Microparticles of healthy origins improve endothelial progenitor cell dysfunction via microRNA transfer in an atherosclerotic hamster model.

AIM: In this study, we aimed: (i) to obtain and functionally characterize the cultures of late endothelial progenitor cells (EPCs) from the animal blood; (ii) to investigate the potential beneficial effects of circulating microparticles (MPs) of healthy origins on EPC dysfunctionality in atherosclerosis as well as involved mechanisms. METHODS: Late EPCs were obtained and expanded in culture from peripheral blood isolated from two animal groups: hypertensive-hyperlipidaemic (HH) and control (C) hamsters. In parallel experiments, late EPC cultures from HH were incubated with MPs from C group. RESULTS: The results showed that late EPCs display endothelial cell phenotype: (i) have ability to uptake 1,1-dioctadecyl-3,3,3,3 tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein and Ulex europaeus agglutinin lectin-1; (ii) express CD34, CD133, KDR, CD144, vWF, Tie-2. Late EPCs from HH exhibited different morphological and functional characteristics compared to control: (i) are smaller and irregular in shape; (ii) present decreased endothelial surface marker expression; (iii) display reduced proliferation, migration and adhesion; (iv) lose ability to organize themselves into tubular structures and integrate into vascular network; (v) have diminished function of inward rectifier potassium channels. The incubation of late EPCs with MPs improved EPC functionality by miR-10a, miR-21, miR-126, miR-146a, miR-223 transfer and IGF-1 expression activation; the kinetic study of MP incorporation into EPCs demonstrated MP uptake by EPCs followed by the miRNA transfer. CONCLUSION: The data reveal that late EPCs from atherosclerotic model exhibit distinctive features and are dysfunctional, and their function recovery can be supported by MP ability to transfer miRNAs. These findings bring a new light on the vascular repair in atherosclerosis.

Renal inflammatory myofibroblastic tumor - a new case report.

Renal inflammatory pseudotumor is uncommon, benign tumor that has been classified into separate group but there is a risk that this lesion could be misdiagnosed. The aim of this work is to report a new case of 57-years-old man presented in our hospital with hematuria, minimal grade fever and right flank pain. Magnetic resonance imaging (MRI) and sonography revealed a tumor of the right mediorenal parenchyma, 2.5 cm in diameter. The patient underwent right nephroureterectomy under the diagnosis of renal cell carcinoma. Macroscopically examination carried out on the removed kidney showed a 2/2/1.5 cm yellowish, gelatinous, well circumscribed, mediorenal and pericaliceal mass. Fragments of the tumor were fixed in 10% formaldehyde, included in paraffin, and the sections were stained with HE, VG and immunohistochemically with vimentin (VIM), MNF116, SyN, smooth muscle actin (ACT), desmin, CD68, S100, HMB45, and CD117. The histological examination revealed a compact spindle cell proliferation, a hypocellular fibrous area in an edematous myxoid background infiltrated by small lymphocytes, histiocytes, some plasma cells and small bone area. The spindle cells were diffuse positive for VIM, ACT, CD68 and negative for desmin, MNF116, SyN, S100, HMB45, and CD117. The pathologic diagnosis was renal inflammatory pseudotumor, raising the problem of differential diagnosis, as the clinical and imagistic aspects are similar to those of a renal carcinoma and the problem in establishing a preoperative correct diagnosis.

Small cell carcinoma of the urinary bladder--a new case report.

Primary pure small cell carcinoma of the urinary bladder is an extremely rare and highly aggressive tumor with an average five-year survival rate of less than 10% as cited by multiple case reports. It accounts for about 0.5-1% of all bladder tumors. We present the case of a 44-years-old man, smoker (10 cigarettes/day) hospitalized in the Department of Urology, from the "Prof. dr. Th. Burghele" Hospital, Bucharest, for one month intermittent hematuria. Ultrasonography showed a sessile tumoral mass, sized 37/30mm. Transurethral resection of the tumor mass was performed and tissue fragments were sent to the pathologic lab to establish the histologic type, the degree of differentiation and invasion. Fragments of the tumor were fixed in 10% formaldehyde, paraffin embedded and processed as standard technique; the sections were stained with HE, VG and immunohistochemically with: CROMO, EMA, NSE, CD56, NK1, p53 and betaHCG. The microscopic examination reveled a tumor proliferation composed of two distinct components: extensive small cells areas and foci of typical low grade (G2) papillary urothelial carcinoma. The small cell are uniformly, round, with increased nucleo-cytoplasmic ratio, eosinophyl cytoplasm, hyperchromatic nuclei, finely granular chromatin and inconspicuous nucleoli. Immunohistochemical stains showed diffuse positive staining of the small cell component for CROMO, EMA, NSE, CD56, NK1 and urothelial carcinoma component stained focally for betaHCG. The rate of cell proliferation was increased (p53 - 80% positive reaction). Conclusions. A diagnosis of small cell carcinoma coexisting with low-grade urothelial carcinoma was established. Because of aggressive behavior and distinct treatment, the pathologist should watch out for the presence of small cell carcinoma component.

Immunohistochemical detection of p53 protein as a prognostic indicator in prostate carcinoma.

UNLABELLED: THE AIM of our study was to evaluate the prognostic significance of p53 protein immunoreactivity for prostate cancer and to determine whether p53 immunoreactivity correlates with the Gleason tumor grade in primary adenocarcinoma. Prostate fragments were fixed in 10% formalin, paraffin-embedded, sectioned and standard Hematoxylin-Eosin stained, then examined using histological grade (Gleason system). P53 expression was studied using immunohistochemistry with monoclonal antibody anti-p53, 1 : 100 (BIOX) on tissue samples obtained during transurethral electroresection, adenomectomy or needle biopsy in 30 patients with prostate carcinoma: group 1 (n = 7) Gleason score 5, group 2 (n = 10) Gleason score 6, group 3 (n = 11) Gleason score 7, group 4 (n = 2) Gleason score 8. Also, we noted the cases with high grade prostatic intraepithelial neoplasia (high grade PIN). All specimens prior to initiation of any treatment were submitted for this study. Staining was defined as positive for p53 whenever any specific nuclear staining was detected. We considered tumors to overexpress p53 protein only when strong nuclear staining was present. Cases exhibiting weak or equivocal nuclear staining were classified as negative, as were cases with extremely rare isolated positive nuclei. A semiquantitative scoring system was employed to assess the level of p53 reactivity. Six of 17 (35.2%) moderately differentiated tumors (Gleason score 5-6) and five of 13 (38.4%) moderate to poorly differentiated (Gleason score 7 and above) revealed strong nuclear positivity for p53. In addition, we noted occasional p53 reactivity in high-grade PIN. CONCLUSIONS: We interpret these data to demonstrate a positive association between p53 reactivity and higher Gleason grade tumors; p53 might be an independent prognostic indicator among metastatic risk cases.

Primary malignant lymphoma of the testis.

UNLABELLED: Primary testicular lymphomas are rare entities representing 1-2% of non-Hodgkin lymphoma (NHML) and 1-7% of malignant testicular tumors and they are the most common testicular tumors in men older than 50 years of age. This study included 8 cases of inpatients diagnosed by echography and NMR with testicular tumors. The age of patients was between 46 and 81 (with a mean of 52). The tumors were unilateral, with disease limited to testicle and accompanied by pain except 1 case with bone involvement. Orchectomy was performed as first therapeutic and diagnostic purpose. All patients were clinically staged according to the Ann Arbor criteria in IE and IIID stage and received a doxorubicin based chemotherapy regimen (CHOP, MTX, CVP, and Leukeran). A standard chemotherapy protocol has not been used because of reduced number of patients. Tumor fragments were fixed in 10% formallin, paraffin embedded, sectioned and standard H.E. stained. Immunohistochemistry for L26, Alphafetoprotein, NK1, CD30, and CLA was performed. Microscopy revealed in all cases a stromal proliferation with medium size cells, monomorphic shape and prominent nucleoli. Alphafetoprotein was positive in seminal tubes and negative in tumor, NK1 in small lymphocyte and negative in tumor and L26 diffuse positive in tumor. CLA diffuse positive in tumor. We were able to follow up only four patients. CONCLUSIONS: The diagnosis was of NHML in 6 cases and for 2 secondary involvement of hematopoietic malignancy (myeloid sarcoma and leukemia). Lymphoma cases were typed using REAL classification as small and large B-cell lymphoma. Unfavorable evolution with 6 months relapse and one death prove a more aggressive evolution of primitive testicular lymphoma.

Cell cycle regulatory factors in juxta-tumoral renal parenchyma.

The aim of this study was to evaluate regulatory cell cycle factors in juxta-tumoral renal parenchyma in order to obtain information regarding early primary changes occurred in normal renal cells. Specimens of juxta-tumoral renal parenchyma were harvested from the tumoral kidney in 10 patients with no history of treatment before surgery. The expression of p53, Bcl-2, Rb and PCNA was studied by immunohistochemical methods in paraffin-embedded tissues. The apoptotic status was evaluated by flow-cytometry analysis following propidium iodide incorporation. The p53 protein expression was recognized in most of the cases (80%) with different intensities. High intensity apoptotic process detected in juxta-tumoral parenchyma seemed to be p53 dependent and well correlated with the low Bcl-2 expression. 70% of cases were Rb positive. In this type of tissue Rb has only an anti-proliferative and anti-tumoral role. PCNA was present in half of the cases being low expressed due to the tissue regenerating mechanism. Our data suggest that the high intensity of programmed cell death in this type of tissue is supported by the status of cell regulatory factors that control this process. Previous studies have demonstrated that healthy renal tissue has neither apoptosis nor mitotic activity. Juxta-tumoral renal tissue is also displaying normal morphology and DNA content (diploidy) but the microenvironmental status induced by the tumor presence prompts cells to choose death rather than malignant transformation. Further studies are necessary to emphasize if these results have a clinical relevance for the outcome of therapeutical approaches in renal carcinomas.

Economic evaluation of oral valdecoxib versus diclofenac in the treatment of patients with rheumatoid arthritis in a randomized clinical trial.

In contrast to economic models that provide probabilistic estimates of economic impact, data extracted from clinical trials may be used to evaluate and compare actual resource utilization and costs. Health-care resource utilization and the costs of these resources were compared from the perspective of the UK National Health Service using data obtained in a 6-month clinical trial of oral valdecoxib 20 mg once daily and diclofenac 75 mg twice daily for the symptomatic treatment of rheumatoid arthritis. However, calculated health-care costs were exclusive of drug acquisition costs because the price of valdecoxib was not available at the time of analysis. While the efficacy of the two treatments was similar, use of valdecoxib was associated with a reduction in total health-care costs amounting to approximately 200 British pounds per patient. This lower cost was associated with reduced use of health-care resources for gastrointestinal serious adverse events (gastrointestinal SAEs). In particular, the incidence of hospitalization and number of hospital days for gastrointestinal SAEs was lower in the valdecoxib group. Analysis of cost per gastrointestinal SAE favoured valdecoxib (cost savings of 742 British pounds), suggesting that even when these events did occur they were less severe. When costs of gastrointestinal SAEs were averaged over the entire population, valdecoxib was suggested to have lower total costs per patient compared with diclofenac (cost savings of 115 British pounds per patient), mainly resulting from significant savings in hospitalization costs (76.49 British pounds per patient). These data are consistent with economic models and suggest that the favourable gastrointestinal profile of valdecoxib observed in clinical trials will be of economic benefit.

The total peroxyl radical trapping potential in serum - an assay to define the stage of atherosclerosis.

Lipid peroxides were identified among the factors that contribute to the atherosclerotic plaque formation in the arterial wall. We hypothesised that a correlation may exist between the content of antioxidant constituents in the serum and the gravity of atherosclerosis. To this purpose, we have determined the serum total peroxyl radical- trapping potential (TRAP), which is the combined capacity of all antioxidants to neutralize free radicals in serum and followed its variation in hyperlipemic animals in correlation with the stage of atherosclerosis. In addition, we compared TRAP values in the sera of coronary heart disease (CHD) patients, with or without type II diabetes mellitus. Results showed that after 18 weeks of hyperlipemic diet, the mean TRAP values measured in sera isolated from hyperlipemic hamsters exhibited an about 44% decrease, in good agreement with the increase of serum cholesterol and triglycerides. In the 3 groups of CHD patients, TRAP values decreased with about 10% in sera of stable angina patients, 20% in unstable patients, as compared with normal subjects. The lowest TRAP values were detected in the sera of patients with acute myocardial infarction. The results obtained for different experimental animals and for CHD patients sera indicate that the TRAP method, as adapted in our laboratory, is a reliable and reproducible assay, fit to be used in clinical studies as an ex vivo measurable parameter that correlates with the stage of the atherosclerosis.

Mannitol 1-phosphate mediates an inhibitory effect of mannitol on the activity and the translocation of glucokinase in isolated rat hepatocytes.

When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.

Investigation on the mechanism by which fructose, hexitols and other compounds regulate the translocation of glucokinase in rat hepatocytes.

In isolated hepatocytes in suspension, the effect of sorbitol but not that of fructose to increase the concentration of fructose 1-phosphate and to stimulate glucokinase was abolished by 2-hydroxymethyl-4-(4-N,N-dimethylamino-1-piperazino)-pyrimidine (SDI 158), an inhibitor of sorbitol dehydrogenase. In hepatocytes in primary culture, fructose was metabolized at approximately one-quarter of the rate of sorbitol, and was therefore much less potent than the polyol in increasing the concentration of fructose 1-phosphate and the translocation of glucokinase. In cultures, sorbitol, commercial mannitol, fructose, D-glyceraldehyde or high concentrations of glucose caused fructose 1-phosphate formation and glucokinase translocation in parallel. Commercial mannitol was contaminated by approx. 1% sorbitol, which accounted for its effects. The effects of sorbitol, fructose and elevated concentrations of glucose were partly inhibited by ethanol, glycerol and glucosamine. Mannoheptulose increased translocation without affecting fructose 1-phosphate concentration. Kinetic studies performed with recombinant human beta-cell glucokinase indicated that this sugar, in contrast with N-acetylglucosamine, binds to glucokinase competitively with the regulatory protein. All these observations indicate that translocation is promoted by agents that favour the dissociation of the glucokinase-regulatory-protein complex either by binding to the regulatory protein (fructose I-phosphate) or to glucokinase (glucose, mannoheptulose). They support the hypothesis that the regulatory protein of glucokinase acts as an anchor for this enzyme that slows down its release from digitonin-permeabilized cells.