From Primary MSC Culture of Adipose Tissue to Immortalized Cell Line Producing Cytokines for Potential Use in Regenerative Medicine Therapy or Immunotherapy.

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.

Autotransplantation of the Adipose Tissue-Derived Mesenchymal Stromal Cells in Therapy of Venous Stasis Ulcers.

Adipose tissue is a reliable source of mesenchymal stromal cells (MSC) for use in regenerative medicine. The aim of this pilot study was to describe the method, and assess the safety and the potential efficacy of transplantation of autologous adipose tissue-derived MSC for the treatment of chronic venous stasis ulcers. Study group consisted of 11 patients (mean age: 66.6 ± 9.5 years) with chronic venous stasis ulcers. Adipose tissue was harvested by tumescent-aspiration method. Stromal cells were separated using a dedicated closed system in a real-time bedside manner. The phenotype of cells was determined immediately after separation. Cell concentrate was implanted subcutaneously around the wound and the wound bed. All ulcers were assessed planimetrically before autotransplantation and every two weeks during the six-month follow-up. During the study all patients received standard local and general treatment. The preparation contained an average of 5.6 × 106 ± 4 × 106 cells per milliliter. The phenotype of 65-82% of transplanted cells expressed MSC markers: CD73+ CD90+ and CD34+. An improvement was observed in 75% of ulcers. The data showed highly significant negative correlation (p < 0.0001) between wound size and wound closure degree. There was no correlation of ulcer healing with other parameters evaluated, including age of the patients. No serious side effects were observed. Autotransplantation of adipose tissue stromal cells may be a safe and promising treatment method for chronic venous ulcers.

Early Detection of Active Glomerular Lesions in Dogs and Cats Using Podocin.

In veterinary medicine, sensitive and specific markers of the early stages of renal failure still remain to be established. Podocytes could be a promising diagnostic tool in veterinary nephrology, especially in the differentiation of active pathological disease and glomerulopathies. Podocin is one of the robust proteins exploitable in detection of podocyturia. This article presents podocyte detection in urine for diagnostic purposes in veterinary medicine using a variety of methods. We describe the advantages and disadvantages of the immunohistochemical technique currently used, and of scanning microscopy, chromatography, and immunostaining. The identification of podocin-positive cells is a promising diagnostic tool in the detection of the early stages of glomerular basement membrane damage. The detection of renal failure prior to the occurrence of azotaemia is of high clinical importance from the clinical and scientific points of view.

Circulating ectosomes: Determination of angiogenic microRNAs in type 2 diabetes.

Ectosomes (Ects) are a subpopulation of extracellular vesicles formed by the process of plasma membrane shedding. In the present study, we profiled ectosome-specific microRNAs (miRNAs) in patients with type 2 diabetes mellitus (T2DM) and analyzed their pro- and anti-angiogenic potential. Methods: We used different approaches for detecting and enumerating Ects, including atomic force microscopy, cryogenic transmission electron microscopy, and nanoparticle tracking analysis. Furthermore, we used bioinformatics tools to analyze functional data obtained from specific miRNA enrichment signatures during angiogenesis and vasculature development. Results: Levels of miR-193b-3p, miR-199a-3p, miR-20a-3p, miR-26b-5p, miR-30b-5p, miR-30c-5p, miR-374a-5p, miR-409-3p, and miR-95-3p were significantly different between Ects obtained from patients with T2DM and those obtained from healthy controls. Conclusion: Our results showed differences in the abundance of pro- and anti-angiogenic miRNAs in Ects of patients with T2DM, and are suggestive of mechanisms underlying the development of vascular complications due to impaired angiogenesis in such patients.

Expression of Cell Membrane Antigens in Cells Excreted in the Urinary Sediment Predicts Progression of Renal Disease in Patients with Focal Segmental Glomerulosclerosis.

BACKGROUND/AIMS: A link between the number of podocytes excreted in the urine and activity of glomerular disease has been established. The aim of this study was to investigate possible correlations between urinary cells' phenotype and the progression of focal segmental glomerulosclerosis (FSGS). METHODS: Forty patients with newly diagnosed FSGS were included. Cells were isolated from urine by adherence to collagen-coated cover slips and assessed for the expression of podocalyxin (PDX), CD68 and Ki67 antigens by indirect immunofluorescence. In addition, double-staining procedures were performed in combinations of the above antigens plus cytokeratin, WT1 and CD-105. Twenty-two patients in whom urinary protein to creatinine ratio exceeded 2.0 at diagnosis were followed for 36 months, with assessments of renal function and proteinuria every 3 months. During observation, patients were subjected to standard therapy. RESULTS: Significantly higher numbers of Ki67 positive cells at the onset of the study were observed in patients who have doubled serum creatinine (SCr) in follow-up, than in those who have not (p = 0.0149). By logistic regression analysis, both CD68 and Ki67, but not anti-PDX positive cell numbers at diagnosis were found to be predictors of doubling SCr concentration in 36 months' follow-up. Results of double staining indicate that PDX positive cells could be identified as podocytes or their precursors and parietal epithelial cells. CONCLUSION: Urinary sediment PDX positive cell numbers do not predict the progression of FSGS, whereas CD68 and Ki67 phenotype of urinary podocytic lineage clearly has a prognostic significance in 36 months' observation of primary FSGS.

[Biological role of Interleukin 33 and its importance in pathophysiology of cardiovascular system]. / Biologiczna rola interleukiny 33 i znaczenie w patofizjologii ukladu sercowo-naczyniowego.

Interleukin 33 (IL-33) is a member of the IL-1 cytokin family. It is expressed by various cells and tissues, mainly epithelial and endothelial cells. It is a cytokine with dual function. It may act both as a traditional cytokine and as intracellular nuclear factor, functioning as transcription regulator. Its biological effect via interaction with membrane-bound ST2 receptor and IL-1 receptor accessory protein (IL-1RAcP) is associated with the induction of Th2-type immune response and IL-5 and IL-13 synthesis. IL-33 has a strong immunoregulatory properties. Depending on the type of activated cells, microenvironment, and costimulatory factors, IL-33 can act either as a pro- or anti-inflammatory cytokine. Recent studies indicate various protective effect of IL-33/ST2 sygnaling in atherosclerosis, obesity, disorders in glucose homeostasis and in heart diseases. The paper presents current state of knowledge about the structure and biological function of IL-33 and its receptor ST2, with particular emphasis on its role in pathophysiology of cardiovascular system.

[Assessment of osteopontin and osteoprotegerin levels in abdominal aortic aneurysm patients]. / Ocena stezenia osteopontyny i osteoprotegryny u chorych z tetniakiem aorty brzusznej.

Abdominal Aortic Aneurysm (AAA) is multifactorial disease with unknown ethiology. Among the theories on the pathogenesis of AAA are some ge. netic factors, infections, disorders in connective tissue (collagenosis), arteriosclerosis, inflammation, incorrect immune response (autoimmunity). It was discovered that crucial for AAA development is intense inflammatory reaction combined with high proteolytic activity. Recent evidence confirmed the association between osteopontin (OPN) and osteoprotegerin (OPG) levels and cardiovascular diseases and arterio. sclerosis. The aim of this work was assessment of plasma levels of OPN and OPG in the group of the patients with AAA and correlation of results with clinical parameters, "classical" risk factors for development of AAA, arteriosclerosis and morbidity. The reference group consist of the patients with Leriche Syndrome (LS). The OPG level was assessed in plasma and OPN levels were assessed in plasma and urine. Plasma OPG levels were higher in AAA group than in LS group (difference not statistically significant, p = 0.0549). It was statistically significant positive correlation between plasma OPN levels and CRP levels in the groups of AAA and LS patients. It was not any association between plasma OPG and OPN levels and abdominal aortic diameter. Plasma OPG levels correlated positively with the existence of coronary artery disease in AAA patients. Insignificant, but higher levels of this protein were found also in a group of AAA patient with myocardial infarction. In LS group we found statistically significant positive association between plasma OPG levels and patient with stroke. However, in AAA patients with incidence of stroke, we found higher plasma levels of OPN. Interestingly, there was not any association between OPN levels in the urine and clinical parameters, risk factors and morbidity, including kidney diseases. inflammatory role of OPN and depicts better reflection of inflammatory reaction of OPN than OPG in both group of patients. Plasma OPG levels in AAA patients are more associated with coronary artery disease than with peripheral artery disease, what is characteristic for LS patients. Lack of association of urine OPN levels with above mentioned parameters suggest minor importance of this urine protein in clinical condition evaluation of patients with AAA and advanced arteriosclerosis.

[Significance of podocytes isolated from urine as a marker of glomerulonephritis activity--is the game worth of a candle?]. / Znaczenie podocytów izolowanych z moczu, jako wskaznika aktywnosci klebuszkowych zapalen nerek--czy gra jest warta swieczki?

The significance of the native urine sediment in the differential of glomerular disease needs no further comments. However the question arises whether it could be useful to develop a more specific diagnostic approach to identify the origin of renal epithelial cells that can be detected in the urine sediments as well. Especially the detection of podocytes in the urine could be a valuable non-invasive method to get information about the disease activity or disease type and could be used as a follow up. So far there are only a few studies that analyzed the clinicaI relevance of renal epithelial cells in the urine. This review summarizes the available information on marker proteins that have been successfully used in the diagnostic of podocytes in the urine. Furthermore it shows possible diagnostic usefulness of epithelial urinary cells assessment and future research directions.

Podocytes in urine, a novel biomarker of preeclampsia?

Preeclampsia is a disorder occurring during pregnancy typically after 20 weeks of gestation. It affects both mother and unborn baby in at least 5-8% of all pregnancies. It is a rapidly progressive condition characterized by high blood pressure and the presence of protein in the urine. The symptoms, such as swelling, sudden weight gain, headaches and vision disturbances, are important signs of preeclampsia which can lead to maternal and infant illness and death. It is estimated that this disorder is responsible for 76,000 maternal and 500,000 infant deaths each year. The main hypothesis explaining the development of preeclampsia is the theory of placental hypoxia/ischemia. An imbalance between vascular endothelial growth factor (VEGF) and soluble fms-like tyro-sine kinase-1 (sFlt-1) seems to play a crucial role. Currently there is no way to predict, with certainty whether preeclampsia will develop during a given pregnancy. There is a need for a diagnostic tool which can help to identify and monitor women at risk. There is growing evidence that podocyturia - urinary excretion of viable podocytes may be a useful predictor of preeclampsia. This paper presents facts supporting such a hypothesis.

Quality of medical follow-up of young women with Turner syndrome treated in one clinical center.

For Turner syndrome (TS) patients, smooth transition from pediatric to adult health care is a critical point. The study objective was to evaluate the medical follow-up of young women with TS in one clinical center 3 years after the latest guidelines had been introduced by the TS Study Group. A questionnaire study was performed in 59 TS adults selected from a database of 117 patients. Twenty-two of them, aged 23.0 ± 2.8 years, consented to participate. Nineteen responders (86.4%) were followed up by general practitioners who were not aware of the TS diagnosis in 14 (63.6%) cases. Eight (36.4%) were seen regularly by the relevant specialists. Adequate medical assessment varied from 5% (celiac serology) to 74% (gynecology assessment) and 82% (ear-nose-throat) of participants. None of the patients had undergone all of the recommended investigations according to recommendation. Height deficiency, body mass index, age at TS diagnosis and level of education did not correlate with the number of assessments performed (p = 0.687, p = 0.810, p = 0.641, and p = 0.568, respectively). Three years after the introduction of the current guidelines, medical follow-up in the transition phase is still inadequate. Improvement in transitional health care is warranted through better patient education, referring to physicians caring for adults with TS and better cooperation with general practitioners with wider popularization of the TS recommendations among them.

Blood pressure regulation and resting heart rate abnormalities in adolescent girls with polycystic ovary syndrome.

OBJECTIVE: To assess the risk of cardiovascular disease on the basis of biochemical, echocardiographic, and 24-hour blood pressure (BP) monitoring parameters in adolescent girls with polycystic ovary syndrome (PCOS). DESIGN: Cross-sectional study. SETTING: Academic and research institution. PATIENT(S): Thirty-four obese and nonobese girls with PCOS were evaluated and compared with body mass index-matched girls with regular menses. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Androgens, gonadotropins, lipids, and fasting and oral glucose tolerance test-stimulated glucose and insulin concentrations were measured. Echocardiographic assessment and 24-hour BP monitoring were done. RESULT(S): Compared with obese controls, obese girls with PCOS had significantly higher 24-hour mean BP, day mean BP, day diastolic BP, and diastolic BP nighttime dip (75.5 ± 4.5 mm Hg vs. 71.7 ± 3.7 mm Hg; 78.2 ± 5.0 mm Hg vs. 73.6 ± 4.0 mm Hg; 67.6 ± 4.9 mm Hg vs. 63.7 ± 3.7 mm Hg; and 20.2% ± 5.2% vs. 15.0% ± 6.6%, respectively). Obese girls with PCOS had significantly higher night heart rate than obese controls (60.4 ± 5.6 beats per minute vs. 61.7 ± 4.8 beats per minute). Left ventricle end-diastolic (4.6 ± 0.3 cm vs. 4.2 ± 0.2 cm) and end-systolic diameter (3.0 ± 0.3 cm vs. 2.7 ± 0.2 cm) were also significantly higher in nonobese girls with PCOS than in nonobese controls; however, all values were still within the accepted range of normal limits. CONCLUSION(S): Higher night heart rate in obese girls with PCOS and higher day BP but preserved diastolic nocturnal dip in nonobese girls with PCOS may be regarded as early cardiovascular disease risk factors.

[Role of mast cells in the pathogenesis of atherosclerosis]. / Rola mastocytów w patogenezie miaidzycy.

Atherosclerosis is chronic, inflammatory disease. In artery inflammation main role play cells of the immune system, including lymphocytes and macrophages. This circle of the cells modulating atherogenesis enclose also mast cells. Activated mast cell degranulate and release many types of mediators, including cytokines, chemokins, growth factors, vasoactive substances and proteolytic enzymes. This mediators can modulate inflammatory reaction in artery wall directly, by releasing proinflammatory cytokines or indirectly, influencing the activity of the other cells of the immune system, taking a part in the process of atherogenesis. Due to the ability of secretion and activation of proteolytic enzymes (chymase, tryptase, metalloproteinases), mast cells are prone to degrade various components of pericellular and extracellular matrices, including collagen, main protein of the fibrous cap of atherosclerotic plaque, rendering to plaque destabilization, increasing the onset of the atherothrombotic complications. Mast cell proteases can also modulate proliferation and apoptosis of the endothelial and smooth muscle cells of the artery wall. In spite of the fact, that most of the studies about mast cells are performed ex vivo on human tissues or cell cultures and rodents, this findings lead to recognize mechanisms by which mast cell can intervene in atherogenesis process. Moreover, there are some trials of using some of the mast cell mediators in differentiation of cardiovascular diseases and prediction the clinical condition of the patients.

Are metabolic syndrome and its components in obese children influenced by the overweight status or the insulin resistance?

INTRODUCTION: The aim of this study was to determine which factors increase the risk of metabolic syndrome (MS) and its components in obese children and adolescents. MATERIAL AND METHODS: In 78 obese children (42 girls, 36 boys), mean age 14.6 ± 3.5 years, blood pressure, total cholesterol, triglycerides, HDL-cholesterol (HDL), insulin and glucose at fasting state as well as in OGTT were measured. Body mass index (BMI) Z-score, LDLcholesterol, and insulin resistance indices (FIGR, R-HOMA) were calculated. RESULTS: Metabolic syndrome was diagnosed in ten (12.8%) children. Hyperinsulinaemia was present in 42 (53.8%) subjects, increased FIGR in eight (10.3%) and increased R-HOMA in 49 (62.3%). Significant correlations between BMI Z-score ≥ 2.5 and MS occurrence and its components (hypertriglyceridaemia, isolated systolic and diastolic hypertension) were found. Hypertriglyceridaemia, low HDL and hypertension, as well as MS occurrence, correlated significantly with stimulated hyperinsulinaemia and increased FIGR. Risk of hypertension was increased 5.6 times by fasting hyperinsulinaemia. Stimulated hyperinsulinaemia increased the risk of hypertriglyceridaemia 3.7 times, risk of low HDL 14.4 times and risk of MS 10.3 times. These risks did not change significantly when adjusted for BMI Z-score. CONCLUSIONS: Our study results show that both BMI Z-score and OGTT stimulated hyperinsulinaemia are good predictors of MS occurrence in obese children and adolescents. The risk of dyslipidaemia and hypertension increase significantly with hyperinsulinaemia and insulin resistance, with low HDL cholesterol being the most affected.

[The role of osteopontin in cardiovascular diseases]. / Rola osteopontyny w chorobach sercowo-caczyniowych.

Osteopontin (OPN) is an acid phosphorylated glycoprotein secreted by cells of the immune system, epithelial cells, smooth muscle cells, osteoblasts, osteoclasts, tumor cells and many more. Osteopontin is a multifunctional protein. Due to characteristic molecular structure containing integrin bining domains, OPN can interact with several integrin receptors, thus it play a role in activation, adhesion and migration of many cell types in such processes as inflammation, tissue mineralization and tumor genesis. This broad biological action of osteopontin underlie its presumed role in the pathogenesis of cardiovascular diseases, including atherosclerosis and abdominal aortic aneurysm formation.

Association of HLA-*08:DRB1*03 with immunoglobulin A-deficiency.

OBJECTIVE: Susceptibility to IgA deficiency (IgAD) is strongly associated with alleles of HLA, but it is not equally strong in different human populations. Therefore, the goal of this study was to determine the HLA-A, -B and -DRB1 antigenic and haplotypic frequencies in unrelated Polish Caucasian IgA-deficient patients who had never been examined so far in this respect. METHODS: The HLA alleles were determined by means of low resolution polymerase chain reaction with sequence specific primers (PCR-SSP) method in a group of IgA-deficient patients and control subjects from the same area. RESULTS: The HLA-DRB1*03 allele showed the strongest association with IgA deficiency in the Polish population (OR=6.6, p cor=0.0084). The HLA-B*08 allele was also associated with predisposition to the disease (OR=6.22, p cor=0.033). These significant associations could be explained in the context of a positive association of IgAD with the HLA-B*08:DRB1*03 haplotype, previously reported in other Caucasoid populations from Northern and Central Europe. In our group the HLA-B*08:DRB1*03 haplotype was present in 52.9% of IgA-deficient patients comparing to 9.9% in controls (p< 0.00011). A positive association of HLA-B*08 and DRB1*03 was stronger in IgA-deficient males than in females from the same group. CONCLUSION: Immunoglobulin A deficiency in Polish population is strongly associated with HLA-B*08:DRB1*03 haplotype rather than with single alleles.

[Causes of precocious puberty in children referred for evaluation in hospital conditions]. / Przyczyny przedwczesnego dojrzewania dzieci kierowanych do dianostyki w warunkach szpitalnych.

INTRODUCTION: Symptoms of precocious puberty (PP) in children always arouse anxiety in their parents. Many children with PP are being hospitalized for the detailed diagnostic work-up. The aim of our study was to analyze the frequency of the variants of PP in children referred to our department. MATERIAL: Retrospective analysis of 119 children (103 girls and 16 boys) referred for hospitalization in the years 2003-2005 due to signs of precocious puberty was performed. RESULTS: Premature thelarche, benign variant of puberty, was diagnosed in 62 (53%) girls, in the mean age of 3.39 (+/- 2.33) years. Their mean height was within 0.7 +/- 1.1 SD. Premature pubarche was diagnosed 30 (25%) children--22 girls and 8 boys in the mean age was 7.24 (+/- 0.81) years. Their mean height was 1.3 +/- 1.0 SD and was significantly higher than normal (p < 0.0001). Premature menarche was diagnosed in 8 (7%) girls in the mean age 4.81 +/-2.26 years. Mean height in this group was normal for age (0.9+/-0.8 SD). PP was diagnosed in 19 (16%) children (11 girls and 8 boys) in the mean age 5.91 +/- 1.63 years. Mean height in this group was 1.6 +/- 0.7 SD, and was significantly higher than the mean for age (p<0.0005). GnRH-dependent type was present in 15 children, diagnosed as idiopathic in 9 girls and 1 boy. In 5 children (4 boys and 1 girl) pathology of central nervous system was found. In 4 children GnRH-independent precocious puberty was diagnosed--in 3 caused by congenital adrenal hyperplasia and in 1 boy by tumour of testis (leydigioma). CONCLUSIONS: Girls with precocious thelarche without growth acceleration present the benign variant of puberty and need clinical follow up only. Boys with clinical signs of precocious puberty should be carefully evaluated to rule out the organic cause.

[IgA, an essential part of the immune system: selected issues]. / IgA istotny element ukladu odpornosci--wybrane zagadnienia.

Immunoglobulin class A is the main protein of the mucosal immune system. The daily production of this isotype exceeds the synthesis of all other classes of immunoglobulins. Because the mucosal surface area of the human organism is about 400 m2, the role of IgA in the defense of this surfaces from harmful agents from the external environment is extremely essential. Any changes connected with a lack or an overproduction of this immunoglobulin can manifest as different clinical diseases. In this paper some selected issues on the heterogeneity of the IgA are presented, its structure and significance for the human organism, the formation of IgA immunity during ontogenesis on one hand and antigenic stimulation on the other, as well as disorders connected with abnormal synthesis or structure of these molecules.

Purification and characterization of beta-mannosidases from white rot fungus Phlebia radiata.

A beta-mannosidase was purified from Phlebia radiata grown in a medium containing wheat bran or galactomannan as a carbon source. Maximal activity was observed at pH 5.5 and at 50 degrees C. Highly purified isoforms of beta-mannosidase (GM-1, GM-2) isolated from media containing galactomanan and (OT-1) media with wheat bran were obtained by means of column chromatography on Q-Sepharose, SP-Sepharose and chromatofocusing on Polybuffer Exchanger PBE-94.

[Influence of fat tissue distribution on metabolic complications in children and adolescents with simple obesity]. / Wplyw rozmieszczenia tkanki tluszczowej na zaburzenia metaboliczne u dzieci i mlodziezy z otyloscia prosta.

BACKGROUND: Increased prevalence of obesity in children and adolescents results in more common metabolic complications characteristic for adults, particularly those with abdominal obesity. The objective of the study was to determine the relation between the fat tissue distribution and metabolic complications and to estimate the prevalence of the metabolic syndrome in obese children and adolescents. MATERIAL AND METHODS: We studied 64 children (42 girls and 22 boys) with simple obesity (BMI =97 pc) in the mean age 12.4+/-3.4 years. The fat tissue distribution was assessed on the basis of waist circumference, hip circumference, waist to hip ratio (WHR) and skinfold thickness (suprailiac, subscapular, biceps and triceps). In all children plasma concentrations of total cholesterol, HDL and LDL cholesterol as well as triglycerides were estimated. Plasma glucose and insulin levels were measured in fasting state and during the oral glucose tolerance test (OGTT). Fasting insulin to glucose ratio (FIGR) was calculated. Blood pressure was measured in triplicate. RESULTS: In 33 (51.6%) of children dyslipidemia, in 10 (15.6%) hyperinsulinemia or impaired glucose tolerance and in 12 (18%) hypertension was diagnosed. The Metabolic syndrome was present in 9 (14%) children. The anthropometric predictor for the risk of metabolic complications was a greater waist circumference, while greater hip circumference decreased the risk. CONCLUSIONS: The metabolic complications characteristic of metabolic syndrome, previously diagnosed exclusively in adults, may occur also in obese children and adolescents. As in adults, abdominal obesity is the most relevant risk factor of the metabolic syndrome.