Time-dependent reduction of structural complexity of the buccal epithelial cell nuclei after treatment with silver nanoparticles.

Recent studies have suggested that silver nanoparticles (AgNPs) may affect cell DNA structure in in vitro conditions. In this paper, we present the results indicating that AgNPs change nuclear complexity properties in isolated human epithelial buccal cells in a time-dependent manner. Epithelial buccal cells were plated in special tissue culture chamber / slides and were kept at 37°C in an RPMI 1640 cell culture medium supplemented with L-glutamine. The cells were treated with colloidal silver nanoparticles suspended in RPMI 1640 medium at the concentration 15 mg L⁻¹. Digital micrographs of the cell nuclei in a sample of 30 cells were created at five different time steps: before the treatment (controls), immediately after the treatment, as well as 15 , 30 and 60 min after the treatment with AgNPs. For each nuclear structure, values of fractal dimension, lacunarity, circularity, as well as parameters of grey level co-occurrence matrix (GLCM) texture, were determined. The results indicate time-dependent reduction of structural complexity in the cell nuclei after the contact with AgNPs. These findings further suggest that AgNPs, at concentrations present in today's over-the-counter drug products, might have significant effects on the cell genetic material.

Prognostic value of survivin expression in Wilms tumor.

PURPOSE: To determine survivin expression patterns in Wilms tumor (WT) and compare it with the expression in normal renal tissue. Also, to analyse cytoplasmic and nuclear survivin expression in relation to histological type, prognostic group and tumor stage. METHODS: Immunohistochemical expression of survivin was analysed in 59 cases of primary WT and in 10 normal kidney specimens, taken from the same patients, but distant from the tumor. RESULTS: 51 out of 59 cases of WT (86.44%) showed decreased cytoplasmic survivin expression and 4 out of 59 cases of WT (6.78%) showed nuclear overexpression of survivin. There was statistically significant difference in the frequency of decreased cytoplasmic expression of survivin in individual components of WT (p=0.005). Decreased cytoplasmic expression of survivin in epithelial, blastemal and stromal component was found significantly more often in low stage WT compared to high stage WT (Fisher exact test, p=0.0002, p=0.002, p=0.002, respectively). There was no statistically significant difference in the frequency of survivin nuclear overexpression between different stages of WT (Fisher exact test, p=0.564), histological types (Fisher exact test, p=0.915), or between different prognostic groups (Fisher exact test, p=1). CONCLUSION: Decreased survivin cytoplasmic expression or nuclear overexpression may be related to favorable prognosis of WT.

Immunohistochemical detection of cyclin E in transitional cell carcinoma.

PURPOSE: It is known that expression disorders of cell cycle regulators play an important role in the development and prognosis of various malignant tumors. Cyclin expression changes during the cell cycle. This work aimed to analyse the expression of cyclin E in transitional cell carcinoma (TCC) and also to compare the expression of cyclin E with tumor stage and histological grade as well as to determine possible existence of differences in the expression of cyclin E in TCCs of the upper and lower urothelium. METHODS: Twenty-four cases of TCC of the urinary tract were retrospectively analysed (6 cancers of the renal pelvis, 2 of the ureter and 15 of the bladder; 4 were infiltrative). Immunohistochemical staining for cyclin E of the analysed transitional cancer cells was assessed semiquantitatively: diffuse cyclin E expression + + + (> 50% of all cells), expression in larger groups of cells: + + (up to 50% of all cells), expression in individual cells or small cell clusters: + (<10% of all cells), and absence of expression. Tumor stage was based on clinical and morphological criteria. WHO classification (Lyon 2004) was used for determination of the histological grade. RESULTS: Non-parametric Spearman's correlation showed that there was no statistically significant correlation between tumor stage and expression of cyclin E (ρ = -0331, p> 0.05). Also, no statistically significant correlation between grade and the expression of cyclin E (ρ = -0077, p> 0.05) was found. x2 test results showed no statistically significant difference (x2 = 2.136, p = 0.775) in the expression of cyclin E between upper and lower urothelium. CONCLUSION: This study showed non significant decreased expression of cyclin E with poor differentiation, muscle invasion and upper/lower urothelium. Expression of cyclin E decreased with increasing histological grade and stage of the tumor.

Survivin expression in odontogenic keratocysts and correlation with cytomegalovirus infection.

OBJECTIVES: The aim of this study was to investigate the expression of survivin, an inhibitor of apoptosis, in odontogenic keratocysts and to compare it to the findings in non-neoplastic jaw cysts - periapical cysts, as well as to establish a possible relationship between survivin expression and human cytomegalovirus presence within these cysts. MATERIALS AND METHODS: Samples of 10 odontogenic keratocysts (five positive and five negative for the presence of cytomegalovirus, as determined by polymerase chain reaction) and 10 periapical cysts (five positive and five negative for the cytomegalovirus presence) were analysed. The expression of survivin was assessed by immunohistochemical methods, using monoclonal antibody that selectively recognizes the cytoplasmic form of survivin. RESULTS: All 10 odontogenic keratocysts showed immunostaining for survivin, while all 10 periapical cysts were negative for its presence. There was no correlation between cytomegalovirus presence and expression of survivin within odontogenic keratocysts. CONCLUSION: Survivin may contribute to the aggressive behavior of odontogenic keratocysts, and thus support the emerging opinion of their neoplastic nature.

Does the pathohistological pattern of renal biopsy change during time?

Biopsy registries are one of the most important sources of accurate epidemiological data and the clinical presentation of renal diseases. A detailed analysis of clinicopathologic correlations over a period of 20 years (1987-2006) was performed earlier by our centre. The aim of this study was to check the current state and to register possible changes in clinicopathologic findings recorded under better socioeconomical circumstances and new management. Records of 665 renal biopsies performed at our institution were prospectively followed from 2007 to 2014. The results were compared with our previously published data. The average annual incidence of renal biopsies increased by 10% and included more elderly patients. Nephrotic syndrome (NS) remained the most common clinical indication for biopsy, while acute kidney injury participated more frequently than in the previous study (p < 0.001). Membranous nephropathy (MN) was still the most common cause of NS. Primary glomerulonephritis (PGN) remained the most prevalent disease, while MN was the most prevalent PGN. In comparison with the earlier period, MN was a more common diagnosis (p = 0.002), while the prevalence of mesangioproliferative non-IgA nephropathy decreased significantly during the time (p = 0.012). LN remained the most frequent secondary glomerulonephritis. The pathohistological pattern of renal biopsy remained largely unchanged during time. However, acute kidney injury was more frequently an indication for biopsy in the current study. The significant increase of biopsied elderly patients is due to the rise in their relative numbers in our population.

Regulation of selectivity and translocation of aquaporins: an update.

All living beings need to solve the problem of controlled transport of water. To this purpose, a special group of integral membrane proteins called aquaporins has evolved. There are 13 known members of this family that act as channels for water and small solutes, such as glycerol and urea. Although they allow large flux of water, they successfully prevent passage of protons. Here, we present the review of the data from the literature on the selectivity mechanism of aquaporins. The regulation of aquaporin activity occurs through regulation of expression of their genes, changing the localization of the already existing proteins in the cells and direct regulation of the activity in situ. We present the review of new data on the mechanisms of direct regulation. Special emphasis is on the advances in comprehension of aquaporin-2 translocation in collecting tubule cells of the kidney. Four elements of this process are described: 1) the role of protein kinase A and phosphorylation of serine 256 on aquaporin-2, 2) the transport of vesicles along the microtubules toward the apical membrane, 3), the removal of cytoskeletal subapical obstruction and the role of Rho GTPase and ezrin-radixin-moesin proteins in this, and 4) elevation of the cytosolic Ca2+ concentration, the fusion of the vesicle with the apical membrane and the role of SNARE proteins in exocytosis.

Acute Renal Failure - A Serious Complication in Patients After Kidney Transplantation.

Free radical-mediated injury releases proinflammatory cytokines and activates innate immunity. It has been suggested that the early innate response and the ischemic tissue damage play roles in the development of adaptive responses, which may lead to acute kidney rejection. Various durations of hypothermic kidney storage before transplantation add to ischemic tissue damage. The final stage of ischemic injury occurs during reperfusion that develops hours or days after the initial insult. Repair and regeneration processes occur together with cellular apoptosis, autophagy and necrosis and a favorable outcome is expected if regeneration prevails. Along the entire transplantation time course, there is a great demand for novel immune and nonimmune injury biomarkers. The use of these markers can be of great help in the monitoring of kidney injury in potential kidney donors, where acute kidney damage can be overlooked, in predicting acute transplant dysfunction during the early post-transplant periods, or in predicting chronic changes in long term followup. Numerous investigations have demonstrated that biomarkers that have the highest predictive value in acute kidney injury include NGAL, Cystatin C, KIM-1, IL-18, and L-FABP. Most investigations show that the ideal biomarker to fulfill all the needs in renal transplant has not been identified yet. Although, in many animal models, new biomarkers are emerging for predicting acute and chronic allograft damage, in human allograft analysis they are still not routinely accepted and renal biopsy still remains the gold standard.

Acute Renal Failure in Transplanted Kidneys.

Free radical-mediated injury releases proinflammatory cytokines and activates innate immunity. It has been suggested that the early innate response and the ischemic tissue damage play roles in the development of adaptive responses, which may lead to acute kidney rejection. Various durations of hypothermic kidney storage before transplantation add to ischemic tissue damage. The final stage of ischemic injury occurs during reperfusion that develops hours or days after the initial insult. Repair and regeneration processes occur together with cellular apoptosis, autophagy and necrosis and a favorable outcome is expected if regeneration prevails. Along the entire transplantation time course, there is a great demand for novel immune and nonimmune injury biomarkers. The use of these markers can be of great help in the monitoring of kidney injury in potential kidney donors, where acute kidney damage can be overlooked, in predicting acute transplant dysfunction during the early post-transplant periods, or in predicting chronic changes in long term followup. Numerous investigations have demonstrated that biomarkers that have the highest predictive value in acute kidney injury include NGAL, Cystatin C, KIM-1, IL-18, and L-FABP. Most investigations show that the ideal biomarker to fulfill all the needs in renal transplant has not been identified yet. Although, in many animal models, new biomarkers are emerging for predicting acute and chronic allograft damage, in human allograft analysis they are still not routinely accepted and renal biopsy still remains the gold standard.

Conversion of azathioprine to mycophenolate mofetil and chronic graft failure progression.

The purpose of the study was to evaluate the impact of conversion from azathioprine (AZA) to mycophenolate mofetil (MMF) on graft function in 35 renal transplant recipients with chronic allograft nephropathy (CAN). The immunosuppressive regimen originally consisted of AZA, cyclosporine (CsA), and prednisone (Pr). At the onset of the study (mean period = 39 posttransplant months), a graft biopsy was performed on all patients who were randomly divided into group 1 (n = 17) in whom MMF was introduced instead of AZA. The remaining 18 subjects (group 2) were maintained on the previous regimen. Two periods were analyzed: period I: 12 months before, and period II: 12 months after biopsy and therapy conversion. Graft function was assessed monthly by measurements of the 24-hour creatinine clearance (CCr). Analysis of variance (ANOVA) was used to compare the differences in CCr and proteinuria between the two groups. No difference was observed in the baseline characteristics, in the incidence of delayed graft function and acute rejection, or in the mean CsA dose. Pathohistological analysis revealed advanced CAN in the majority of patients in both groups. The morphological changes negatively correlated with graft function. The graft function showed parallel deterioration in the two groups; no significant difference was observed in the mean CCr values in the periods studied. Proteinuria was similar for both groups throughout the study. Conversion of AZA to MMF in recipients with CAN, albeit safe, was without significant benefit on the progression of chronic graft failure over the period of a year.

Morphometric analysis of glomerular basement membranes (GBM) in thin basement membrane disease (TBMD).

We measured the thickness of glomerular basement membrane in 46 patients with thin basement membrane disease (TBMD), (age range 15-50 years, almost equal M:F ratio), and compared with that in a control group of 5 patients (age range 5-38 years) with normal glomerular morphology. The measurements of glomerular basement membrane taken from electron micrographs (magnification x 12,500) were analyzed using an interactive image analysis system assembled around an INTEL 10 microcomputer, with a high resolution touch sensitive screen as the interactive peripheral. Calculation was done by printing on an electron micrograph a grating replica (21,600 lines/cm), with the same magnification as the electron micrographs of the glomeruli and calibrating the arithmetic (AM) and harmonic (HM) mean for each case. Comparing the results of TBMD cases (AM 129-202 nm; HM 128-213 nm) with those of the control group consisting of 5 cases of "minimal change nephrotic syndrome" (AM 287-317 nm; HM 300-333 nm) it was found that GBM in TBMD is remarkably thin. The thinning was caused mainly by the decreased width of the lamina densa (TBMD group: 71.4-147.0 nm; HM 72.4-154.4 nm in comparison with the control group: AM 174.4-235.5 nm; HM 184.2-249.6 nm). This finding allows us to differentiate thin basement membrane disease from other glomerulopathies presenting primarily with isolated or recurrent hematuria.

Application of artificial neural networks in estimating predictive factors and therapeutic efficacy in idiopathic membranous nephropathy.

Idiopathic membranous nephropathy (IMN) is one of the most frequent causes of the nephrotic syndrome in adults and one of the most common cause of chronic renal failure among primary glomerular diseases. The aim of this study was to develop artificial neural networks (ANN) to investigate factors of poor outcome for IMN and to evaluate the efficacy of different therapeutic protocols. Data were collected retrospectively for 124 patients with IMN (82 males, mean based on the received therapy patients were divided into three groups: corticosteroids only (group 1), cyclophsophamide with corticosteroids (group 2), and so called Ponticelli protocol (group 3). After achieving satisfactory truthfulness of the transcription function of ANN through clustering, we have applied the efficacy analysis to all patients and then compared them to each group separately, and evaluated the influence of initial characteristics on disease outcome as well as the therapy efficacy. The greatest therapy inefficiency was recorded for isolated corticosteroid therapy (29.41%) and the smallest inefficiency for Ponticelli protocol, for which the greatest accuracy of prognosis was recorded (82.09%). The greatest negative prognostic influence had kidney insufficiency (22%), quantitative proteinuria (15%) and index of interstitial infiltration (14%). Based on our results, we can recommend that patients diagnosed with IMN with renal insufficiency, nephrotic syndrome or a high degree of interstitial infiltration at the time of diagnosis should be treated concomitantly with cytotoxic drugs and corticosteroids, particularly with the Ponticelli protocol.

Umbilical metastasis (Sister Joseph's nodule) as a first sign of a disseminated ovarian carcinoma: comparative immunohistochemical analysis of primary tumor and its metastases.

The case of a 46-year-old female with umbilical metastasis as a first sign of an ovarian carcinoma is reported with the results of immunohistochemical analysis of primary tumor and lymph node and umbilical metastases. All specimens were positive for cytokeratin 7, CA 125, E-cadherin, alpha-, beta-, and gamma-catenin, as well as for MSH2. Staining with cytokeratin 20 and MLH1 was negative, and Ki-67 labeled from 5% (in the center of the lesions) to over 25% (at the periphery of the lesions) of the nuclei. Beta-catenin showed membranous positivity in the central parts and absence of staining at the periphery of ovarian tumor and umbilical metastasis, whereas lymph node metastasis presented with uniform reaction throughout. The results of immunohistochemical staining could point to the mechanisms employed by malignant tumors during invasion and growth of metastasis and suggest the possible role of the microenvironment in the expression of some adhesion molecules on tumor cells.

Spontaneous blastogenesis profiles and IL-2 receptor expression on kidney allograft.

To examine immune activation rate of interstitial and glomeruli infiltrating MNC in different conditions of human renal allograft function deterioration, 33 renal transplant biopsies were performed 1-30 months after transplantations. Forty-one patients observed were on immunosuppressives: Pr, Aza, CsA following renal transplantation from a living-related donor parent. The patients were divided according to their histologic diagnosis into the following groups: 1, 15 pts in acute rejection attack (AR); 2, 10 pts with cyclosporine nephrotoxicity (CsN); 3, 10 pts with chronic vascular rejecting kidney (ChR). A conventional histologic investigation and immunohistochemical analyses of CD3 and CD25 molecules were performed in groups 1-3. Spontaneous blastogenesis (SB) of peripheral lymphocytes was simultaneously determined and compared with the controls (C)-30 healthy people, and with patients with stable renal allograft function (S)-8 pts. The highest IL-2R expression on diffuse or focal dense MNC infiltrates in interstitium was observed during AR, without IL-2R+ MNC in glomeruli. Low to moderate focal interstitial infiltrates in damaged areas of renal parenchyma due to CsN, were IL-2R negative. In ChR, moderate IL-2R expression was observed on interstitial spare mild or focal dense MNC infiltrates with IL-2R expression present on glomeruli infiltrating MNC. Significant increases of SB values were recorded during the first week after transplantation and AR in comparison to C. The highest SB values were in ChR group. Values of SB in CsN and S were on the C and before transplantation levels.(ABSTRACT TRUNCATED AT 250 WORDS)

[Adhesion molecules in kidney diseases (part I)]. / Adhezioni molekuli u bolestima bubrega (I deo).

Adhesive molecules are (glyco)proteins of the cellular membranes. All of them have their extramembranous, transmembranous and intracytoplasmatic parts. As receptor molecules, their extracellular parts bind the specific ligand. The ligand can be found on the surface of the other cell or in the extracellular matrix (basal membranes). The following families of adhesion molecules are: cadherins, selectins, integrins and members of immunoglobuline supergene family. Different members of the same family could have different times (in ontogenesis, in adult form) and space distribution (in different tissues, different tissue structures). The contact between the cells and basal membranes with these molecules is important for cell division, maintaining the tissue architecture, polarization and function of cells, migration of cells, endo- and exo-cytosis as well as for maintaining the structure and function of basal membranes. As above stated all this is important in the occurrence morphogenesis, haemostasis, inflammation, malignant cell transformation and metastasis. This knowledge is important for the better understanding of renal diseases.

[Adhesion molecules in kidney diseases (part II)]. / Adhesioni molekuli u bolestima bubrega (II deo)]

Different adhesion molecules are involved in the maintenance of tissue architecture, morphogenesis, immunosurveillance, inflammation, tumour growth, etc. Thus, this review will be directed to the role of cadherins, selectins, integrins and members of the immunoglobuline supergene family in the pathogenesis of glomerulonephritis, acute renal failure, reaction of renal rejection, development of renal tumours, their invasion and metastases. A better understanding of the role of adhesion molecules in nephropathology may provide new aspects of treatment of different forms of renal diseases including tumours.

Correlation of glomerular basement membrane alterations with clinical data in progressive hereditary nephritis (Alport's syndrome).

Electron microscopic examination of glomerular basement membrane (GBM) was performed in 19 patients whose morphological changes as well as clinical features indicated the diagnosis of progressive hereditary nephritis (Alport's syndrome). The percentage of characteristically thickened and split and of thin GBM portions was determined in all the cases. The clinical course was more severe in males, which corresponded to higher rate of GBM alterations. In males, 58% of GBM was thickened and split and 24% was thin, while in females, the reverse was true, 28% was split and 48% of GBM was thin. There was a positive correlation of the split lesions and age in males, but not in females. The degree of splitting was directly proportional to the grade of proteinuria, while GBM thinning did not significantly correlate with proteinuria.

Laminin, fibronectin, and Goodpasture antigen detection in patients with Alport's syndrome.

Indirect immunofluorescence study with laminin and fibronectin monoclonal antibodies on paraffin sections, as well as with serum from a patient with Goodpasture's syndrome with high titer of autoantibodies that recognize the antigenic determinants in human glomerular and tubular basement membrane, was performed on 14 patients with Alport's syndrome and 5 specimens of normal renal tissue obtained from donors in cases of renal transplantation (control group). We found no binding of Goodpasture antigen to glomerular and distal tubular basement membranes in renal biopsy tissue from all 14 patients with Alport's syndrome. In contrast, there was bright linear fluorescence of Goodpasture antigen on glomerular and tubular basement membranes of normal renal material. There was no difference in laminin and fibronectin binding in patients with Alport's syndrome and controls. In all the cases binding was strongly positive. These results suggest an abnormality or absence of immunoreactive autoantigen in the glomerular and distal tubular basement membrane in patients with Alport's syndrome. Therefore, Goodpasture antigen detection could be an important diagnostic method in early stages of Alport's syndrome when characteristic morphological changes are not yet developed.

[Ultrastructural changes in transplantation glomerulopathy]. / Ultrastrukturne promene transplantacione glomerulpatije.

Twenty-eight biopsy specimens were obtained from patients 3-95 months after kidney transplantation and studied by light, electron and in some cases also by immunofluorescence microscopy. Electron microscopic studies showed that the most frequent glomerular lesion was widening of lamina rata interna which is accompanied with subendothelial accumulation of finely granular material, formation of new subendothelial basement membrane and deposition of microfibrils and fine filaments. The mesangial changes were mainly those of mesangiolysis and mesangial sclerosis with deposition of mesangial matrix and microfibrils, but little cellular proliferation. Fragmented red blood cells were seen in nearly half of the patients. Arterial intimal thickening and occasionally also thrombosis produced ischaemic changes in the kidney and in the glomeruli and contributed to the process of transplant rejection.

[Electron microscopic analysis of eosinophilic renal cell carcinoma]. / Elektronosko-mikrospopska analiza eosinofilnog karcinoma celija bubrega.

Renal cell carcinoma with cytoplasmic eosinophilic globules visualized on routine histologic preparations was analyzed. Eosinophilic globules in cytoplasm of the cells in renal cell carcinoma are very rate and till today we have not heard or found in the literature an attempt to analyze and describe them and that was the aim of our study. By electron microscopy, the globules most closely resembled non-membrane bound filamentous material that normally constitutes the cytoskeleton of normal and neoplastic renal epithelium.

[Intercellular adhesion molecule-1 expression in renal cell carcinoma]. / Ispoljavanje intercelulne adhezivne molekule-jedan u karcinomu celija bubrega.

The presence of intercellular adhesion molecule-1 (ICAM-1) was analyzed using indirect immunoperoxidase technique in frozen sections of 27 renal cell carcinomas (RCC)--18 clear cell, 6 granular, 2 chromophobe and 1 sarcomatoid. Great variations in ICAM-1 expression were observed in clear and granular cell RCC, without correlation with mononuclear (lympho-monocytic) cell infiltration. Sarcomatoid type displayed widespread ICAM-1 distribution and intense mononuclear infiltrate, while chromophobic RCC, despite better prognosis, expressed ICAM-1 weakly and focally, almost without mononuclear infiltrate. With such results, no relation between ICAM-1 expression on tumor cells and behavior of various RCC types could be established and possible explanations are: different histogenesis of some RCC (chromophobe), nonspecific upregulation of ICAM-1 as a result of ischaemic-necrotic processes, or negative effect of ICAM-1 expression on complement action.