Label-free quantitative SWATH-MS proteomic analysis of adult myocardial slices in vitro after biomimetic electromechanical stimulation.

A special in vitro model maintained with ultrathin cardiac slices with a preserved architecture, multi-cellularity, and physiology of the heart tissue was used. In our experiments, we performed label-free quantitative SWATH-MS proteomic analysis of the adult myocardial slices in vitro after biomimetic electromechanical stimulation. Rat myocardial slices were stretched to sarcomere lengths (SL) within the physiological range of 1.8-2.2 µm. Electromechanically stimulated slices were compared with slices cultured without electromechanical stimulation (unloaded and nonstimulated-TW) on a liquid-air interface and with fresh myocardial slices (0 h-C). Quantitative (relative) proteomic analyses were performed using a label-free SWATH-MS technique on a high-resolution microLC-MS/MS TripleTOF 5600+ system (SCIEX). The acquired MS/MS spectra from the DDA LC-MS/MS analyses of the rat heart samples were searched against the UniProt Rattus norvegicus database (version of 15.05.2018) using the Paragon algorithm incorporated into ProteinPilot 4.5 (SCIEX) software. The highest number of differential proteins was observed in the TW group-121 when compared to the C group. In the 1.8 and 2.2 groups, 79 and 52 proteins present at a significantly different concentration from the control samples were found, respectively. A substantial fraction of these proteins were common for two or more comparisons, resulting in a list of 169 significant proteins for at least one of the comparisons. This study found the most prominent changes in the proteomic pattern related to mitochondrial respiration, energy metabolism, and muscle contraction in the slices that were stretched and fresh myocardial slices cultured without electromechanical stimulation.

Acute fright induces onset of symptoms in vanishing white matter disease-case report.

Vanishing white matter disease is a newly recognised leukoencephalopathy of identified genetic background, characterised by cystic degeneration and progressive vanishing of white matter. The characteristic clinical symptoms are spasticity and ataxia with relatively preserved cognitive functions. A characteristic feature of the disease is the occurrence of the symptoms after a physical stress situation such as mild head trauma or febrile infection. We would like to present a case of a 6-year-old girl whose first symptoms of the disease occurred after being frightened by a horse.

Expression of EBV encoded latent membrane protein 1 (LMP-1) and bcl-2 protein in childhood and adult Hodgkin's disease: application of microwave irradiation for antigen retrieval.

Surgical biopsies obtained from 32 children, and 34 adults with Hodgkin's disease (HD) were investigated for the expression of the EBV encoded Latent Membrane Protein 1 (LMP-1), bcl-2 protein, markers for HD; LeuM1 (CD15), BerH2 (CD30) and the new BLA.36, as well as for B (L26) and T lymphocytes (UCHL1). Before immunostaining, sections were subjected to an Antigen Retrieval (AR) procedure based on microwave irradiation in citrate buffer. In 13 cases staining with and without the AR procedure was compared. Immunoreactivity for LMP-1 was found in 44% of the biopsies from adults and 53% from children. We also found reactivity for the bcl-2 protein in Hodgkin's and Reed Sternberg (HRS) cells in 48% of the biopsies from adults and 45% from children. Immunoreactivity with BLA.36 was found in 94% of the biopsies from adults and 100% from children, with LeuM1 in 83% from adults and 93% from children and with BerH2 in 24% from adults and 84% from children. Nuclear PCNA staining was seen in HRS in all cases both adult and childhood. The T cell marker (UCHL1) displayed no reactivity with HRS cells. In 21% of the adult and 9% cases from the childhood cases we observed reactivity with the B cell marker (L26) in HRS cells. We can conclude that antigen retrieval improves immunostaining results of paraffin sections which were previously negative for bcl-2, LeuM1 and BerH2 antibodies. The high percentage of LMP-1 positive cases, both in adults and in children, indicates that the potential pathogenetic effect of EBV may be of similar importance both in childhood and in adult HD. The new MAb BLA.36 gave consistent immunostaining with HRS cells but also with other cell types. In a panel of markers for HRS cells BLA.36 together with LeuM1 (CD15) and BerH2 (CD30) are useful.

Is P-glycoprotein a sufficient marker for multidrug resistance in vivo? Immunohistochemical staining for P-glycoprotein in children and adult leukemia: correlation with clinical outcome.

Seventy-eight patients: 45 children, 33 adults and 27 normal healthy donors were enrolled in the study. Expression of P-glycoprotein (P-gp) was evaluated with three monoclonal antibodies (MAb's) directed to intra-(C219, JSB-1) and extra-cellular (MRK-16) epitopes of P-gp and immunocytochemical (IC) APAAP staining method. Twenty-seven healthy donors peripheral blood mononuclear cells (PBMC) were investigated by means of IC and FACScan analysis. Positive staining for P-gp was detected in 31% children's and 33% adults' leukemia samples. No reactivity of three MAb's was observed with peripheral blood mononuclear cells (PBMC) by means of IC. Flow cytometry analysis with C219 MAb revealed staining for P-gp present on sub-population of lymphocytes and monocytes. P-gp (+) as well as P-gp (-) cases were compared in respect to clinical outcome, FAB classification and blood group. Complete remission (CR) was achieved in 12/14 (85%) children's and 9/11 (81%) adults' P-gp (+) leukemia cases. Within the P-gp (-) leukemia cases CR was observed in 24/29 (82%) and 18/22 (81%), respectively. Partial remission, relapse, resistance and death were noticed in 14% children's and 18% adults' P-gp (+) samples. In P-gp (-) cases these parameters were observed in 17% and 18%, respectively. These results raise the question whether the expression of P-gp can be used as single prognostic marker to detect multidrug resistance (MDR phenomenon) in vivo?

EEG abnormalities preceding the epilepsy onset in tuberous sclerosis complex patients - a prospective study of 5 patients.

Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder characterized by multiple hamartomas development. Epilepsy is the most common symptom appearing in 80-90% of the patients mainly in the first year of life. A prompt and early seizure control is crucial and can prevent development of an epileptic encephalopathy and secondary mental retardation. Therefore the very early identification of seizures seems to be of a great importance. We present the cases of 5 patients diagnosed with TSC prenatally or perinatally and regularly monitored (at 4-6 weeks intervals) with EEG before the epilepsy onset. The patients' age at baseline varied from 9 days to 9 weeks. In all of the patients epileptiform discharges preceded the epilepsy onset. The time interval between abnormality detection on EEG and the epilepsy onset varied from 1 to 8 days. The patient's age at the epilepsy onset ranged from the 17th day to the 5th month of life. In one patient the EEG was abnormal from the beginning and in this patient the epileptic seizures started from the neonatal period. In the rest of the patients (4/5) the EEG remained normal throughout the first months of life. In all of the children epilepsy started with focal motor seizures. Our study is the first prospective one showing the results of the EEG monitoring in TSC patients and the natural evolution of the EEG patterns in patients with the seizures types other than infantile spasms.

Altered expression of immune surface markers in children with recurrent infections of respiratory tract.

Infections of the respiratory tract are very common in young children. They may affect the quality of life and have a great economic impact. On the other hand, respiratory tract infections through MALT system play a positive role in the maturation and development of the immune system. The aim of the study was to examine quantitative and qualitative changes of T and B cells and granulocytes surface molecules in 24 children with recurrent (more than 8 episodes per year) infections of the respiratory tract and in 18 healthy children. The expression of CD2, CD3, CD4, CD8 on T cells; HLA-DR, CD19, CD5/CD20 on B cells, and CD11a/CD18, CD11b/CD18, CD62L, CD16 on granulocytes from peripheral blood was evaluated by a flow cytometry method. We observed a significant increase in the CD5+/CD20+ positive cells on B cells. We also observed a decreased expression of CD11c+/CD18+ cells, CD11a, and CD62L on granulocytes. The expression of other structures on lymphocytes and the CD11b/CD18+ on granulocytes remained unchanged. CD5+/CD20+ cells constitute a filogenetically old population responsible for the production of IgM of low specificity and affinity for specific antigens. The prevalence of this fetal phenotype population may be explained as a delayed maturation of the humoral immune system leading to increased susceptibility to infections. A lower percentage of CD11a may be related to the blockade of that molecule by rhinoviruses.

[Is standardization possible in immunohistochemical examination?]. / Czy mozliwa jest standaryzacja w badaniach immunohistochemiczynch.

Immunohistochemistry has become an important tool in research and in surgical pathology. Rapid growth of a new methods in immunohistochemistry supplement traditional histochemical and histological light microscopy investigations. Immunohistochemistry has given pathologists a chance to location different antigens on the cell surface as well as in the cell compartments. The expression of antigens are mostly influenced by factors connected with tissue processing; fixation and embedding. The aim of present article is to show the role of these factors and their influence on some immunohistochemical staining results. Not all the problems are discussed here, the main goal which authors would like to obtain is to show the way how to solve problems which can occur during immunohistochemical staining procedure. They want also to delineate the importance of standardization in immunohistochemistry to make the results more reliable between different laboratories.

[P glycoprotein and multidrug resistance]. / P-glikoproteina a opornosc wielolekowa.

Drug resistance has been shown to be associated with the expression of P-glycoprotein (P-gp), the product of mdr-1 gene. One of the suggested mechanisms for the phenomenon called Multidrug Resistance (MDR) is related to the overexpression and amplification of the mdr-1 gene. The product of this gene is called P-gp and it has been considered as a potential marker for drug resistance. Transfection of the mdr-1 gene into drug-sensitive cells confers the role of mdr-1 gene in developing MDR phenotype. Structural analysis of homologous cDNA, responsible for production of transport proteins, from: MDR cell lines, bacteria and yeast, revealed high similarity. Molecular structure analysis indicate that P-gp has nucleotide binding sites. It has been established that P-gh has an internal ATP-ase activity, thus it can act as energy dependent transport protein. The expression of P-gp has been found in neoplastic and normal tissues (as adrenal glands, kidney, liver, pancreas, jejunum and large intestine), as well as in several cell lines which have been induced to become resistant to cytostatics. The aim of present study was to review the role of P-gp expression in laboratory and clinic.