First report of clear cell renal carcinoma metastasizing to the ischiorectal fossa 17 years after radical nephrectomy: an additional reason for lifelong follow-up.

Renal cell carcinoma has a high propensity for metastatic spread. There are several case reports of metastatic renal cell carcinomas associated with rare metastatic sites, in many cases more than ten years after the initial diagnosis. We present a 60-year-old man with perianal pain and a mass in the ischiorectal space, revealed by computed tomography. The patient had a history of clear cell renal carcinoma operated on 17 years ago. A wire localization surgical excision of the ischiorectal fossa mass was performed. The pathological report revealed a metastatic clear cell renal carcinoma. To our knowledge, this is the first case of a clear cell renal carcinoma metastasizing to the ischiorectal fossa reported in the literature. We therefore recommend that any newly discovered mass in any site of a patient with a history of renal cell carcinoma should be carefully explored and biopsied.

Haematocrit and haemoglobin decrease following image-guided percutaneous core needle biopsies.

AIM: To determine the clinical significance of variation in haematocrit (Ht) and haemoglobin (Hb) values before and after image-guided percutaneous core needle biopsies (PCNBs) and evaluate its clinical significance. MATERIALS AND METHODS: This single-centre, retrospective study included all the patients who underwent image-guided PCNBs between November 2012 and September 2018. In total, 105 cases (56 male; 53.3%; mean age 72±8 years) were available for analysis. Biopsies included lesions of the liver, lung, kidney, bone, paravertebral and soft-tissue masses, peritoneal implantations, and retroperitoneal neoplasms. The study's primary outcome was to compare the pre- and post-procedural Ht and Hb values and to evaluate their clinical significance. RESULTS: A significant decrease of the mean Hb and Ht values was detected post-biopsy (12.79±1.85 g/dl versus 12.03±1.72 g/dl and 38.75±4.93% versus 36.49±4.73%; p<0.0001). A decrease in the Ht and/or Hb level was noted in 93/105 (88.6%) and 94/105 (89.5%) of the patients; respectively. Four minor bleeding complications were noted (4/105; 3.8%), which resolved without any further treatment. An >4% decrease in Ht value was noted in 17/105 cases (16.2%) and an Hb decrease of ≥1.5 mg/dl was noted in 10/105 cases (9.5%), all without any haemodynamic compromise. CONCLUSIONS: A moderate post-PCNB decrease in Ht and Hb values compared to baseline should be expected, but should not raise concerns regarding an ongoing bleeding event, if not correlated with haemodynamic and clinical signs of haemorrhage.

Development of rapidly dissolving pellets within the Quality by Design approach.

The purpose of this study was the development of novel, fast disintegrating, effervescent pellets by employing the direct pelletization technique as a single step process. In line with the Quality by Design (QbD) regulatory framework, statistical experimental design was extensively applied to correlate significant formulation and process variables with the critical quality attributes of the product. Pellets were studied with regards to sphericity, size and size distribution. In contrast to the existing multiparticulate platforms, this development integrated only water-soluble excipients to facilitate the multifunctional use of the final dosage form. The application of a screening fractional factorial design augmented to a full factorial design set the roadmap for the rational selection of the composition and process parameters, revealing in parallel the positive contribution of the powder feeder on the CQAs, when the critical process and formulation factors were properly adjusted. The response surface methodology was exploited for the final process optimization phase, which allowed the construction of appropriate mathematical models connecting the input variables and the CQAs under study. The implementation of the desirability function, lead to the optimum formulation and process settings for the production of pellets with narrow size distribution and geometric mean diameter of approximately 800 µm. In conclusion, using a lean approach supported by design of experiments (DoE) techniques within the QbD framework, a novel multifunctional formulation platform has been developed.

Sexual dysfunction in testicular cancer patients subjected to post-chemotherapy retroperitoneal lymph node dissection: a focus beyond ejaculation disorders.

Post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND) represents an integral part of multidisciplinary treatment of advanced germ cell cancer; however, it is associated with a high complications rate. The present study aimed to describe sexual disorders in 53 patients with testicular cancer who underwent full bilateral, non-nerve-sparing PC-RPLND in our institution, focusing beyond ejaculatory dysfunction. The International Index for Erectile Function (IIEF) questionnaire was used as diagnostic tool of male sexual functioning pre-operatively and three months after RPLND, while post-operatively patients were asked to describe and evaluate changes in selected sexual parameters. Study findings demonstrate mixed pattern of changes in sexual functioning, with no difference in erectile functioning before and after operation. However, orgasmic function and intercourse and overall sexual satisfaction were found significantly impaired post-operatively. Sexual desire and frequency of attempted sexual intercourses were found significantly increased post-operatively, in comparison with pre-operative levels. With regard to patients' subjective perception on sexual functioning alterations after PC-RPLND, a significant number of patients reported higher levels of sexual desire, no difference in erectile function and worse orgasmic function and satisfaction post-operatively. Thus, patients subjected to PC-RPLND should be closely and routinely evaluated due to close relationship of sexual dissatisfaction with secondary psychological disorders.

Molecular pathogenesis of non muscle-invasive bladder cancer: implications for novel targeted therapies.

Approximately 70% to 80% of patients with urothelial carcinomas of the bladder are initially diagnosed with non-muscle invasive disease. Superficial, non-muscle invasive bladder cancers (NMIBCs) are managed with cystoscopic transurethral resection of all visible lesions followed by intravesical chemotherapy and/or immunotherapy. Despite this treatment, up to 70% of these tumors will recur within five years and 15% will ultimately progress to muscle-invasive disease, suggesting that novel therapeutic strategies are necessary. Recent studies have greatly advanced our understanding of urothelial carcinogenesis and have highlighted the distinct molecular pathogenesis of NMIBCs versus muscle-invasive bladder tumors. It is now clear that diverse genetic and epigenetic events are driving the oncogenesis of NMIBCs, thereby attesting to their potential as therapeutic targets for these tumors. This article reviews the molecular pathogenesis of NMIBCs, discusses recently completed and ongoing clinical trials and anticipates the future direction of molecular targeted agents in this disease.

Extracellular microenvironment and cytokine profile of the ureterovesical junction in children with vesicoureteral reflux.

PURPOSE: Vesicoureteral reflux is caused by a defective valve mechanism of the ureterovesical junction. Previous studies have revealed structural and metabolic changes in the intravesical ureter, impairing its contractile properties. Smooth musculature and nerves are replaced by collagen, while matrix degrading enzymes are over expressed. We investigated the presence of regulating cytokines and the extracellular matrix composition to elucidate further the pathophysiology of vesicoureteral reflux. MATERIALS AND METHODS: Ureteral endings were obtained from 28 children during antireflux surgery, and 14 age matched autopsy specimens served as controls. Routine histological sections were immunostained for insulin-like growth factor-1, nerve growth factor, transforming growth factor-beta1, tumor necrosis factor-alpha and vascular endothelial growth factor. Smooth muscle staining was supplemented by tenascin C, tetranectin and fibronectin detection. Staining patterns were investigated using computer assisted, high power field magnification analyses. RESULTS: Tumor necrosis factor-alpha and transforming growth factor-beta1 were significantly more abundant in vesicoureteral reflux samples, whereas insulin-like growth factor-1, nerve growth factor and vascular endothelial growth factor were more prevalent in healthy controls. Fibronectin was intensely expressed in refluxing ureters, while it was scarce in healthy children. Tenascin C was notable within the urothelium of both groups. Only vesicoureteral reflux samples displayed tenascin C in the musculature and connective tissue. Tetranectin staining was only detected in vesicoureteral reflux. CONCLUSIONS: Several cytokines are differentially expressed in primary refluxing ureters, indicating an ongoing tissue remodeling process in the ureterovesical junction region. Additionally, the smooth muscle coat is widely lacking, while extracellular matrix proteins typical for tissue shrinkage and reorganization are over expressed. These alterations are likely to contribute to the malfunctioning active ureteral valve mechanism in primary vesicoureteral reflux.

The use of tamsulozin as adjunctive treatment after ESWL in patients with distal ureteral stone: do we really need it? Results from a randomised study.

Our study aimed to define the position of tamsulosin as adjunctive therapy in patients with stones of the distal ureter who had undergone extracorporeal shock wave lithotripsy (ESWL). In total, 61 consecutive patients (38 men and 23 women) with single distal radiopaque ureteral stone of > or =6 mm of diameter were enrolled. After ESWL patients were randomized in two groups. Non-steroidal anti-inflammatory drug (supp. diclofenac 50 mg) was given to both groups upon demand. In group B, all patients (30) received additionally tamsulozin 0.4 mg every day. Follow-up visits were performed 1, 2, 3 and 4 weeks after ESWL. Evaluation included a KUB plain film and an ultrasound examination. Efficacy was evaluated in terms of success rate, stone-free rate, expulsion time of the fragments and use of diclofenac. Two patients from the tamsulosin group experienced dizziness and one was withdrawn. The success rate was 58.06 and 66.66% for the control and the tamsulosin group, respectively, while the corresponding values for stone-free rate were 51.6 and 63.33%, respectively. The mean expulsion time of the fragments was 13.22 days for group A and 12.95 days for group B. These results did not achieve statistically significant difference (P > 0.05). The mean diclofenac dose was 118.9 mg in group A and 56.9 mg in group B. This difference was statistically significant (P = 0.02). Despite the relatively small number of patients, our data indicate that the use of tamsulosin after ESWL in this specific subgroup of patients does not result in improved success and stone-free rate and expulsion time. In contrast, a significantly reduced need for analgesics was found.

The combined action of carvacrol and high hydrostatic pressure on Listeria monocytogenes Scott A.

AIMS: The aim of the study was to investigate the combined antimicrobial action of the plant-derived volatile carvacrol and high hydrostatic pressure (HHP). METHODS AND RESULTS: Combined treatments of carvacrol and HHP have been studied at different temperatures, using exponentially growing cells of Listeria monocytogenes, and showed a synergistic action. The antimicrobial effects were higher at 1 degrees C than at 8 or 20 degrees C. Furthermore, addition of carvacrol to cells exposed to sublethal HHP treatment caused similar reductions in viable numbers as simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with L. monocytogenes. CONCLUSION: Carvacrol and HHP act synergistically and the antimicrobial effects of the combined treatment are greater at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP and indicates the potential of these combined treatments in food processing.

Overexpressing eukaryotic translation initiation factor 4E stimulates bovine mammary epithelial cell proliferation.

BACKGROUND AND AIMS: The eukaryotic translation initiation factor 4E regulates the proliferation of many cell types. In the present study, the effect of its overexpression on the growth of an immortalized bovine mammary epithelial cell line, MAC-T, has been investigated. Since involvement of cyclin D1 in growth regulation of other cell types has been suggested previously, the differences in cyclin D1 expression among the 4E-overexpressing and parental cells were also investigated. METHODS: The cDNA of mouse eukaryotic translation initiation factor 4E coding region (either wild-type or mutant, where Trp-56 was mutated to Ala) was transfected into MAC-T cells, and its protein expression was detected by Western blot analysis. Growth rates and saturation densities were calculated based on the cell counting data at desired time points. KEY RESULTS: The cells overexpressing wild-type 4E displayed higher growth rates and saturation densities compared to the parental cells (P<0.05), whereas cells expressing mutant 4E showed lower growth rates and saturation densities than the parental controls (P<0.05). The amounts of cyclin D1 mRNA and protein were higher in the wild-type transfectants than in the parental controls, whereas the mutant transfectants contained lower amounts of cyclin D1 mRNA and protein compared to the parental cells. CONCLUSION: Our results suggest that overexpression of eukaryotic translation initiation factor 4E leads to increased cyclin D1 expression at the transcriptional level, which consequently stimulates the proliferation of MAC-T cells.

Combined action of S-carvone and mild heat treatment on Listeria monocytogenes Scott A.

The combined action of the plant-derived volatile, S-carvone, and mild heat treatment on the food-borne pathogen, Listeria monocytogenes, was evaluated. The viability of exponential phase cultures grown at 8 degrees C could be reduced by 1.3 log units after exposure to S-carvone (5 mmol l-1) for 30 min at 45 degrees C, while individual treatment with S-carvone or exposure to 45 degrees C for 30 min did not result in a loss in viability. Other plant-derived volatiles, namely carvacrol, cinnamaldehyde, thymol and decanal, were also found to reduce the viability of L. monocytogenes in combination with the same mild heat treatment at concentrations of 1.75 mmol l-1, 2.5 mmol l-1, 1.5 mmol l-1 and 2 mmol l-1, respectively. These findings show that essential oil compounds can play an important role in minimally processed foods, and can be used in the concept of Hurdle Technology to reduce the intensity of heat treatment or other individual hurdles.

The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle.

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

Isolated-organ perfusion for local gene delivery: efficient adenovirus-mediated gene transfer into the liver.

Targeted gene delivery is essential for gene therapy involving in vivo gene transfer. In the present study we analyzed the efficiency and tissue-specificity of gene transfer into the liver with recombinant adenoviruses. Adenovirus vectors carrying the E. coli lacZ gene (Ad.RSV.beta-gal) and the firefly luciferase gene (AdCMV-luc) as reporters were administered to the liver of adult Wistar rats, either via infusion into the portal vein (intraportal infusion; IPI) or via perfusion of the vascularity isolated liver (isolated liver perfusion; ILP). Ex vivo liver perfusion experiments with Ad.RSV. beta-gal were used to optimize the conditions for hepatic gene transfer. Ex vivo perfusion of rat livers with 2 x 10(9) plaque forming units (p.f.u) Ad RSV.beta-gal was sufficient to infect about 20% of the liver parenchymal cells. Perfusion with chelating agents (1 mM EGTA, or 2 mM EDTA) prior to the administration of the vector increased the efficiency to at least 40%. Similar efficiencies were obtained in experiments with liver lobes of Rhesus monkeys. In vivo administration of AdCMV-luc via ILP resulted in a significantly more efficient (P = 0.028) and also more reproducible gene transfer when compared to IPI. Although detectable in both groups, extrahepatic luciferase expression was considerably reduced in the ILP group. Our data demonstrate that IPL can be used for efficient and reproducible liver-specific gene delivery. Therefore, we think that the perfusion of vascularly isolated organs is useful as a modality for the tissue-specific administration of recombinant adenovirus vectors.

Translational regulation of the mammalian growth-related protein P23: involvement of eIF-4E.

Synthesis of the mammalian growth-related protein P23 is rapidly induced after serum stimulation of mouse fibroblasts and Ehrlich ascites tumour cells. This induction occurs at the translational level. Growth-induction leads also to an increase in phosphorylation of the rate-limiting initiation factor eIF-4E. Here, we present the following evidence indicating the involvement of eIF-4E in the regulation of P23 synthesis: 1) P23 synthesis is induced by the same mitogenic stimuli which lead to enhanced eIF-4E phosphorylation. 2) Upon heat shock treatment of Ehrlich ascites cells (which results in immediate dephosphorylation and concomitant inactivation of eIF-4E), P23 synthesis is rapidly shut off. 3) In control NIH 3T3 cells, P23 synthesis is readily induced by growth stimulation. This response is strongly diminished in cells overexpressing eIF-4E, and the basal level of P23 synthesis is elevated in these cells. Overexpression of a nonfunctional mutant of eIF-4E diminishes the basal level of P23 synthesis as well as the serum-response of the cells with respect to P23 induction. 4) Cells transformed by overexpression of the ras or src genes in which eIF-4E is highly phosphorylated do not show any inducibility of P23 synthesis. 5) HeLa cells expressing antisense RNA of eIF-4E, have reduced levels of eIF-4E/F and show reduced rates of growth and protein synthesis. In these cells the total amount of P23 protein is about 50% compared with control cells. The results suggest that P23 is one of the gene products, the synthesis of which is regulated by eIF-4E activity.

Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E.

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E.

Cellular eukaryotic mRNAs (except organellar) contain at the 5' terminus the structure m7(5')Gppp(5')N (where N is any nucleotide), termed cap. Cap recognition by eukaryotic initiation factor eIF-4F plays an important role in regulating the overall rate of translation. eIF-4F is believed to mediate the melting of mRNA 5' end secondary structure and facilitate 43S ribosome binding to capped mRNAs. eIF-4E, the cap-binding subunit of eIF-4F, plays an important role in cell growth; its overexpression results in malignant transformation of rodent cells, and its phosphorylation is implicated in signal transduction pathways of mitogens and growth factors. The molecular mechanism by which eIF-4E transforms cells is not known. Here, we report that overexpression of eIF-4E facilitates the translation of mRNAs containing excessive secondary structure in their 5' non-coding region. This effect may represent one mechanism by which eIF-4E regulates cell growth and transforms cells in culture.

Ras mediates translation initiation factor 4E-induced malignant transformation.

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

The mRNA 5' cap-binding protein, eIF-4E, cooperates with v-myc or E1A in the transformation of primary rodent fibroblasts.

We present evidence that eIF-4E, the mRNA 5' cap-binding protein, cooperates with two immortalizing oncogenes, v-myc and E1A, to cause transformation of rat embryo fibroblasts. eIF-4E alone can transform rat embryo fibroblasts when selection is applied. The pattern of transformation by eIF-4E is similar to that of p21 Ras, raising the possibility that eIF-4E shares a common signal transduction pathway with p21 Ras.

Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E.

Ornithine aminotransferase (OAT) is a mitochondrial enzyme expressed at high levels in liver, kidney, and retina. To characterize OAT regulation in retinal lines, we have been studying OAT synthesis in retinoblastomas RB355 and Y79. Our previous data (Fagan, R.J., Sheffield, W.P., and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517) indicated similar OAT mRNA levels in the two strains with 3-fold greater immunoreactive OAT protein and enzyme activity in Y79. To examine the regulatory mechanisms in these cell lines, we performed nuclear runoff experiments and characterized polysome-associated OAT mRNAs. The nuclear runoff data did not reveal any differences in transcription between the two strains. However, OAT mRNA of the RB355 strain was present in the lighter polysome fractions as compared with Y79. Treatment with cycloheximide, which slows the rate of elongation, indicated that initiation was decreased in RB355. Eukaryotic initiation factor eIF-4E mRNA and protein were reduced in RB355, suggesting that eIF-4E might be rate-limiting for OAT translation. Overexpression of a wild-type eIF-4E in RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpression of a mutant eIF-4E had no such effect. We previously identified an alternatively spliced OAT mRNA (containing exon 2) in these cells. This mRNA appeared in the lightest fractions of the gradient in both strains and was not affected by eIF-4E overexpression.