Nanoscale flow cytometry to distinguish subpopulations of prostate extracellular vesicles in patient plasma.

OBJECTIVE: To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance. MATERIALS AND METHODS: Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA). RESULTS: By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer. CONCLUSION: The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate.

FUS-CHOP Promotes Invasion in Myxoid Liposarcoma through a SRC/FAK/RHO/ROCK-Dependent Pathway.

Deregulated SRC/FAK signaling leads to enhanced migration and invasion in many types of tumors. In myxoid and round cell liposarcoma (MRCLS), an adipocytic tumor characterized by the expression of the fusion oncogene FUS-CHOP, SRC have been found as one of the most activated kinases. Here we used a cell-of-origin model of MRCLS and an MRCLS cell line to thoroughly characterize the mechanisms of cell invasion induced by FUS-CHOP using in vitro (3D spheroid invasion assays) and in vivo (chicken chorioallantoic membrane model) approaches. FUS-CHOP expression activated SRC-FAK signaling and increased the invasive ability of MRCLS cells. In addition, FAK expression was found to significantly correlate with tumor aggressiveness in sarcoma patient samples. The involvement of SRC/FAK activation in FUS-CHOP-mediated invasion was further confirmed using the SRC inhibitor dasatinib, the specific FAK inhibitor PF-573228, and FAK siRNA. Notably, dasatinib and PF573228 could also efficiently block the invasion of cancer stem cell subpopulations. Downstream of SRC/FAK signaling, we found that FUS-CHOP expression increases the levels of the RHO/ROCK downstream effector phospho-MLC2 (T18/S19) and that this activation was prevented by dasatinib or PF573228. Moreover, the ROCK inhibitor RKI-1447 was able to completely abolish invasion in FUS-CHOP-expressing cells. These data uncover the involvement of SRC/FAK/RHO/ROCK signaling axis in FUS-CHOP-mediated invasion, thus providing a rationale for testing inhibitors of this pathway as potential novel antimetastatic agents for MRCLS treatment.

Angiotensin-(1-7) prevents systemic hypertension, attenuates oxidative stress and tubulointerstitial fibrosis, and normalizes renal angiotensin-converting enzyme 2 and Mas receptor expression in diabetic mice.

We investigated the relationship between Ang-(1-7) [angiotensin-(1-7)] action, sHTN (systolic hypertension), oxidative stress, kidney injury, ACE2 (angiotensin-converting enzyme-2) and MasR [Ang-(1-7) receptor] expression in Type 1 diabetic Akita mice. Ang-(1-7) was administered daily [500 µg/kg of BW (body weight) per day, subcutaneously] to male Akita mice from 14 weeks of age with or without co-administration of an antagonist of the MasR, A779 (10 mg/kg of BW per day). The animals were killed at 20 weeks of age. Age-matched WT (wild-type) mice served as controls. Ang-(1-7) administration prevented sHTN and attenuated kidney injury (reduced urinary albumin/creatinine ratio, glomerular hyperfiltration, renal hypertrophy and fibrosis, and tubular apoptosis) without affecting blood glucose levels in Akita mice. Ang-(1-7) also attenuated renal oxidative stress and the expression of oxidative stress-inducible proteins (NADPH oxidase 4, nuclear factor erythroid 2-related factor 2, haem oxygenase 1), pro-hypertensive proteins (angiotensinogen, angiotensin-converting enzyme, sodium/hydrogen exchanger 3) and profibrotic proteins (transforming growth factor-ß1 and collagen IV), and increased the expression of anti-hypertensive proteins (ACE2 and MasR) in Akita mouse kidneys. These effects were reversed by A779. Our data suggest that Ang-(1-7) plays a protective role in sHTN and RPTC (renal proximal tubular cell) injury in diabetes, at least in part, through decreasing renal oxidative stress-mediated signalling and normalizing ACE2 and MasR expression.

A high-fat diet modulates iron metabolism but does not promote liver fibrosis in hemochromatotic Hjv⁻/⁻ mice.

Hemojuvelin (Hjv) is a membrane protein that controls body iron metabolism by enhancing signaling to hepcidin. Hjv mutations cause juvenile hemochromatosis, a disease of systemic iron overload. Excessive iron accumulation in the liver progressively leads to inflammation and disease, such as fibrosis, cirrhosis, or hepatocellular cancer. Fatty liver (steatosis) may also progress to inflammation (steatohepatitis) and liver disease, and iron is considered as pathogenic cofactor. The aim of this study was to investigate the pathological implications of parenchymal iron overload due to Hjv ablation in the fatty liver. Wild-type (WT) and Hjv(-/-) mice on C57BL/6 background were fed a standard chow, a high-fat diet (HFD), or a HFD supplemented with 2% carbonyl iron (HFD+Fe) for 12 wk. The animals were analyzed for iron and lipid metabolism. As expected, all Hjv(-/-) mice manifested higher serum and hepatic iron and diminished hepcidin levels compared with WT controls. The HFD reduced iron indexes and promoted liver steatosis in both WT and Hjv(-/-) mice. Notably, steatosis was attenuated in Hjv(-/-) mice on the HFD+Fe regimen. Hjv(-/-) animals gained less body weight and exhibited reduced serum glucose and cholesterol levels. Histological and ultrastructural analysis revealed absence of iron-induced inflammation or liver fibrosis despite early signs of liver injury (expression of α-smooth muscle actin). We conclude that parenchymal hepatic iron overload does not suffice to trigger progression of liver steatosis to steatohepatitis or fibrosis in C57BL/6 mice.

Angiotensin-(1-7): A Novel Peptide to Treat Hypertension and Nephropathy in Diabetes?

The renin-angiotensin system (RAS) plays a pivotal role in mammalian homeostasis physiology. The RAS can be delineated into a classical RAS (the pressor arm) including angiotensinogen (Agt), renin, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) and angiotensin type 1 receptor (AT1R), and a counterbalancing novel RAS (the depressor arm) including Agt, renin, angiotensin-converting enzyme-2 (ACE-2), angiotensin-(1-7) (Ang 1-7) and Ang 1-7 receptor (or Mas receptor (MasR)). Hyperglycemia (diabetes) induces severe tissue oxidative stress, which stimulates the pressor arm of the renal RAS axis and leads to an increase in ACE/ACE-2 ratio, with excessive formation of Ang II. There is a growing body of evidence for beneficial effects of the depressor arm of RAS (ACE-2/Ang 1-7/MasR) axis in diabetes, hypertension and several other diseased conditions. Evidence from in vitro, in vivo and clinical studies reflects anti-oxidant, anti-fibrotic, and anti-inflammatory properties of Ang 1-7. Most of the currently available therapies only target suppression of the pressor arm of RAS with angiotensin receptor blockers (ARBs) and ACE inhibitors (ACEi). However, it is time to consider simultaneous activation of the depressor arm for more effective outcomes. This review summarizes the recent updates on the protective role of Ang 1-7 in hypertension and kidney injury in diabetes, as well as the possible underlying mechanism(s) of Ang 1-7 action, suggesting that the ACE-2/Ang 1-7/MasR axis can be developed as a therapeutic target for the treatment of diabetes-induced hypertension and renal damage.

Seasonality of diarrheagenic Escherichia coli pathotypes in the US students acquiring diarrhea in Mexico.

BACKGROUND: Up to 60% of the US visitors to Mexico develop travelers' diarrhea (TD). In Mexico, rates of diarrhea have been associated with the rainy season and increase in ambient temperature. However, the seasonality of the various diarrheagenic Escherichia coli pathotypes in travelers has not been well described. OBJECTIVE: A study was undertaken to determine if ambient temperature and rainfall have an impact on the acquisition of TD due to different diarrheagenic E coli pathotypes in Mexico. METHODS: We conducted a cohort study of the US adult students traveling to Cuernavaca, Mexico, who were followed during their stay and provided a stool sample with the onset of TD. The presence of E coli was analyzed by a direct fecal multiplex polymerase chain reaction for common E coli pathotypes including enterotoxigenic, enteropathogenic, enteroinvasive, shiga toxin-producing, and enteroaggregative E coli (ETEC, EPEC, EIEC, STEC, and EAEC respectively). The presence of pathotypes was correlated with daily rainfall, average, maximum, and minimum temperatures. RESULTS: A total of 515 adults were enrolled from January 2006 to February 2007. The weekly attack rate of TD for newly arrived travelers was lower in the winter months (range 6.8%-16.3%) than in summer months (range 11.5%-25%; p = 0.05). The rate of ETEC infection increased by 7% for each degree centigrade increase in weekly ambient temperature (p = 0.003). In contrast, EPEC and EAEC were identified in similar proportions during the winter and summer seasons. CONCLUSIONS: Temperature variations in central Mexico influenced the rate of ETEC but not EAEC-associated diarrhea in the US visitors. This epidemiological finding could influence seasonal recommendations for the use of ETEC vaccines in Mexico.

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

Enterotoxigenic Escherichia coli heat-labile toxin seroconversion in US travelers to Mexico.

BACKGROUND AND AIMS: Enterotoxigenic Escherichia coli (ETEC) is the most common bacterial pathogen isolated from travelers suffering of diarrhea. Exposure to heat-labile toxin (LT) produces a high rate of seroconversion. However, the role of LT-producing ETEC (LT-ETEC) as a cause of diarrhea is controversial. We conducted a cohort study in US students traveling to Mexico to assess the ETEC-LT seroconversion rate after natural exposure. METHODS: Participants provided a serum sample on arrival and departure and a stool sample when ill. ETEC-LT immunoglobulin G antibodies were measured by enzyme-linked immunosorbent assay, and LT-ETEC were detected by means of polymerase chain reaction done on fecal DNA. RESULTS: A total of 422 participants with a mean age of 34.5 years were followed a mean of 19.9 days; 304 were females (72.0%), and 319 (75.6%) traveled during the summer months. In total, 177 individuals (41.9%) developed travelers' diarrhea and 33.9% had LT-ETEC identified in their stools. Among individuals having an LT-ETEC strain, 74% seroconverted compared to 11% of those not having diarrhea (p < 0.0001). When analyzed with a logistic regression model, the odds of seroconversion were significantly reduced in participants not having LT-ETEC in their stool (odds ratio = 0.1, p < 0.0001) after adjusting for season, length of stay, age, gender, race, and ethnicity. CONCLUSION: In US young adults traveling to Mexico, ETEC-LT seroconversion reliably identifies individuals naturally exposed to ETEC and correlates with symptomatic illness, length and season of travel.

MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells.

Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) --> MAPK kinase (MKK) --> MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 -->--> p38 kinase -->--> TonE.

AT1A-mediated activation of kidney JNK1 and SMAD2 in obstructive uropathy: preservation of kidney tissue mass using candesartan.

Literature suggests the involvement of the renin-angiotensin system and transforming growth factor (TGF)-beta in the renal injury that follows chronic ureteric obstruction. SMAD proteins and the JNK1 cascade are essential components of TGF-beta signaling machinery, and recent data suggest cooperative interaction between JNK1 and SMAD proteins in TGF-beta-mediated gene expression. We used a rat model of chronic unilateral ureteric obstruction to study the effects of candesartan, an AT(1A)-receptor blocker, on tissue morphology and the activities of JNK1 and SMAD2 protein in the kidney. Ureteric obstruction for 28 days leads to interstitial fibrosis, tubule atrophy, and marked activation of SMAD2 and JNK1, without significant change in p38 kinase or ERK. Candesartan treatment, however, attenuated the chronic tubulointerstitial injury in obstructed kidneys and was associated with significant preservation of kidney tissue mass. Furthermore, treatment with candesartan diminished JNK1 activity and downregulated SMAD2 protein and activity in obstructed kidneys. In conclusion, obstructed kidneys showed chronic tubulointerstitial injury, which was associated with JNK1 and SMAD2 activation. The renoprotective effects afforded by AT(1A)-receptor blockade in obstructive uropathy are consistent with attenuation of JNK1- and SMAD2-mediated renal injury.

Mutagenicity of nitroaromatic degradation compounds.

The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100. A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system. A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds. The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100. This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test. Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic. In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic. These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate.

Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure.

Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart.

Molecular cloning and analysis of the Cryptosporidium parvum aminopeptidase N gene.

Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules found on the surface of sporozoites and merozoites. In this study, we cloned and expressed the C. parvum aminopeptidase N gene by screening a large insert, P1 artificial chromosome library with a probe identified from a Cryptosporidium genome survey-sequencing project. Analysis of the predicted protein encoded by the 2.3 kb gene demonstrated a high degree of homology with prokaryotic and eukaryotic aminopeptidases. The 783 amino acid sequence predicted a M(r) of approximately 89,000. The active site sequence was found to be highly conserved when compared with other Apicomplexan aminopeptidases. Motifs commonly found in aminopeptidases of this class and a unique single Arg-Gly-Asp (RGD) tripeptide motif predictive of cell adhesion were identified. The aminopeptidase N mRNA was expressed in infective sporozoites and during the infection of human HCT-8 enterocytes as revealed by reverse transcription PCR.