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.

[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.

[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.

[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.

[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.