Quantification of cancer risk in glomerulonephritis.

BACKGROUND: The association of increased cancer risk with glomerulonephritis (GN) is well known, but controversy exists concerning which types of GN are involved, and the size of the association. A national registry survey was performed to assess the size of this association, and the temporal relationship of cancer diagnosis to GN diagnosis. METHODS: All patients with biopsy-proven GN between 1985 and 2015 in Denmark were extracted from The Danish Renal Biopsy Registry and the National Pathology Data Bank. Incident cancer diagnoses between 10 years previous and 10 years subsequent to the GN diagnosis were extracted from the Danish Cancer Registry. Residence, birth and death data were obtained from the National Patient Register. Expected cancer incidence, classified according to cohort, age and sex were extracted from the Nordcan database. RESULTS: Nine hundred eleven cancers were diagnosed in 5594 patients. Thirty five percent were prevalent at renal biopsy. Prevalence at biopsy was 5.5% (expected 3.1%), but incidence was not increased < 1 year before biopsy. Increased cancer rates were seen for GN forms: minimal change, endocapillary, focal segmental glomerulosclerosis, mesangioproliferative, membranous, focal segmental, membranoproliferative, proliferative, ANCA-associated vasculitis, lupus nephritis and unclassified. Increased cancer rates were seen for lung, prostate, renal, non-Hodgkin lymphoma, myeloma, leukaemia and skin. The increased incidence was mainly limited to - 1 to 1 year after biopsy, but skin cancer showed an increased risk over time. Some diagnoses showed an increase 5-10 years after biopsy. Incidence was raised for patients with uraemia and nephrosis, but less for proteinuria or haematuria. Cancers in patients < 45 years were rare. The risk of developing cancer 0-3 years after biopsy for patients 45-64 years varied from 7.3% (minimal change) to 15.8% (unclassified GN); > 64 years from 11.8 (endocapillary GN) to 20.3% (unclassified). The diagnosis with the highest risk was membranoproliferative GN (8.6 & 19.6%). CONCLUSIONS: Cancer rates are increased for many cancer and most GN diagnoses. Cancer screening for patients < 45 years and for patients without nephrosis or uraemia may not be necessary. The findings suggest that screening programs for specific GN diagnoses can be extended to other GN forms.

Ultrastructural characterization of mesenchymal stromal cells labeled with ultrasmall superparamagnetic iron-oxide nanoparticles for clinical tracking studies.

INTRODUCTION: To evaluate survival and engraftment of mesenchymal stromal cells (MSCs) in vivo, it is necessary to track implanted cells non-invasively with a method, which does not influence cellular ultrastructure and functional characteristics. Iron-oxide particles have been applied for cell tracking for years, but knowledge regarding possible cytotoxic ultrastructural changes subsequent to iron-oxide particle labeling is limited. Hence, the purpose of this study was to label MSCs with dextran-coated ultrasmall super-paramagnetic iron-oxide (USPIO) particles conjugated with the transduction sequence of trans-activator of transcription (TAT) (IODEX-TAT) and evaluate the effect of labeling on ultrastructure, viability, phenotype and proliferative capacity of the cells. MATERIALS AND METHODS: MSCs were labeled with 5 and 10 µg IODEX-TAT/10(5) cells for 2, 6 and 21 hours. IODEX-TAT uptake and cellular ultrastructure were determined by electron microscopy. Cell viability was determined by propidium iodide staining and cell proliferation capacity by 5-bromo-2-deoxyuridine (BrdU) incorporation. Maintenance of stem cell surface markers was determined by flow cytometry. Results. IODEX-TAT labeling for 2, 6 and 21 h did not influence cellular ultrastructure or viability. Moreover, neither stem cell surface markers nor cell proliferation capacity was affected by labeling with IODEX-TAT. CONCLUSION: Our results demonstrate that labeling of MSCs for 21 h with a clinically relevant dose of 10 µg IODEX-TAT/10(5) cells is feasible and does not affect MSC ultrastructure, viability, phenotype or proliferation capacity.

Ultrastructure of interstitial cells in subserosa of human colon.

We studied the ultrastructure of interstitial cells in the subserosal/adventitial layer in human colon. An interstitial cell type with an ultrastructure intermediate between fibroblast-like cells (FLC) and interstitial cells of Cajal was identified (IC-SS). IC-SS had thin and flattened branching processes, most densely arranged close to the longitudinal muscle cells. Caveolae, bundles of intermediate filaments and membrane-associated dense bands, often with a patchy basal lamina, were characteristic. Secretory organelles (granular endoplasmic reticulum, smooth endoplasmic reticulum, Golgi, coated vesicles) were prominent. The IC-SS ultrastructure was different from that of FLC in the longitudinal layer, which had no caveolae and fewer intermediate filaments. Peg-and-socket junctions between IC-SS and between IC-SS and muscle cells were present, and IC-SS processes had close, selective appositions to muscle cells. Gap junctions were not observed. Small nerve bundles were abundant, but close contacts (<100 nm) between IC-SS or muscle cells and nerves were inconspicuous. Close appositions between IC-SS and mast cells were present; close relations to macrophages were not observed. The myoid features of IC-SS are thus more pronounced in comparison with FLC of other locations in the gastrointestinal muscle. The organization and ultrastructure may suggest a regulatory nature of IC-SS on the colonic muscle layers.

Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA-based detection of bladder cancer.

Increased incidence and improved prognosis of glomerulonephritis: a national 30-year study.

BACKGROUND: While there are many cross-sectional studies of glomerulonephritis (GN) incidence, changes in incidence over time, particularly in the 21st century have received less attention. Similarly, little is known about temporal changes in GN prognosis. The presence in Denmark of comprehensive registries for renal biopsy results, end-stage renal disease (ESRD), comorbidity and mortality permit these questions to be addressed. METHODS: Data for all renal biopsies in Denmark between 1985 and 2014 were extracted from the Danish Renal Biopsy Registry and Patobank registries. The date of first dialysis or transplantation was extracted from the Danish Nephrology Registry for those patients developing ESRD. Dates of death and presence of chronic comorbid conditions at date of biopsy were extracted from the National Patient Registry. The incidence of GN, adjusted to the 2013 European standard population, was calculated. ESRD incidence and mortality were calculated, both in absolute terms and after correction for age, comorbidity and presence of renal tubulointerstitial fibrosis. RESULTS: The incidence rose from 33.3 patients per million (ppm)/year in 1985-94 to 46.5 ppm in 2005-14. The increase could in part be related to changes in renal biopsy policy. Large increases in Anti-neutropil cytoplasmic antibody (ANCA) vasculitis (ANCAV) (3.1-7.7 ppm/year) and focal segmental glomerulosclerosis (FSGS) (1.5-5.7 ppm/year) incidence were noted. The biopsy-proven prevalence of GN in 2014 was 748 ppm of which 155 ppm were being treated with dialysis or transplantation. Adjusted ESRD incidence fell by 25% during the study period, mortality by 62% and combined ESRD/mortality by 46%. The fall in ESRD incidence was limited to minimal change GN, FSGS, membranous GN and lupus nephritis, while reductions in mortality, and the combination of ESRD and/or death, were seen for nearly all GN diagnoses. CONCLUSIONS: This study suggests that the incidence of GN has generally increased between 1985 and 2014, but some of the increase may be related to changes in renal biopsy policy. Major increases in FSGS and ANCAV incidence have occurred. The prognosis of GN, both as regards ESRD and mortality, has improved.

Rituximab Treatment in a Patient with Kimura Disease and Membranous Nephropathy: Case Report.

Kimura disease (KD) is a chronic, inflammatory disorder with slowly developing subcutaneous tumor-like swellings, often occurring in the head and neck region. KD is diagnosed based on histology, elevated levels of immunoglobulin type E, and increased peripheral eosinophil granulocytes. KD may coexist with glomerular renal diseases, and this case report is based on a patient with KD-associated membranous nephropathy. Patients with membranous nephropathy without KD have demonstrated responsiveness to treatment with monoclonal anti-CD20 antibodies. This case report is the first to investigate the effect of rituximab treatment in a patient with KD-associated membranous nephropathy. A 30-year-old Italian man living in Denmark was diagnosed with Kimura's disease based on subcutaneous nodules with eosinophil angiolymphoid hyperplasia. The patient was admitted to the hospital due to nephrotic syndrome. Serology showed eosinophil granulocytosis and negative PLA2-receptor antibody. Renal biopsy showed membranous nephropathy, and the patient was treated with systemic methylprednisolone followed by cyclosporin and then cyclophosphamide with only partial remission. Ultimately, treatment with intravenous rituximab was initiated, which resulted in overall remission and no nephrotic relapses at 30 months of follow-up. Thus, intravenous rituximab effectively decreased proteinuria and prevented nephrotic relapses in a patient with treatment-refractory membranous nephropathy due to KD.

YKL-40 and mast cells are associated with detrusor fibrosis in patients diagnosed with bladder pain syndrome/interstitial cystitis according to the 2008 criteria of the European Society for the Study of Interstitial Cystitis.

AIMS: Bladder pain syndrome/interstitial cystitis (BPS/IC), diagnosed according to the new 2008 criteria of the European Society for the Study of Interstitial Cystitis (ESSIC), may lead to detrusor fibrosis. In some inflammatory diseases, fibrosis is related to YKL-40. The aims were to examine YKL-40 antigenic expression in bladder tissue and levels in serum and urine in BPS/IC and to evaluate whether YKL-40 could be a non-invasive, prognostic biomarker for bladder fibrogenesis and treatment intensity. METHODS AND RESULTS: Immunohistochemistry, immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) analyses in 45 patients showed YKL-40 expression in detrusor mast cell granules and submucosal macrophages, and elevated YKL-40 levels in serum and urine compared to healthy individuals (median 72 versus 7 µg/l, P < 0.001). Clinicopathological parameters showed associations of detrusor fibrosis with YKL-40-positive cells (P = 0.001), mast cells (P = 0.014) and urine YKL-40 (P = 0.009). Bladder capacity correlated inversely with YKL-40-positive cells (P < 0.001) and mast cells (P = 0.029). Treatment intensity was not associated with YKL-40. CONCLUSION: Serum and urine levels of YKL-40 may be used as non-invasive biomarkers in BPS/IC for the evaluation of bladder fibrogenesis.

Bladder pain syndrome/interstitial cystitis in a Danish population: a study using the 2008 criteria of the European Society for the Study of Interstitial Cystitis.

OBJECTIVE: To characterize and evaluate a Danish patient population with bladder pain syndrome/interstitial cystitis (BPS/IC), using a working definition for BPS/IC incorporating six variables, and a set of criteria defined by the European Society for the Study of Interstitial Cystitis (ESSIC); to describe the clinical course and treatment intensity in relation to these variables. PATIENTS AND METHODS: Clinical data were obtained retrospectively from medical records for 349 consecutive patients with IC referred to the Department of Urology, Copenhagen University Hospital Herlev, Denmark between 1966 and 2008. The median (range) age at diagnosis was 53 (16-88) years; 64% were followed for at least 2 years. The outcome was expressed in terms of treatment intensity and was correlated with clinical data (pain, nocturnal frequency, bladder capacity, mucosal glomerulations, detrusor mastocytosis, detrusor intrafascicular fibrosis, IFF). RESULTS: All patients had pain and 75% had nocturia at least twice. The bladder capacity estimated under general anaesthesia was <500 mL in 42%; 53% presented with detrusor mastocytosis (> or =28 mast cells/mm(2)) and 50% with IFF. The detrusor mast cell count (P < 0.001), IFF (P = 0.004) and nocturnal frequency (P = 0.043) had statistically significant prognostic value for treatment intensity, whereas bladder capacity and glomerulations were not significant. CONCLUSION: Nocturia, detrusor mastocytosis and IFF are associated with multiple treatments and presumed failure of standard urological therapy in patients with BPS/IC, while bladder capacity and glomerulations are not. Valid conclusions cannot be drawn because of numerous limitations to the study.

Evaluation of analytical accuracy of HER2 status in patients with breast cancer: Comparison of HER2 GPA with HER2 IHC and HER2 FISH.

Human epidermal growth factor receptor 2 (HER2) gene status and overexpression, occurring in ~ 13.6% of primary breast cancers, is essential for identifying patients likely to benefit from biological treatment. In this method of evaluation study, we tested and compared the HER2 gene-protein assay (GPA) with routine HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The GPA was evaluated using 67 formalin-fixed paraffin-embedded (FFPE) HER2 equivoval IHC (2+) breast cancer tissue samples. Overall, agreement between GPA silver in situ hybridization (SISH) and FISH was 91.9% (57/62). Regression analysis revealed slightly higher, but non-significant difference in HER2/chromosome enumeration probe 17 (CEP17) ratio for GPA as compared to FISH (p = 0.074). Intraclass correlation coefficients (ICCs) of 0.94 and Spearman´s rank correlation coefficients of 0.93 (p < 0.0001) for FISH and GPA SISH suggested strong inter-observer association for methods with one observer counting on average 0.23 significant higher for GPA SISH (p = 0.014). Intra-observer IHC method reproducibility was 52.6% (κ = 0.3122, p = 0.004) and 79.7% (κ = 0.6428, p = 0.9197), suggesting fair significant and substantial non-significant difference between tests for reviewers. Inter-observer reproducibility for IHC methods was 53%. While inter-observer reproducibility for experienced IHC interpretation suggested significant differences (κ = 0.3636, p = 0.0332), unexperienced interpretation of IHC GPA suggested fair non-significant difference between reviewers (κ = 0.3101, p = 0.0747). Using FISH as reference, the diagnostic indices for GPA SISH were as follows: sensitivity 100%, specificity 95% and accuracy 92%. Inaccuracy between tests was in 80% of cases due to ISH categorization as equivocal by one of the methods. IHC results highlight that it may be beneficial with a method for simultaneously visualization of HER2 gene and protein status.

Ultrastructure of Cajal-like interstitial cells in the human detrusor.

The aim of this ultrastructural study was to examine the human detrusor for interstitial cells of Cajal (ICC)-like cells (ICC-L) by conventional transmission electron microscopy (TEM) and immuno-transmission electron microscopy (I-TEM) with antibodies directed towards CD117 and CD34. Two main types of interstitial cells were identified by TEM: ICC-L and fibroblast-like cells (FLC). ICC-L were bipolar with slender (0.04 microm) flattened dendritic-like processes, frequently forming a branching labyrinth network. Caveolae and short membrane-associated dense bands were present. Mitochondria, rough endoplasmic reticulum and Golgi apparatus were observed in the cell somata and cytoplasmic processes. Intermediate filaments were abundant but no thick filaments were found. ICC-L were interconnected by close appositions, gap junctions and peg-and-socket junctions (PSJ) but no specialised contacts to smooth muscle or nerves were apparent. FLC were characterised by abundant rough endoplasmic reticulum but no caveolae or membrane-associated dense bands were observed; gap junctions and PSJ were absent and intermediate filaments were rare. By I-TEM, CD34 gold immunolabelling was present in long cytoplasmic processes corresponding to ICC-L between muscle fascicles but CD117 gold immunolabelling was negative. Thus, ICC-like cells are present in the human detrusor. They are CD34-immunoreactive and have a myoid ultrastructure clearly distinguishable from fibroblast-like cells. ICC-L may be analogous to interstitial cells of Cajal in the gut.

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon.

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer.

Expression and function of toll-like receptor 8 and Tollip in colonic epithelial cells from patients with inflammatory bowel disease.

OBJECTIVE: Growing evidence indicates that innate immunity, including toll-like receptor (TLR) signalling, plays a role in inflammatory bowel disease (IBD). This may also apply in the case of TLR-8, which has recently been shown to reverse the immunosuppressive function of regulatory T cells. However, the role of TLR-8 in IBD is currently unknown, and therefore we investigated the expression of TLR-8 and its natural antagonist, Tollip, in normal and inflamed human gut, and examined whether the receptor is functionally active. METHODS: TLR-8 and Tollip mRNA expression were measured in colonic epithelial cells (CEC) and lamina propria mononuclear cells (LPMNC) by quantitative polymerase chain reaction. TLR-8 protein expression was visualized in whole biopsy specimens by indirect immunofluorescence microscopy. Cellular localization of TLR-8 protein was assessed by immuno-electron microscopy. IL-8 secretion was measured by ELISA after stimulation with TLR-8 ligand. RESULTS: TLR-8 mRNA and protein expression were substantially up-regulated in CEC from inflamed mucosa from patients with ulcerative colitis (approximately 350-fold, p<0.01) and Crohn's disease (approximately 45-fold, p<0.05) compared to controls. TLR-8 proteins resided on the luminal surface membrane and in intracellular organelles. Tollip was not increased in CEC from IBD patients. CEC from normal mucosa responded to TLR-8 stimulation by secreting IL-8. TLR-8 was expressed only on the mRNA level in LPMNC with no differences between IBD patients and controls. CONCLUSION: Expression of TLR-8, but not Tollip, is highly up-regulated in the colonic epithelium from patients with active IBD. Since the receptor is functionally active, our data suggest that TLR-8 signalling is important in the pathogenesis of IBD.

Hypertensive nephropathy is associated with an increased risk of myeloma, skin, and renal cancer.

Previous studies suggest an increased cancer risk in hypertension. Patients with hypertensive nephropathy have not been studied. A national registry study was performed to assess the presence and size of this association. Clinical data and cancer diagnoses for all patients with biopsy-proven hypertensive nephropathy between 1985 and 2015 in Denmark were extracted from four national registries and compared with age- and sex-adjusted national cancer rates. The risk of cancer was twice the background population. It was raised for renal cancer (odds ratio 10.4), myeloma (13.2), skin cancer (7.9), and other/unspecified (1.8). No increase in incidence was seen until 1 year before renal biopsy and then rose rapidly. It was again normal 5 years after biopsy. Hypertensive nephropathy is associated with an increased risk of myeloma, skin, renal, and other cancers. Screening of patients with hypertensive nephropathy, in the presence of reduced renal function or significant proteinuria, may be indicated.

Evidence of Augmented Intrarenal Angiotensinogen Associated With Glomerular Swelling in Gestational Hypertension and Preeclampsia: Clinical Implications.

Background AGT (angiotensinogen) synthesis occurs in renal proximal tubular epithelial cells, independent from systemic AGT , as a component of the intrarenal renin-angiotensin system. We investigated urinary AGT , as a biomarker for renin-angiotensin system activation, and electrolyte concentrations, in relation to glomerular volume, as a proxy for glomerular endotheliosis in renal biopsy tissue from pregnant normotensive control and hypertensive women. Methods and Results Urine samples were collected from normotensive control (n=10), gestational hypertensive (n=6), and pre-eclamptic (n=16) women at the time a renal biopsy was obtained. Samples were collected from Lund University Hospital between November 1999 and June 2001. Urinary AGT , potassium, and sodium were measured, normalized to urinary creatinine. Mean glomerular volume was estimated from biopsy sections. AGT protein expression and localization were assessed in renal biopsies by immunohistochemistry. Urinary AGT concentrations were higher in hypertensive pregnancies (median, gestational hypertension: 11.3 ng/mmol [interquartile range: 2.8-13.6]; preeclampsia: 8.4 ng/mmol [interquartile range: 4.2-29.1]; normotensive control: 0.6 ng/mmol [interquartile range: 0.4-0.8]; P<0.0001) and showed a positive relationship with estimated mean glomerular volume. Urinary potassium strongly correlated with urinary AGT ( P<0.0001). Although numbers were small, AGT protein was found in both glomeruli and proximal tubules in normotensive control but was present only in proximal tubules in women with hypertensive pregnancy. Conclusions This study shows that pregnant women with gestational hypertension or preeclampsia have increased urinary AGT and potassium excretion associated with signs of glomerular swelling. Our data suggest that the kidneys of women with hypertensive pregnancies and endotheliosis have inappropriate intrarenal renin-angiotensin system activation, which may contribute toward the pathogenesis of hypertension and renal injury.

CD34-positive interstitial cells of the human detrusor.

Interstitial cells of Cajal (ICC) are well described in the bowel wall. They are c-kit positive and play a role as pacemaker cells. Similar c-kit-positive cells have recently been described in the human bladder. The aim of this study was to characterize interstitial cells of the bladder detrusor using a panel of antibodies directed against CD117/c-kit, CD34, CD31, S100, tryptase, neurofilament, NSE, Factor-VIII and GFAP. A striking finding was an interstitial type of cell which is CD34 immunoreactive (CD34-ir) but CD117/c-kit negative. The cells have a tentacular morphology, enveloping and intermingling with individual muscle fasicles. Morphologically and immunohistochemically, they show no neurogenic, endothelial or mast cell differentiation. Transmission electron microscopy (TEM) showed the presence of interstitial cells with a round-to-oval nucleus, sparse perinuclear cytoplasm and long flattened processes, ramifying primarily in a bipolar fashion. Using immunoelectron microscopy (I-TEM) it was possible to view CD34 gold labelling of cells corresponding to interstitial cells. Although similar CD34-positive cells have been demonstrated in the bowel wall, they have never been described in the detrusor. The ontogeny and function of CD34-ir, a kit-negative cell, is unknown, but it may be involved in smooth muscle contraction.

Influence of angiogenesis inhibitors on endothelial cell morphology in vitro.

Human umbilical vein endothelial cells (HUVEC) propagated in co-culture with fibroblasts form capillary-like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co-cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co-culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary-like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co-culture untreated HUVEC formed capillary-like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel-Palade bodies and transport vesicles. Immuno-EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell-cell contacts, and at the luminal and abluminal side of the capillary-like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor-treated co-cultures. Our results demonstrate that treatment with levamisole or anti-VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP-470 results in formation of short endothelial cell cords. We discuss the implications of these findings.

Transitional cell bladder tumor: predicting recurrence and progression by analysis of microsatellite loss of heterozygosity in urine sediment and tumor tissue.

Transitional cell bladder tumors (TCT) is prone to recurrence (60-80%) after tumor resection. Up to 25% of these patients will progress, so it is important to find reliable predictive markers. We analyzed for loss of heterozygosity (LOH) with respect to 13 microsatellites located on 10 different chromosomal arms. This analysis was performed on the urine sediment and tumor tissue from 59 patients with bladder TCT and on the urine and normal-looking mucosa from 25 patients with a history of bladder TCT but no evidence of disease at the time of the study inclusion. The median follow-up period was 23.1 months (range, 2-48 months) for the 59 patients with bladder TCT and 25 months (range, 4-57 months) for the 25 patients without evidence of ongoing active disease. Correlation between LOH and eventual recurrence, progression, and mortality was investigated. In patients with noninvasive TCT, correlation between 11p tumor tissue LOH and recurrence was found. Similarly, 8p LOH in both urine sediment and tumor tissue correlated with progression. Finally, in the group of patients with a history of bladder TCT, normal tissue 8p and/or 11p LOH correlated with recurrence.

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2.

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Under-representation of bladder transitional cell tumour 9q, 11p and 14q LOH in urine and impact on molecular diagnosis.

BACKGROUND: To investigate whether the recently reported evidence of differences in the overall loss of heterozygosity (LOH) frequency between urine and tumour tissue in patients with transitional cell tumours (TCT) of the urinary bladder involved specific chromosomal sites, and their impact in diagnosis. MATERIALS AND METHODS: Blood, tissue and urine specimens were obtained from 55 patients and 25 controls. Sixteen microsatellites were PCR-amplified and blindly analyzed for LOH through a laser-based capillary electrophoresis system. RESULTS: Significant frequence differences between tumour tissue and urine sediment LOH were found in 9q and 11p in non-invasive disease and 14q in invasive disease. There was no significant difference for all the other chromosomal arms analyzed. CONCLUSION: The contribution in the urine sediment of cells belonging to tumours of the same histological classification differs according to the specific genetic alterations these cells carry. Furthermore, the location regarding these differences could indicate regions involved in tumour exfoliation or apoptosis.

Breast carcinoma metastasis to the lacrimal gland: Two case reports.

A 77-year-old female, with proptosis, reduced eye motility and diplopia which had developed over two to three months and a 69-year-old female with proptosis, oedema of the eyelid, reduced motility and ptosis, which had developed over three weeks, are presented in the present study. Computed tomography scans revealed irregular lacrimal gland tumours in the two patients. The two patients had history of breast cancer. The first breast cancer metastasis in the lacrimal gland demonstrated a cribriform growth pattern containing ductal elements. The epithelial tumour cells stained positive for cytokeratin (1-8, 10, 14-16, 18 and 19), oestrogen receptor, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and gross cystic disease fluid protein 15 (GCDFP-15). The second metastatic tumour was positive for EMA and estrogen receptor, but variably positive for CEA and GCDFP-15. The metastasis in the lacrimal gland was a pleomorphic tumour. The tumour cells were positive for EMA and variably positive for oestrogen and CEA. Metastases to the lacrimal gland are extremely rare, and metastases to the lacrimal gland should be considered in the diagnoses of lacrimal gland tumours. The present study aimed to describe two such cases and draw attention to breast carcinomas as a differential diagnosis and the most frequent cause of lacrimal gland metastasis.