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.

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.

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.

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.

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.

The resistance of delayed xenograft rejection to alpha(1,3)-galactosyltransferase gene inactivation and CD4 depletion in a mouse-to-rat model.

Critical to the prevention of xenograft loss is the prevention of delayed xenograft rejection (DXR), due to its resistance to conventional immunosuppression. The role of the carbohydrate galactose-alpha1,3-galactose (alpha1,3Gal) has been a matter of great debate and it has been proposed that the reaction between alpha1,3Gal epitopes on donor endothelial cells and recipient anti-alpha1,3Gal antibodies (Abs) may damage the graft during DXR. Recipient anti-alpha1,3Gal Abs are produced by CD4-dependent B cells. To test the above-mentioned hypothesis, hearts from alpha1,3Gal-free mice (GT-Ko mice), generated by alpha1,3-galacto-syltransferase gene disruption, were transplanted to anti-alpha1,3Gal antibody-free Lew/Mol rats. This model consists of an alpha1,3Gal/alpha1,3Gal-antibody-free environment, eliminating a possible influence of this specific system on DXR. A subgroup of recipients were furthermore CD4 depleted in order to inhibit CD4-dependent B-cell antibody production. Rejected hearts were evaluated by light- and immunofluorescence microscopy. Treatment effects on recipient T-cell subsets and cytokine expression were analyzed by flow cytometry, while antibody production was measured by ELISA. All recipients developed DXR with no differences among the groups. DXR was related to thrombosis with IgG and IgM desposition in vessel walls, as well as macrophage and granulocyte accumulation in the myocardium. No complement C3, CD4 cells or NK cells were found. Flow cytometric analysis confirmed peripheral blood CD4 depletion and IFN-gamma suppression in CD4 Ab-treated recipients. Finally, ELISA showed that specific anti-alpha1,3Gal Ab production was absent. However, Ab(s) against an unidentified Galalpha 1 were found among recipients. In our model, DXR is resistant to alpha1,3-galactosyltransferase gene inactivation and CD4 depletion. However, other Galalpha 1 epitopes and antibodies may play a role during DXR. Further studies are needed to elucidate the precise pathways leading to DXR.

Microsatellite analysis of urine sediment versus urine cytology for diagnosing transitional cell tumors of the urinary bladder.

The aim was to evaluate microsatellite analysis of urine sediment (MAUS) as an alternative method to urine cytology for routine diagnosis of patients with transitional cell tumors (TCT) of the urinary bladder. Urine cytology has the advantage of being non-invasive, fast and cheap, but is of limited value because of its low sensitivity. MAUS has previously been found to be a successful alternative method. However, the experimental set-up of such investigations implied exclusion of samples with unfavorable characteristics and use of a large number of markers. In the present study, MAUS was tested on all samples routinely available and a small panel of markers was selected. The urine sediments of 66 TCT patients and 24 controls were analyzed by MAUS with 16 fluorescent markers and by urine cytology. All samples were analyzed, including the ones of later micturition, with gross hematuria, leukocyturia or absence of visible sediment. In patients with tumors of low grade (grades I-II), MAUS was significantly more sensitive than urine cytology. The two methods were of equivalent diagnostic power in high-grade (grades III-IV), high-stage (pT1-pT4) tumors. A panel of the six most informative markers for MAUS was selected. Although MAUS has an advantage over routine cytology in low-grade, low-stage tumors, an overall sensitivity of 45% is not sufficient for routine clinical use.

New evaluation of plasma DNA microsatellite analysis in patients with TCC of the urinary bladder.

BACKGROUND: To determine the diagnostic value of plasma DNA microsatellite analysis in patients with transitional cell carcinoma (TCC) of the urinary bladder, by redefining plasma LOH from the equivalent analysis in controls. The method was further tested for MSI (microsatellite instability) and compared with tissue DNA analysis. MATERIALS AND METHODS: Sixteen microsatellites were amplified in leukocyte, plasma and tissue DNA from 40 patients and 28 controls, and analysed in a laser-based, capillary electrophoresis system. Plasma LOH was determined from the controls' cut-off values. RESULTS: The difference between plasma LOH frequency in patients (25% (10/40)) and controls (14% (4/28)) was not significant. Nevertheless, it occurred significantly more often in low rather than high-grade tumors (p=0.03) and controls (p=0.04). Plasma MSI was dependent upon the number of PCR cycles. Tissue LOH was present in 78% (31/40) of the patients and in none of the controls. Tissue MSI was uncommon. CONCLUSION: The results of plasma DNA microsatellite analysis in TCC need cautious interpretation.

Genetic analysis of an orbital metastasis from a primary hepatic neuroendocrine carcinoma.

A 71-year-old female with a known history of primary hepatic neuroendocrine carcinoma, presented with a visual defect, proptosis and restricted eye movements of the right eye. Biopsies from the orbit and from the primary hepatic neuroendocrine carcinoma showed similar morphological and immunohistochemical features, and high-resolution, array-based comparative genomic hybridization demonstrated loss of one copy each of chromosomes 3 and 18, and gain of 1q both in the primary hepatic neuroendocrine carcinoma and in the orbital tumour. The orbital mass was diagnosed as a metastasis from the primary hepatic neuroendocrine carcinoma. Primary hepatic neuroendocrine tumours are extremely rare, and the orbit is an extremely rare location for a neuroendocrine carcinoma metastasis. This is the first reported case of an orbital metastasis with origin from a primary hepatic neuroendocrine carcinoma.

Custom-designed MLPA using multiple short synthetic probes: application to methylation analysis of five promoter CpG islands in tumor and urine specimens from patients with bladder cancer.

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.