High CD39 expression is associated with the non-muscle-invasive phenotype of human bladder cancer

Background: An accurate prediction of progression is critical to define the management of bladder cancer (BC). The ectonucleotidases CD39 and CD73 play strategic roles in calibrating purinergic signals via an extracellular balance between ATP and adenosine. The altered expression of these enzymes plays a potential role in tumor invasion and metastasis, therefore, has been proposed to be used for prognosis of solid tumor. In BC this is not yet clear.Objective: This study aimed to evaluate CD39 and CD73 expression in a cohort of patients with non-muscle-invasive (NMI) and muscle-invasive (MI) BC regard to its association with clinicopathological features.Materials and Methods: Retrospective clinical follow-up data and primary urothelial BC specimens of 162 patients were used (87 from patients who underwent transurethral resection and 75 from cystectomized patients). Tissue microarrays were constructed, and immunohistochemistry for CD39 and CD73 was performed to make associations with clinicopathological data.Results: Overall, 96 were NMI (59.3%) and 66 MI (40.7%). CD39 immunoreactivity in BC cells was found in 72% of the cases, while CD73 was found in 97%. High CD39 expression alone was more frequent in NMI BC (p < 0.001), while CD73 expression was not powerful to predict the stage of BC. The association of both markers confirmed that only CD39 has potential in BC prognosis.Conclusions: The altered expression of CD39 presented herein supports the idea that this ectonucleotidase may be involved in bladder tumorigenesis. High expression of CD39 in tumor cells is correlated with the early stage of BC.     

Interspike interval analysis in a patient with peripheral nerve hyperexcitability and potassium channel antibodies

Neuromyotonia or Isaacs' syndrome is a rare peripheral nerve hyperexcitability disorder caused by antibodies against potassium channels of myelinated axons. We present the high-density surface electromyographic (EMG) recordings of a patient with fasciculations and cramps due to neuromyotonia. To characterize the time course of hyperexcitability, we analyzed the interspike intervals (ISIs) between fasciculation potentials, doublet, and multiplet discharges. ISI duration increased within each burst. The ISI histograms found can be explained by the recovery cycle of the myelinated axon and its dependency on the slow potassium conductance. We conclude that ISI analysis is a useful tool to understand the membrane dynamics underlying abnormal motor unit activity.     

Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.     

Modulating glypican4 suppresses tumorigenicity of embryonic stem cells while preserving self-renewal and pluripotency

Self-renewal and differentiation of stem cell depend on a dynamic interplay of cell-extrinsic and -intrinsic regulators. However, how stem cells perceive the right amount of signal and at the right time to undergo a precise developmental program remains poorly understood. The cell surface proteins Glypicans act as gatekeepers of environmental signals to modulate their perception by target cells. Here, we show that one of these, Glypican4 (Gpc4), is specifically required to maintain the self-renewal potential of mouse embryonic stem cells (ESCs) and to fine tune cell lineage commitment. Notably, Gpc4-mutant ESCs contribute to all embryonic cell lineages when injected in blastocyts but lose their intrinsic tumorigenic properties after implantation into nude mice. Therefore, our molecular and functional studies reveal that Gpc4 maintains distinct stemness features. Moreover, we provide evidence that self-renewal and lineage commitment of different stem cell types is fine tuned by Gpc4 activity by showing that Gpc4 is required for the maintenance of adult neural stem cell fate in vivo. Mechanistically, Gpc4 regulates self-renewal of ESCs by modulating Wnt/β-catenin signaling activities. Thus, our findings establish that Gpc4 acts at the interface of extrinsic and intrinsic signal regulation to fine tune stem cell fate. Moreover, the ability to uncouple pluripotent stem cell differentiation from tumorigenic potential makes Gpc4 as a promising target for cell-based regenerative therapies. Stem Cells2012;30:1863-1874.