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Lanthanide-based beta-tricalcium phosphate (β-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped β-TCP nanoparticles (βTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further

Approximately 75% of breast cancer cells express estrogen receptor positive, being the second leading cause of cancer death among women worldwide with an incidence of 25% per year. 16α-[18F]-fluoro-17β-estradiol, FES-18F, is a radiopharmaceutical that binds to estrogen receptors enabling the acquisition of molecular images for non-invasive diagnosis of primary and metastatic breast cancer using PET-CT. The objective of this work was to study the synthesis of FES-18F in the GE TRACERlab® MXFDG module, using the chemical kit and the ABX® disposable cassette, and to determine the process yield and the analytical parameters to be used in the routine production of this radiopharmaceutical. Automated synthesis occurs in 75 minutes and includes percolation of [18F-] fluoride into an anion exchange cartridge, cartridge elution, 3-step azeotropic drying, labeling using precursor 3-methoxymethyl-16β,17β-epiestriol-O-cyclic sulfone (MMSE) and a hydrolysis step. Product purification is done in the module using solid phase extraction (SPE) cartridges. The radiochemical yield was reproductive, regardless of fluor-18 activity at entry into the module, and the results of quality control tests suggest that the radiopharmaceutical meets the criteria established for other fluor-18-labelled radiopharmaceuticals that have monographs in official compendiums. In vivo biodistribution studies in healthy animals and with a tumor model developed with MCF-7 cells, demonstrated radiopharmaceutical uptake in excretory organs and specificity for breast tumor cells.

Renal cell carcinoma (RCC) is a highly deadly urological tumor due to its high metastatic incidence and its notorious chemoresistance. The nuclear transcription factor kappa B (NF-κB) family has been associated with apoptosis resistance and cellular invasion in RCC. The purpose of this study was to evaluate the impact of NF-κB1 gene silencing on the colony formation, cell migration and invasion abilities of the RCC cell line. Renca-mock and Renca-shRNA-NF-κB1 cells were used in this work. NF-κB1 downregulation was assessed by western blotting. The mRNA expression levels of interleukin-1 beta (IL-1β) and MMP-9 were assessed by real-time quantitative polymerase chain reaction (RT-qPCR). The IL-1β levels in the culture media were determined by a commercial ELISA kit. The MMP-9 protein expression and gelatinolytic activity were evaluated by western blotting and zymography, respectively, and the migration and invasion abilities were analysed. The expression levels of p105 and p50 in Renca-shRNA-NF-κBmoc1 cells were significantly reduced compared with those in the Renca-mock cells. The colony numbers of shRNA-NF-кB1 cells were lower than the colony numbers of the Renca-mock cells. NF-κB1 knockdown inhibited the cell migration and invasion of Renca-shRNA-NF-κB1 cells. These cells also exhibited reduced levels of IL-1β. The MMP-9 expression and activity levels were significantly reduced in Renca-shRNA-NF-κB1 cells. Taken together, these results indicate that the downregulation of NF-κB1 suppresses the tumourigenicity of RCC by reducing MMP-9 expression and activity; thus, NF-κB1 could be a molecular target for RCC treatment.

Background: Due to the loss of von Hippel-Lindau tumor suppressor function, clear renal cell carcinoma (ccRCC) deregulates hypoxia pathways. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes: GeNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable, and therefore, no agreement was observed for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes to be used in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To our knowledge, this is the first study to evaluate reliable genes that can be used in gene expression analysis in ccRcc under a hypoxic environment.

Background: Cardiovascular complications are common in chronic kidney disease (CKD) patients. Endothelial nitric oxide synthase (eNOS) is very important for the homeostasis of the cardiovascular system, and its gene polymorphism at position 894 (G>T) has not been investigated with QTc interval in patients on dialysis. Objective: This study evaluated the association of the 894G>T polymorphism with QTc prolongation and endothelial dysfunction risk in dialysis patients. Methods: Predialysis blood samples were collected for eNOS gene polymorphism, nitric oxide (NO), thiobarbituric acid reactive substances (TBARS), total antioxidant, asymmetric dimethylarginine (ADMA) and L-arginine, and 12-lead electrocardiograms were analyzed in these patients. Statistics were based on continuous and categorical variables using Fisher’s exact or Chi-square or one-way ANOVA or KruskalWallis tests. The results were considered significant when P < 0.05. Results: The study showed that the GG genotype was prevalent, with 54% of patients, followed by 41% GT and 6% TT, and the genotypic distribution was not associated with QTc prolongation. Furthermore, patients with the T allele showed increased ADMA, L-arginine and peroxidation lipid levels with reduced NO synthesis. Conclusion: Our study showed a lack of association between QTc interval and eNOS polymorphism; however, it was found that patients with the T allele had a greater risk of developing endothelial dysfunction by ADMA, which could contribute to future cardiovascular complications and worsening of CKD.

Renal cell carcinoma (RCC) represents 3% of human malignant tumors and approximately 90% of malignant renal neoplasms. Despite great therapeutic advances in the last decade, metastatic RCC (mRCC) is still considered an incurable disease. In this study, we examined the potential of essential elements as biomarkers of mRCC using an orthotropic metastatic mouse model. Frozen lung and plasma samples from healthy and mRCC-induced mice were lyophilized, digested, and analyzed using inductively coupled plasma mass spectrometry. In metastatic lungs, a significant increase in Ca concentration (268%) was observed, whereas a significant decrease in Cu (23.2%), Fe (17.4%), Mn (38.8%), and Na (11.7%) was observed. The plasma of mRCC-induced mice showed decreased concentrations of Mn (53%), Na (19.7%) and Zn (49,50%) and increased levels of Ca (53%), Cu (39.5%), Our findings revealed marked differences in the concentrations of essential elements in the lung and plasma of the metastatic mouse model. The circulating levels of Ca, Cu, Mn, Na, and Zn could be utilized as diagnostic and therapeutic response biomarkers.

Aim: To evaluate the role of endostatin (ES) gene therapy on myeloid-derived suppressor cells (MDSC) in a metastatic model of renal cell carcinoma (RCC). Materials and Methods: Balb/C mice bearing orthotopic Renca tumors were treated with NIH/3T3LendSN or, as a control, with NIH/3T3-LXSN cells. At the end of in vivo experiment, plasma and tissue lung samples were collected. Plasma ES and granulocyte colony stimulating factor (G-CSF) levels were measured by ELISA and Milliplex, respectively. Quantification of CD11b+Gr-1+ cells and their subsets was performed by flow cytometry. Reactive oxygen species (ROS) production was measured in CD11b+Gr-1+ MDSC using the DCFDA marker by flow cytometry. Results: Metastatic RCC (mRCC) induced expansions of CD11b+Gr-1+ MDSC and promoted accumulation of these cells and their subtypes in lymphoid organ and metastases. ES treatment promoted low G-CSF plasmatic levels which were produced by the tumor microenvironment, reflecting the reduced metastatic accumulation of CD11b+Gr-1+ MDSC in the lungs. However, the therapy was selective for granulocytic cells, thus reducing the production of ROS. Conclusion: These findings confirm the expansion of MDSC during metastatic progression of RCC and indicate the important role of ES in reducing MDSC and possible use of ES therapy in combined anticancer treatment.

The phenolic compound [6]-Gingerol, isolated from Zingiber officinale, has been demonstrated to have antitumor activity for different types of malignant tumours. Prostate cancer is the most common malignancy among males worldwide, being the second leading cause of cancer death in men. In the present study, we investigated the antitumor action of [6]-Gingerol on a human prostate cancer cell line (LNCaP). Our data shows that [6]-Gingerol treatment induced a dose-dependent decrease in the cell viability. Compared with the vehicle control, the cell viabilities were 79.90 ± 3.56% and 53.06 ± 7.82% when the LNCaP cells were exposed to 150 μg/mL and 300 μg/mL of [6]-Gingerol, respectively. The treatment of LNCaP with 300 µM of [6]-Gingerol led to a significant reduction (~25%) on the clonogenic survival of these cells. Furthermore, [6]-gingerol acted as a radiosensitizer for LNCaP cells. The pretreatment of these cells with [6]-Gingerol significantly enhanced the killing effects of ionizing radiation with a dose enhancement ratio of 1.25. Our results demonstrate the anti-tumour activities of [6]-Gingerol. Further studies are needed to elucidate the mechanisms involved.

Acute kidney injury (AKI) is an important health problem and can be caused by number of factors. The use of aminoglycosides, such as gentamicin, is one of these factors. Recently, an effort has been made to find biomarkers to guide treatment protocols. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to estimate the contents of Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn in serum and urine of the healthy, AKI, and spontaneous recovery (SR) groups of animals. The animal model of AKI and SR was validated by measuring serum and urinary urea and creatinine. The quantitative determination of the elements showed a decrease in serum levels of Ca, and Fe in the AKI group (P

Renal cell carcinoma (RCC) remains one of the greatest challenges of urological oncology and is the third leading cause of death in genitourinary cancers. Surgery may be curative when patients present with localized disease. Our previous results demonstrated the autofluorescence of blood PpIX in primary RCC mouse model and an increase in fluorescence intensity as a function of growth of the subcutaneous tumor mass. In another work, a nice correlation between the growth of the tumor mass and tissue fluorescence intensity was found. The aim of this study was to evaluate the expression profile of porphyrin biosynthesis pathway-related genes of human kidney cells. We used two kidney cell lines, one normal (HK2) and another malignant (Caki-1). Endogenous and 5-aminolevolinic acid (ALA) induced protoporphyrin IX (PpIX) HK2 and Caki-1 cells were analyzed by fluorescence spectroscopy. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure mRNA of those genes. Emission spectra were obtained by exciting the samples at 405 nm. For ALA untreated cells the maximum fluorescence intensity was detected at 635 nm. The mean peak area of emission spectra in both cells types increased linearly in function of cell number. Besides, basal levels of PpIX autofluorescence of each cell concentration of HK2 samples were significantly lower than those of Caki-1 samples. For ALA-treated cells the mean PpIX spectra shows PpIX emission peak at 635 nm with a shoulder at 700 nm. Analysis of PpIX fluorescence intensity ratio between tumor cells and HK2 cells showed that fluorescence intensity was, on average, 26 times greater in tumor cells than in healthy cells. qRT-PCR revealed that in Caki-1 ALA-treated cells, PEPT gene was significantly up-regulated and FECH and HO-1 genes were significantly down regulated in comparison with HK2 ALA-treated cells. In conclusion, our results demonstrate the preferential accumulation of ALA-induced PpIX in human RCC and also indicate that PEPT1, FECH and HO-1 genes are major contributors to this accumulation.

Renal cell carcinoma (RCC) accounts for approximately 2%-3% of human malignancies and is the most aggressive among urologic tumors. Biological heterogeneity, drug resistance, and chemotherapy side effects are the biggest obstacles to the effective treatment of RCC. The NF-κB transcription factor is one of several molecules identified to be responsible for the aggressive phenotype of this tumor. In the past decade, several studies have demonstrated the activation of NF-κB in RCC, and many have implicated NF-κB1 (p50) as an important molecule in tumor progression and metastasis. In the present study, a lentivirus was used to deliver shRNA targeting NF-κB1 into mouse RCC (Renca) cells. It was determined that the knockdown of the NF-κB1 gene led to a reduction in cell proliferation and late apoptosis/necrosis in vitro. Flow cytometry analysis demonstrated G2/M arrest in the cells. In addition, immunoblotting analysis revealed a significant increase in cyclin B1 and Bax. In vivo experiments showed that Renca-shRNA-NF-κB1 cells have significantly diminished tumorigenicity. Moreover, immunohistochemical analysis revealed an increase in necrotic areas of Renca-shRNA-NF-κB1 tumors. Thus, this study indicates that downregulation of NF-κB1 can suppress RCC tumorigenesis by inducing late apoptosis/necrosis. Therefore, NF-κB1 may be a potential therapeutic target for RCC.

Following physical examination and radiography analysis, cystotomy was performed to remove a rabbit’s single bladder stone. This rabbit urolith was analyzed by X-ray fluorescence spectrometry (EDXRF) and scanning electron microscopy (SEM). The EDXRF technique was successful for the determination of major elements (Ca, Mg, P, K and S) and presented sufficient sensitivity to also trace elements (Sr, Fe, Cu, V, Cr, Mn, Zn and Pb) determination. The results showed significant quantitative and structural variations among the urolith regions. The EDXRF technique using the fundamental parameters method and SEM attend as complementary techniques that can be useful in the management of urinary stone analysis.

Normal prostate tissue contains high levels of citrate. In the presence of prostate cancer, the citrate level is diminished. In this paper we show that it is possible to use europium–oxytetracycline complex as a citrate fluorescent probe and consequently as a prostate cancer probe. We analyzed normal nude male mice urine and urine from nude male mice in which prostate cancer was induced by intraprostatic inoculation of DU145 cells. The urine samples were collected from the animals at the 7th, 14th, 21st, and 35th days after the surgery procedures. The intensity of europium emission at 615 nm in europium–oxytetracycline complex in the presence of citrate increases linearly. The citrate concentrations were determined from a calculated calibration curve. A concentration decrease in malignant prostate urine from the normal (PBS group) urine value from ∼8.0 mM to ∼2.4 mM (tumor group at 35th day) was found. The obtained results indicated that europium–oxytetracycline provides a significant biomarker for prostate cancer detection with a direct, accurate, noninvasive, and non-enzymatic method for measurement of citrate in biological fluids.

Despite recent advances in targeted therapy, renal cell carcinoma (RCC) remains one of the most lethal urologic malignancies. Approximately 30% of patients with localised RCC will develop metastases after curative surgery. Presurgical therapy has been explored for treatment of localised RCC. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential use of an antiangiogenic agent as a neoadjuvant therapy in an orthotopic metastatic mouse model of RCC. BALB/c mice bearing Renca cells were treated before nephrectomy with NIH/3T3-LendSN cells. At the end of the experiment, ES serum levels were measured. Primary and metastatic tumour area and microvascular area were determined. In the survival studies, mice were monitored daily until they died. ES serum levels in treated mice were higher in the control group (P < 0.05). The median primary tumour area and the mean microvascular area were significantly lower in the ES-treated group compared to control group (P < 0.05). The proliferation of Renca cells in the ES-treated group was significantly reduced compared with the control group (P < 0.01). ES therapy led to a significant reduction in the number of pulmonary metastatic nodules compared with the control group (P < 0.01). Kaplan–Meier survival curves showed that the probability of survival was significantly higher in mice receiving ES therapy (P = 0.0243, Log-Rank test). Our results indicated that neoadjuvant ES gene therapy has the potential to decrease tumour burden, extend survival, and may have clinical benefit in the management of RCC.

Summary Background The objective of this paper was to verify if the oral administration of δ-aminolevulinic acid (ALA) in animals with prostate tumor can increase the sensitivity of cancer diagnosis by protoporphyrin IX blood autofluorescence. In this study, the autofluorescence of blood porphyrin was analyzed using fluorescence spectroscopy on healthy male NUDE mice and in those with prostate cancer induced by the inoculation of DU145 cells. Methods A total of 18 male NUDE mice, ∼8 weeks old on arrival were divided into 3 groups: Control, Tumor and ALA Tumor. The autofluorescence of blood porphyrin of the groups was analyzed using fluorescence spectroscopy at different days after tumor induction, to monitor the tumor progression. Emission spectra were obtained by exciting the samples at 405 nm. The animals inoculated had their blood collected with and without oral ALA solution administration to compare PPIX endogenous (Tumor group) and exogenous (ALA Tumor group) signal intensity and to establish a method to diagnosis early prostate cancer. Results Significant differences were observed in autofluorescence intensities measured in the 575–725 nm spectral regions for the studied groups. Conclusions The results showed an enhancement of almost 100% in blood PPIX fluorescence, using the oral administration of δ-aminolevulinic acid on male NUDE mice with prostate cancer, making fluorescence measurements more accurate and sensitive since the first week after tumor induction.

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. In the survival studies, 10 mice/group daily, mice were monitored daily until they died. The presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). The significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). The IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.

Renal cell carcinoma (RCC) remains one of the greatest challenges of urological oncology and is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and are amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of erythrocyte PpIX fluorescence spectroscopy for monitoring the efficacy of antiangiogenic therapy in metastatic renal cell carcinoma (mRCC), using an orthotopic metastatic mouse model. Balb/C-bearing Renca cells were treated with NIH/3T3-LendSN cells. Lung weight, nodule area, microvascular area (MVA), and erythrocyte PpIX fluorescence were evaluated. Emission spectra were obtained by exciting the samples at 405 nm. There was a significant decrease in lung wet weight, lung nodule area and MVA in the treated group compared to the control group (P

Prostate cancer is one of the most common types of cancer in men, and unfortunately many prostate tumours remain asymptomatic until they reach advanced stages. Diagnosis is typically performed through Prostate-Specific Antigen (PSA) quantification, Digital Rectal Examination (DRE) and Transrectal Ultrasonography (TU). The antigen (PSA) is secreted by all prostatic epithelial cells and not exclusively by cancerous ones, so its concentration also increases in the presence of other prostatic diseases. DRE and TU are not reliable for early detection, when histological analysis of prostate tissue obtained from a biopsy is necessary. In this context, fluorescence techniques are very important for the diagnosis of cancer. In this paper we explore the potential of using endogenous phorphyrin blood fluorescence as tumour marker for prostate cancer. Substances such as porphyrin derivatives accumulate substantially more in tumours than in normal tissues; thus, measuring blood porphyrin concentration by autofluorescence intensity may provide a good parameter for determining tumour stage. In this study, the autofluorescence of blood porphyrin was analyzed using fluorescence and excitation spectroscopy on healthy male NUDE mice and in those with prostate cancer induced by inoculation of DU145 cells. A significant contrast between the blood of normal and cancer subjects could be established. Blood porphyrin fluorophore showed an enhancement on the fluorescence band around 632 nm following tumour growth. Fluorescence detection has advantages over other light-based investigation methods: high sensitivity, high speed and safety. However it does carry the drawback of low specificity of detection. The extraction of blood porphyrin using acetone can solve this problem, since optical excitation of further molecular species can be excluded, and light scattering from blood samples is negligible.

Protoporphyrin (PpIX), a porphyrin derivative, is the intermediate metabolic precursor of the heme molecule. Abnormal metabolism of total erythrocyte PpIX has been observed in diseases such as cancer, lead poisoning, psoriasis, iron deficiency anemia and acute porphyries. Diabetes mellitus (DM) is a complex metabolic syndrome in which hyperglycemia is the primary clinical manifestation and contributes to the diabetic complications. The aim of this study was to evaluate the utility of fluorescence spectroscopy of erythrocyte PpIX for monitoring the early stages of diabetes. A total of 14 male C 57BL mice, 6 weeks old, were divided into two groups: diabetic and non-diabetic. Diabetes was induced by intraperitoneal injection of streptozotocin (SZT). Blood cells were cultured with standard and 50 mM supplemented RPMI medium. Blood smears were prepared and stained for qualitative morphology analysis under optical microscopy. Blood porphyrin autofluorescence was analyzed by fluorescence spectroscopy. Characteristic PpIX emission spectra were obtained by exciting the samples at 405 nm. Average blood glucose was lower in the control group than in the diabetic group (156.50 ± 8. 11 mg/dL vs. 371.10 ± 14.43 mg/dL, P, < 0.05). Both diabetic and glucose-cultured erythroblasts showed a significant decrease (around 30.5% and 40%, respectively) in the emission band intensity at 635 nm. Our results indicate that the erythrocyte PpIX profile could be used as a biological monitor for diabetes.

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600-700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.

The present study investigated the influence of 241Am on microbial growth and the degradation of organic waste. Leachate samples collected in a lysimeter were periodically analyzed for bacterial growth, under both aerobic and anaerobic conditions. 241Am inhibited bacterial growth, and the degradation of organic matter was delayed in comparison with the control. Minimal inhibitory concentration assays and survival curves revealed that it inhibits the growth of Pseudomonas putida F1. The assay also revealed that 241Am is more toxic than 238U, Zn2+ and Cd2+. This study further led to the finding of four new radionuclide-tolerant bacterial strains: Flavobacterium spp., Pseudomonas gladioli, Chryseobacterium indologenes and Ochrobactrum anthropi. The survival curves of P. gladioli, C. indologenes revealed that these bacteria are resistant to metal as consortia.

OBJECTIVE: Investigate the possible association of insertion/deletion (2G/G) polymorphism at nucleotide -1607 of the MMP-1 promoter with the development and progression of renal cancer MATERIALS AND METHODS: In this study, we genotyped 217 individuals, 99 patients with renal cell carcinoma (RCC) and 118 controls without cancer. DNA specimens were extracted from epithelial buccal cells and paraffin-embedded tissue of RCC patients and from epithelial buccal cells and blood cells of healthy controls RESULTS: The difference in frequency of 2G/2G genotype between controls (22.9%) and RCC patients (28.6%) was not statistically significant (p = 0.461). We also did not find correlation between 2G/2G and histological type of RCC. The comparison of genotype distribution and frequency of 2G allele in different populations showed a strong variability of 2G allele frequency among the different ethnic groups. This fact may influence on the collaboration of this 2G allele in RCC or others diseases CONCLUSION: Our data suggest that the matrix metalloproteinase-1 (MMP-1) promoter polymorphism may not play a significant role in renal cell carcinoma patients in Brazil.

SUMMARY: Background: Renal ischaemia-hypoxia is a leading cause of acute renal failure, a clinical condition associated with rapid loss of renal function and high rates of mortality. Renal proximal tubular cells are the most severely injured during renal ischaemia, caused by the breakdown of the extracellular matrix of the tubular basement membrane. Endostatin is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage and it is well-known as being an inhibitor of angiogenesis. In vitro, endostatin inhibits endothelial cell proliferation and migration, as well as tubule formation. In vivo, it has a potent inhibitory effect on tumour growth. In this study, we analysed endostatin gene expression in C57BL/6 mouse kidneys subjected to ischaemia/reperfusion. Methods: Ischaemic renal failure was induced via 45 min of bilateral occlusion of the renal artery and vein, followed by 12 h or 24 h of reperfusion. Whole-kidney homogenate and total RNA were extracted for examination by western blot analysis and quantitative polymerase chain reaction. The immunohistological examination revealed increased endostatin expression in injured kidney, mainly in the proximal tubule and collecting ducts. Results: Endostatin/collagen XVIII mRNA and protein expression increased during ischaemia and within 12 h of reperfusion. In the western blot assay, we identified increased expression of the 30 kDa endostatin-related fragment and of matrix metalloproteinase-9. CD31 was significantly expressed during reperfusion (P < 0.05). Immunohistological examination revealed glomerular and tubulointerstitial expression of endostatin. Conclusion: These data suggest the local synthesis of a 30 kDa endostatin-related fragment following acute renal failure and suggest its role in the modulation of renal capillary density.

Radioactive lightning rods were manufactured in Brazil until 1989, when the licenses for using radioactive sources in these products were lifted by the national nuclear authority. Since then, these rods have been replaced by the Franklin type and collected as radioactive waste. However, only 20% of the estimated total number of installed rods has been delivered to the Brazilian Nuclear Commission. This situation causes concern, since there is the possibility of the rods to be disposed as domestic waste. In Brazil, 64% of the municipal solid waste is disposed at garbage dumps without sufficient control. In addition, (241)Am, the radionuclide most commonly employed, is classified as a high-toxicity element, when incorporated. In the present study, (241)Am migration experiments were performed by means of a lysimeter system, in order to evaluate the risk of contamination caused by radioactive lightning rods disposed as common solid waste. (241)Am sources removed from lightning rods were placed inside lysimeters filled with organic waste that was collected at the restaurant of the Instituto de Pesquisas Energéticas e Nucleares. The generated leachate was periodically analyzed, and characteristics such as pH, redox potential, solid content and the concentration of the radioactive material were determined. The equivalent dose for members of the public was calculated considering ingestion of contaminated drinking water as the major path of exposure. Estimated doses were about 20-times below the effective dose limit of 1 mSv year(-1) for members of the public as recommended by the International Commission on Radiological Protection. This suggests the radiation risk caused by lightning rods disposed at uncontrolled garbage dumps to be low. It should be noted, however, that the number of investigated lightning rods was quite small. The results of this study might therefore not be entirely representative and should be interpreted with care. They provide, however, a very first basis for characterizing the transfer of (241)Am from lightning rods to the human food chain.

SUMMARY: Background: Nitric oxide (NO) released from endothelial cells is related to the maintenance of physiological vascular tone. The impairment of endothelial NO generation brought about by gene polymorphism is considered one of the deterioration factors in progressive renal disease. In the endothelial nitric oxide synthase (eNOS) intron 4 polymorphism, the presence of the aa genotype has been associated with cardiovascular and renal disease. The aim of this study was to investigate the presence of eNOS gene intron 4 polymorphism in patients with end-stage renal disease (ESRD). Methods: A total of 114 patients and 94 controls were studied. DNA specimens were extracted from blood and amplified by polymerase chain reaction. The alleles were separated by agarose gel electrophoresis. Genotype distribution and allele frequencies were compared between groups using the chi-squared test. Results: Statistical analysis revealed that the frequency of the eNOS4 genotype aa was significantly different in ESRD patients and in controls (P = 0.016, OR = 2.07, CI 95%: 1.14‐3.74). There was also a statistically significant difference between ESRD patients and controls regarding allele carriers (P = 0.004; OR = 2.26; CI 95%: 1.29‐3.96). When the frequencies of allele carriers in the diabetic nephropathy group and in the control group were compared, a significant difference was found (P = 0.034, OR = 2.28; CI 95%: 1.04‐5.00). Conclusion: This study showed a strong correlation between eNOS4a polymorphism and end-stage renal disease.

Background Renal cell carcinoma (RCC) is a highly vascularized cancer resistant to chemotherapy and radiotherapy. RCC is frequently infiltrated with immune cells, with macrophages being the most abundant cell type. Alternatively activated M2 macrophages are known to contribute to tumor progression. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we investigated the impact of ES gene therapy on the polarization of tumor-associated macrophages (TAMs) in lung metastases from tumor-bearing mice. Methods BALB/c mice divided into three groups: Normal, Control and ES-treated. Tumor-bearing mice were treated with ES-transduced cells or control cells over ten days. At the end of the study, plasma was collected, and pulmonary macrophages were isolated and used for FACS or RT-PCR. ELISA tests were used to analyze plasma and cell culture supernatant cytokines. Results ES treatment significantly reduced the levels of anti-inflammatory and pro-angiogenic cytokines, including IL4, IL-10, IL-13 and VEGF. Gene expression of M2 markers, such as IL-10, Arg-1, VEGF and YM-1, declined significantly. Flow cytometry showed a reduction in the number of M2 F4/80 + CD36 + CD206 + CD209+ macrophages and in IL-10 secretion by these cells. Reduced levels of IL-10 were also found in the culture supernatants of the ES-treated group. Conclusions Our research corroborates previous observations that ES has an important anti-tumoral role. However, aside from promoting interferon-ɤ secretion and an effective T cell response, we show here that this switch is extended to TAMs, complicating the maintenance of pro-tumorigenic M2 macrophages and thus favoring tumor elimination.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.

Renal cell carcinoma (RCC) represents approximately 2-3% of human malignancies. Nuclear transcription factor кB (NF-кB) is composed of a family of transcription factors that have been associated with the development and progression of RCC. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we evaluated the expression of NF-кB in metastatic tumor cells from animals treated with ES. Balb/c-bearing Renca-EGFP cells were treated with NIH/3T3-LendSN or NIH/3T3-LXSN cells as a control. At the end of the in vivo experiment, plasma Renca-EGFP-sorted cells and tissue lung samples were collected. A real-time PCR array for NF-κB target genes revealed that ES therapy led to down regulation of Bcl-3 (P

Renal cell carcinoma (RCC) is characterized by high vascular endothelial growth factor (VEGF) production and, consequently, excessive angiogenesis. Several strategies have been developed to target angiogenesis as a method for treating metastatic RCC (mRCC). Endostatin (ES) is a C-terminal fragment of collagen XVIII that has antiangiogenic activity. The aim of this study was to investigate the predictive value of circulating VEGF-A in a murine model of mRCC after ES gene therapy. ES therapy did not affect the levels of collagen XVIII/ES or ES in the tissue. The circulating level of ES was increased in the control and ES-treated groups (normal vs. control, P

This paper describes the elimination of porphyrins by feces. It was demonstrated that porphyrin accumulates substantially more in tumors than in normal tissues, and consequently more PPIX reaches the blood of patients and animals with tumors, and then, it needs to be eliminated. The fluorescence of feces revealed that there are large amounts of PPIX in the excreta of animals with cancer comparing with healthy animals. The autofluorescence of feces porphyrin extracted with acetone was analyzed using fluorescence spectroscopy of animals inoculated with DU145 cells into the prostate and healthy animals to monitor the PPIX concentration. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences were observed in autofluorescence intensities measured in the 575–725 nm spectral regions for the studied groups. The results showed a noninvasive, simple, rapid and sensitive method to detect cancer by feces analysis.

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers and is highly resistant to both radiotherapy and chemotherapy. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor, was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick. This paper reports the biological effect of Amblyomin-X on inducing cell death by apoptotic process in vitro. For this purpose, the changes in morphological aspects of cells, the phosphatidylserine exposition and DNA degradation were evaluated after treatment with Amblyomin-X. We found that Amblyomin-X was able to induce apoptosis in Renca cells in a dose-dependent manner. So, the results presented here open perspectives for new researches and developing for Amblyomin-X in the treatment of RCC.

Background Metastatic renal cell carcinoma (mRCC) is one of the most treatment-resistant malignancies. Despite all new therapeutic advances, almost all patients develop resistance to treatment and cure is rarely seen. In the present study, we evaluated the antitumor effect of a bicistronic retrovirus vector encoding both endostatin (ES) and interleukin (IL)-2 using an orthotopic metastatic RCC mouse model. Methods Balb/C-bearing Renca cells were treated with NIH/3T3-LendIRES-IL-2-SN cells. In the survival studies, mice were monitored daily until they died. At the end of the in vivo experiment, serum levels of IL-2 and ES were measured, the lung was weighed, and the number of metastatic nodules, nodule area, tumor vessels and proliferation of tumor-infiltrating Renca cells were determined. Results Inoculation of NIH/3T3-LendIRES-IL-2-SN cells resulted in an increase in ES and IL-2 levels in the treated group (p < 0.05). There was a significant decrease in lung wet weight, lung nodule area and tumor vessels in the treated group compared to the control group (p < 0.001). The proliferation of Renca cells in the bicistronic-treated group was significantly reduced compared to the control group (p < 0.05). Kaplan–Meier survival curves showed that the probability of survival was significantly higher for mice submitted to bicistronic therapy (log-rank test, p = 0.0016). Bicistronic therapy caused an increase in the infiltration of CD4, CD4 interferon (IFN)γ-producing, CD8, CD8 IFNγ-producing and natural killer (CD49b) cells. Conclusions Retroviral bicistronic gene transfer led to the secretion of functional ES and IL-2 that was sufficiently active to: (i) inhibit tumor angiogenesis and tumor cell proliferation and (ii) increase the infiltration of immune cells. Copyright © 2011 John Wiley & Sons, Ltd.

The erythrocyte protoporphyrin IX was used to monitor the antiangiogenic treatment with endostatin in animal models of renal cancer. In the treated group there was a significant reduction in erythrocyte PpIX fluorescence levels.

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys.Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 microg (p0.05); 12.5 versus 3.15 microg (p0.05). Immobilization of KES28kDa supports endothelial cell survival over the control (p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers.These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival.

The development of cancer is a complex, multistage process during which a normal cell undergoes genetic changes that result in phenotypic alterations and in the acquisition of the ability to invade other sites. Inductively coupled plasma optical emission spectroscopy was used to estimate the contents of Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, and Zn in healthy kidney and renal cell carcinoma (RCC), and significant differences were found for all elements. Along with the progression of the malignant disease, a progressive decrease of Cd and K was observed. In fact, for Cd, the concentration in stage T4 was 263.9 times lower than in stage T1, and for K, the concentration in stage T4 was 1.73 times lower than in stage T1. Progressive accumulation was detected for P, Pb, and Zn in stage T4. For P, the concentration in stage T4 was 11.1 times higher than in stage T1; for Pb, the concentration in stage T4 was 232.7 times higher than in T1; and for Zn, the concentration in T4 was 8.452 times higher than in T1. This study highlights the marked differences in the concentrations of selected trace metals in different malignant tumor stages. These findings indicate that some trace metals may play important roles in the pathogenesis of RCC.

We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the NIH/3T3 fibroblast cell line could inhibit renal tumor growth in vivo. NIH/3T3 cells were transduced with retroviral vectors containing the murine endostatin (ES) gene. SCID mice bearing CaKi-1 derived tumors were given a subcutaneous injection of either ES-transduced cells or control cells and were monitored for tumor growth. At the end of the in vivo experiment, the mean tumor volume of treated mice was 51.6 +/- 2.4 mm(3), while the tumor volume of control was 234.5 +/- 14.8 mm(3). Microvascular density was significantly decreased on treatment (control 9.79 vs. ES 2.53%, < 0.001) accompanied by a 23-fold increase in intraturmoral necrotic area and a 2.94-fold increase in the apoptotic index, determined by immunohistochemistry with anti- activated caspase-3. Apoptotic cells were found in foci enriched in infiltrating leukocytes. in conclusion, retroviral endostatin gene transfer led to secretion of functional endostatin that was sufficiently active to inhibit tumor angiogenesis and tumor growth. A second mechanism may also be implied in endostatin-dependent tumor regression, associated with tumor infiltration of leukocytes. Besides its antiangiogenic properties, endostatin may be a promising adjuvant to immunotherapy. IPEN CNEN SP, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, Brazil CEPID FAPESP, Ctr Terapia Celular, São Paulo, Brazil Univ São Paulo, Fac Med, Dept Radiol, Expt Oncol Lab, São Paulo, Brazil Universidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, Brazil Web of Science

S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer. The T47D breast carcinoma cell line, a human breast epithelial cell that expresses high levels of S100P, was a tool used in this study to investigate the alteration in cell cycle induced by a retrovirus-mediated transfer of antisense-S100P. First we used the real-time PCR technique to quantify the gene expression. The results showed a reduction of 63% of expression within the T47D-S100P-A/S infected population compared with control T47D-LXSN clones. To determine the impact of the S100P antisense technique on protein expression in T47D cells, we performed immunofluorescence staining and analyzed the resulting images using a confocal microscope. The images showed much less pronounced antibody marking of the S100P protein in the T47D-S100P-A/S compared with control cells. To evaluate whether the antisense approach caused any alteration in the cell cycle, we concluded the study with flow cytometric analysis of the cell distribution. Our findings indicated that, in our model, S100P-antisense cells showed a 23% reduction of cells at the S-phase. Using transduction techniques with an S100P antisense-retroviral construct we were able to demonstrate a significant reduction in S-phase of the T47D cell cycle. To the best of our knowledge, this is the first time that an antisense approach has been used against S100P mRNA in breast cancer epithelial cells. The results showed here seem to further classify S100P as a protein that might be involved in the cell cycle imbalance observed during breast carcinogenesis.

Renal cell carcinoma (RCC) accounts for approximately 3% of new cancer incidence and mortality in the United States. Unfortunately many RCC masses remain asymptomatic and nonpalpable until they are advanced. Diagnosis and localization of early carcinoma play an important role in the prevention and curative treatment of RCC. The autofluorescence of blood porphyrin of healthy and tumor induced in male SCID mice was analyzed using fluorescence and excitation spectroscopy. A significant contrast between normal and tumor blood could be established. Blood porphyrin fluorophore showed enhanced fluorescence band (around 630 nm) in function of the tumor growth. This indicates that either the autofluorescence intensity of the blood fluorescence may provide a good parameter for the “first approximation” characterization of the tumor stage.

We introduce the use of a lanthanide complex, tetracycline–europium, for the clinical diagnosis of urea hydrogen peroxide in human whole blood. The values obtained agree with the urea concentration variation verified in 49 patients, including 12 predialysis, 12 peritoneal, and 15 dialysis subjects, and 10 controls. This method is noninvasive and can help in the identification of renal and cardiac diseases.

The tetracycline-europium (Tc-Eu) complex is known to show emission at 615 nm. On addition of hydrogen peroxide (HP), a strongly fluorescent complex is formed. In this paper it is reported that the europium fluorescence intensity is increased when urea hydrogen peroxide is added to the tetracycline-europium aqueous solution. It was conceived that this enhancement could be used to determine urea hydrogen peroxide (UHP) levels. This method is simple, practical, and relatively free of interference from coexisting substances, and it can be applied successfully to assess urea peroxide in biological samples, for example, on human whole blood. The values obtained for whole blood agree with the urea concentration variation verified in 50 patients, including 25 pre-dialysis, 15 dialysis subjects and 10 controls. This method is non invasive and can help in the identification of renal and cardiac diseases.

SPECIFIC AIMSIn this study we aimed to demonstrate that grafts of primary human keratinocytes, genetically modified to secrete hGH, can lead to sustained levels of circulating hGH in immunodeficient dwarf mice (lit/scid) resulting in phenotypic alterations, e.g., increases in their weight. As such, the grafted mice could serve as a model for hGH gene therapy.PRINCIPAL FINDINGS1. In vitro secretion of hGH by genetically modified primary human keratinocytesThe retroviral LXSN vector containing hGH cDNA under the control of the LTR promoter was used to produce a replication-defective retrovirus generated by transfecting GP+E86 ecotropic packaging cells with hGH vector (pLhGHSN) DNA via the CaCl2 method and subsequently infecting amphotropic GP+env+AM12 packaging cells. Eighteen colonies of the infected cells were grown separately and hGH virus-producing clones #1 and 6, designated AM12pLhGHSN-1 and -6, were selected on the basis of the viral titer (1.0×105 and 2.3×106 CFU/mL) and hGH concentration in the cul...

Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 μg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.

Homozygous 40- to 90-day-old "little" mice (lit/lit), derived from the C57BL/6J strain, have been used to set up an in vivo body weight gain bioassay for GH whose performance has been compared to the widely used hypophysectomized rat assay. A log dose-response curve has been analyzed in order to choose doses in the linear range that are suitable for setting up useful, precise, and economical 2 x 2 factorial assays. A comparison between the response in the two sexes has also been carried out, showing no significant difference between male and female little mice of this age. Three assays have been carried out in these animals for the potency determination of a local human GH (hGH) reference preparation in terms of First International Standard of GH (human) for bioassay (WHO 80/505), comparing them to a classical assay in hypophysectomized rats performed with the same preparations. The calculated potency values were in good agreement, while the statistical parameters indicated a comparable assay precision, even in a practical and rapid 4-day assay. We suggest the substitution of hypophysectomized rats with little mice for this in vivo test, still required for potency and bioidentity determination of clinical preparations of recombinant hGH. This avoids the highly invasive, costly, and time-consuming surgery, providing a faster, more flexible, and economical assay method, while still directly measuring parameters of in vivo linear growth in an animal model closely related to isolated hGH deficiency type I.

Autofluorescence of blood porphyrin was analyzed using fluorescence spectroscopy on healthy and prostate cancer male NUDE mice induced by DU145 cells. An enhancement of PPIX was observed for animals that received aminolevulinic acid solution.