive pulmonary disease (COPD). Higher TNF-alpha and sTNFR1 concentrations were also associated with the presence of heart failure (HF), lower ejection and shortening fraction, the presence of diabetes or metabolic syndrome, lower serum HDL cholesterol, and higher uric acid. In multivariate analysis the common independent predictors of higher TNF-alpha, sTNFR1, and sTNFR2 were lower GFR, lower HDL cholesterol, higher BNP, and the presence of asthma or COPD. There were no associations between AMPD1 C34T or ADA G22A genotypes and TNF-alpha or its receptors. In conclusion, the concentrations of TNF-alpha, sTNFR1, and sTNFR2 reflect the impairment of cardiac and renal function in patients with CAD. Metabolic syndrome and diabetes are associated with higher plasma concentrations of TNF-alpha and its receptors.

Diagnostic value of fasting capillary glucose, fructosamine and glycosylated haemoglobin in detecting diabetes and other glucose tolerance abnormalities compared to oral glucose tolerance test

Abstract New diagnostic criteria for diabetes mellitus recommend lowering of the fasting plasma glucose to 7.0 mmol/l. In contrast to recommendations of the American Diabetes Association (AA). WHO recommends using the oral glucose tolerance test (OGTT) in clinical practice. In this study, based on OGTT results and WHO 1998 criteria, we determined if measuring fasting capillary glycaemia (FCG) along with fructosamine and/or glycosylated haemoglobin allows the detection of glucose tolerance abnormalities better than FCG alone. OGTT was performed in 538 patients. Serum fructosamine was determined in 480 of the patients, and glycosylated haemoglobin in 234 of the patients. According to WHO 1998 criteria, the patients were divided into groups due to glucose tolerance abnormalities. Fructosamine correlated stronger with 2-h post-load glucose concentrations than with FCG. HbA1c correlated stronger with FCG than with 2-h post-load glucose. Combined use of fructosamine and FCG predicted 2-h post-load glucose better than combined use of FCG and HbA1c. Receiver operating characteristic curve analyses showed that FCG was the best criterion in discriminating diabetes. Combined use of FCG and fructosamine slightly improved the ability to discriminate glucose tolerance abnormalities from normal glucose tolerance. FCG is the most effective predictor of 2-h post-load glucose and the best criterion for discriminating diabetes and other glucose tolerance abnormalities from normal glucose tolerance. Fructosamine is a potentially useful post-load glycaemia index. OGTT is irreplaceable in identification of patients with high post-load glycaemia.

Effect of CYP2C19 and MDR1 polymorphisms on cure rate in patients with acid-related disorders with Helicobacter pylori infection

A proton pump inhibitor (PPI) plus two antibiotics (amoxicillin and either clarithromycin or metronidazole) are recommended for treatment of acid-related disorders with Helicobacter pylori (H. pylori) infection. The aim of this pharmacogenetic study was to evaluate the efficacy of triple therapy with PPIs on eradication of H. pylori infection in relation to cytochrome P450 2C19 (CYP2C19) and P-glycoprotein (MDR1) gene polymorphisms. The retrospective study involved 70 Polish Caucasian patients with H. pylori infection, diagnosed and treated with one of the two different triple therapy regimens [omeprazole, amoxicillin, and clarithromycin (OAC) or pantoprazole, amoxicillin, and metronidazole (PAM)]. Using genomic DNA, CYP2C19 (*2 and *3) and C3435T MDR1 alleles were determined by means of polymerase chain reaction-restriction fragment length polymorphism assays. A significantly higher prevalence (P<0.05) of heterozygous extensive metabolizers (hetEM) with CYP2C19*1/*2 genotype (32.4% versus 8.3%) and homozygous with 3435TT MDR1 genotype (38.2% versus 13.9%) was found in patients cured after the first cycle of triple therapy than in patients with failure of eradication after the first cycle. CYP2C19*1/*2 and 3435TTMDR1 genotypes as well as PAM regimen of treatment were also predictive of successful eradication of H. pylori infection after the first cycle of triple therapy at univariate/multivariate logistic regression analysis. This pharmacogenetic study on the influence of different CYP2C19 and C3435TMDR1 genotypes on H. pylori eradication suggests that CYP2C19 and MDR1 polymorphisms may be independent predictable determinants of the efficacy of triple therapy including PPI. The PAM regimen of treatment seems to be more effective after the first cycle of the therapy than the OAC regimen.

Comparison of embryological and clinical outcome in GnRH antagonist vs. GnRH agonist protocols for in vitro fertilization in PCOS non-obese patients. A prospective randomized study

PurposeEmbryological and clinical efficacy of gonadotropin-releasing hormone (GnRH) antagonist and agonist stimulation protocols in non-obese women with polycystic ovarian syndrome (PCOS) were compared.MethodsA prospective randomized study. Setting: Medical University Hospital. Patients: 70 infertile PCOS patients; 33 in GnRH antagonist and 37 in GnRH agonist group.ResultsSimilar mature metaphase II oocyte rate (76% vs. 76%) was observed in both protocols. Optimal pronuclear morphology zygotes dominated in both groups (64% vs. 66%). Transferred embryo quality did not differ in both protocols. No significant differences between both protocols were found in delivery rate (p = 0.481), pregnancy rate (p = 0.810), multiple pregnancy rate (p = 0.501), miscarriage rate (p = 0.154), fertilization rate (p = 0.388) and implantation rate (p = 1.000). Duration of stimulation and total follicle-stimulating hormone (FSH) dose were significantly lower in GnRH antagonist protocol (p = 0.0005).ConclusionsGnRH antagonist and agonist protocols in non-obese PCOS patients yield similar embryological and clinical outcomes. Shorter duration of treatment and lower FSH requirement in GnRH antagonist group may be financially beneficial and therefore attractive for patients.

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 μg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.