Assessment of interleukin 17A and 23 in the course of bladder cancer and selected benign urological diseases.

Several cytokines have been indicated to be significantly involved in urological diseases. Interleukin 17A (IL-17A) and interleukin 23 (IL-23) have recently received attention for their involvement in inflammatory diseases and cancers. The aim of the study was to show changes in the level of pro-inflammatory interleukins IL-17A and IL-23 in patients with bladder cancer (BC) and selected urological diseases. An important cognitive aspect was to study the interdependencies between the studied interleukins and to assess their diagnostic value for such diseases. The material for the study was urine sample from patients with BC, urinary tract infection (UTI), urolithiasis, benign prostatic hyperplasia (BPH), US (urethral stricture), which was compared to the urine sample of healthy people without urological disorders. Interleukin concentrations were measured by the immunoenzymatic method. The levels of IL-17A and IL-23 in the urine of patients with BC, UTI, BPH and US were significantly higher compared to the control group. Statistically significant differences were found in the level of both interleukins compared to the control group in all diseases except urolithiasis. IL-17A and IL-23 correlated with each other in patients with all urological diseases except urolithiasis. The results of the conducted studies showed that selected urological diseases changed the levels of IL-17A and IL-23 in the urine of patients. The observations made confirmed the participation of these interleukins in the course of the urological diseases, especially in BC, and allowed to classify them as potentially useful parameters for diagnostic purposes.

The relationship between interleukin-13 and angiogenin in patients with bladder cancer.

Participation of anti-inflammatory interleukin-13 (IL-13) in the process of carcinogenesis was well studied. Angiogenesis plays a key role in the process of tumour growth and metastasis. Higher expression of angiogenin (ANG) have been proven in many types of cancers. The aim of the study was to more fully understand the significance of plasma IL-13 as an immunomodulator and ANG as a stimulator of the angiogenesis process in patients with bladder cancer (BC) and to investigate the relationship between parameters. These parameters were examined in the group of BC patients and in subgroups of BC depending on clinical stage: non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC), histopathologic malignancy low grade (LG), high grade (HG) and in primary and recurrent BC. The level of IL-13 and ANG in the plasma of BC patients and controls were measured by enzyme-linked immunosorbent assay. All calculations were done using the STATISTICA 13.3 (TIBCO software Inc.). Plasma levels of IL-13 and ANG were significantly higher in BC patients and in all patient subgroups examined than in the controls (p < 0.001). A negative significant correlation was found between ANG and IL-13 levels in BC-patients. Based on the receiver operating characteristic curves (ROC), IL-13 had good diagnostic value in BC. The presented results may suggest a relationship between angiogenesis and inflammation in the pathogenesis of bladder cancer and the development of this disease. With the increase of IL-13 level in BC-patients plasma, the ANG level decreased.

A biosensor for determination of the circulating biomarker CA125/MUC16 by Surface Plasmon Resonance Imaging.

CA125/MUC16 is an ovarian tumor cell marker widely used as a biomarker in epithelial ovarian carcinoma. CA125/MUC16 is also used for evaluation of the ROMA (Risk of Ovarian Malignancy Algorithm) value. In this work, a Surface Plasmon Resonance Imaging (SPRI) biosensor for circulating CA125/MUC16 has been developed. The anti-MUC16 antibody was attached to a gold chip via a cysteamine linker. The EDS/NHS protocol was used for the covalent attachment of the antibody. The developed biosensor is specific for CA125/MUC16, and exhibits good recovery and acceptable precision. Its linear response range (2.2-150 U/ml) is well suited to determination of the marker in the blood serum of a healthy control group and, after appropriate dilution, of patients with ovarian cancer. CA125/MUC16 was determined in two series of real samples: blood serum from patients with ovarian cancer and endometrial cysts. The method was validated by parallel determination of the samples using the chemiluminescent Architect i2000 method.

Uroplakin IIIa Is a Marker in Bladder Cancer but Seems Not to Reflect Chemical Carcinogenesis.

BACKGROUND: Uroplakins are glycoproteins investigated as potential markers of urothelial carcinoma. However, their role in chemical carcinogenesis is uncertain. In this study the diagnostic value of plasma and urine uroplakin IIIa (UPIIIa) levels in bladder cancer (BC) was investigated, particularly in the aspect of environmental exposure to chemical carcinogens, measured by DNA damage and detoxification ability in the BC smoking group. The correlation between uroplakin, 8-OHdG, and GSTπ was investigated. MATERIAL AND METHODS: This study included 61 BC patients and 33 healthy controls. UPIIIa, 8-OHdG, and GSTπ levels were estimated by the immunoenzymatic method (ELISA). RESULTS: UPIIIa levels were elevated in BC patients in plasma (p≤0.001) and in urine (p≤0.001), as were 8-OHdG and GSTπ levels in urine. Moreover, the 8-OHdG level was higher in invasive or high grade tumors. A positive correlation between UPIIIa/GSTπ and 8-OHdG/GSTπ was observed, but no UPIIIa/8-OHdG correlation was noted. CONCLUSION: The study showed the diagnostic value of urine and plasma UPIIIa in BC (good sensitivity, specificity, and predictive value). The lack of UPIIIa correlation with 8-OHdG and smoking suggests that UPIIIa does not reflect the environmental exposure. The increased levels of 8-OHdG and GSTπ in the invasive tumor stage indicate their value in BC monitoring.

Preliminary Evaluation of the Diagnostic Usefulness of Uroplakin 2 with an Assessment of the Antioxidant Potential of Patients with Bladder Cancer.

BACKGROUND: Urothelial carcinoma is the most common type of bladder cancer (BC). It makes up more than 90% of all bladder cancers. Uroplakins are tissue-specific, glycoproteins, playing a role in the construction and function of urothelium. The emergence of uroplakins in the urine and/or plasma may be of potential importance in the early detection of BC. In our study, the diagnostic value of plasma and urine uroplakin 2 (UP2) concentration in bladder cancer was investigated, with an assessment of the antioxidant potential of BC patients. The correlation between UP2, total antioxidant capacity (TAC), and concentration of glutathione (GSH) was also examined. MATERIALS AND METHODS: This study included 61 BC patients and 33 healthy controls. UP2 concentration was estimated by the immunoenzymatic method (ELISA). TAC and GSH were determined in spectrophotometrically methods. RESULTS: UP2 concentration in BC patients was significantly higher (p≤0.001) both in plasma and in urine compared to the control groups (C). TAC concentration in urine (p≤0.001) and GSH concentration in plasma (p=0.047) were significantly lower in BC group compared to the C group. The high specificity and sensitivity for UPK2 in plasma (76%, 80%, respectively) and urine (88%, 84%, respectively) were observed. Positive correlations were observed between concentration of UP2 in plasma and TAC concentration in urine and between UP2 concentration in plasma and GSH concentration in the same material. CONCLUSION: The study showed the early diagnostic value of urine and plasma UP2 in BC. There was a decrease in UP2 concentration in the urine of patients with the development of BC. The decrease of antioxidant systems (TAC, GSH) indicates their relationship with the BC process. Based on the obtained results, it is justified to continue the study in a larger group of patients with BC.

Immune checkpoints in aggressive breast cancer subtypes.

Immune checkpoints are molecules referred to inhibitory pathways in the immune system that play a pivotal role in prevention of autoimmunity and oncogenesis. The aim of the study was to evaluate expression levels of selected immune checkpoints- PD-1 (programmed cell death protein 1), and PD-L1 (programmed cell death 1 ligand 1) in breast cancer patients, suitable for breast conservation and sentinel node biopsy and determine their associations with clinicopathological factors.Expression of the genes coding for PD-1 and PD-L1 was analyzed in formalin-fixed paraffin-embedded specimens using real-time PCR. mRNA expression levels were determined using beta actin (ACTB) as an endogenous control. There was a trend towards significance between higher PD-1 and PD-L1 levels in triple negative breast cancers (p=0.1). Higher PD-L1 expression was also found in aggressive breast cancer subtypes e.g. triple negative and HER2 (human epidermal growth factor receptor 2) -positive as compared with subtypes with better prognosis such as luminal A and luminal BHER2-negative (p=0.05). There was a trend towards significance in higher PD-1 levels in triple negative and HER-2 positive breast cancers (p=0.1). A statistically significant difference was found between PD-L1 expression and tumor grade (p=0.01). Elevated PD-L1 levels were noted in G3 tumors. Immunogenicity appears to be gaining importance in triple negative and HER2-positive molecular subtypes of breast cancer, and the results in this study provide a basis for further investigation into the role of immune checkpoints in breast cancer.

Apoptosis-, proliferation, immune function-, and drug resistance- related genes in ER positive, HER2 positive and triple negative breast cancer.

The aim of our study was to examine an association between gene expression assessed using a 23-gene microarray and receptor status of breast cancer samples categorized as ER positive, HER2 positive and triple negative subtypes. The ER positive cohort was subsequently divided into Luminal A, Luminal B HER2 negative and Luminal B HER2 positive subtypes. Core- needle biopsies were collected from 78 female patients with inoperable locally advanced breast cancer or resectable tumors suitable for downstaging, before any treatment. Expressions of 23 genes were determined by means of TagMan Low Density Arrays. Analysis of variance was used to select genes with discriminatory potential between receptor subtypes. We introduced a correction for false discovery rates (presented as q values) due to testing multiple hypothesis. Pairwise post-hoc comparisons of receptor subtypes were performed using Tukey 's HSD test. Five genes out of a 23-gene microarray differed significantly in relation to breast cancer receptor-based subtypes. Among these five genes, we identified: BCL2 (p=0.0002, q=0.0009), MKI67 (p=0.0037, q=0.0064), IGF1R (p=0.0040, q=0.0064), FOXC1 (p=0.0113, q=0.0135) and IRF1 (p=0.0435, q=0.0416) as ones showing ER positive, HER2 positive and triple negative -subtype specific expression profiles. When incorporating Luminal A, Luminal B HER2 negative, Luminal B HER2 positive subtypes into analysis, four genes: BCL2 (p=0.0006, q=0.0034), MKI67 (p=0.0078, q=0.0198), FOXC1 (p=0.0102, q=0.0198) and IGF1R (p=0.0174, q=0.0254) were selected. Elevated levels of IGF1R and BCL2 were significantly linked with Luminal A subtype. Triple negative breast cancer subtype was associated with higher expression of IRF1, FOXC1 and MKI67. In HER2 positive cohort lower expression of all five analyzed genes was noted.

Kidney graft function in long-term cyclosporine and tacrolimus treatment: comparative study with nephrotoxicity markers.

Calcineurin inhibitors improve kidney allograft survival in the posttransplantation period; however, they may cause nephrotoxicity. The objective of this study was to compare the effect of cyclosporine (CsA) and tacrolimus (Tac) treatment on the transplanted kidney. The study included 219 patients aged 21 to 65 years. Of these, 120 (39 women and 81 men) were treated with CsA and 99 (38 women and 61 men) were treated with Tac. Patients were divided into 4 groups depending on the time since kidney transplantation. We evaluated urine markers of nephrotoxicity: proximal tubular cells lysosomal enzymes (N-acetyl-beta-d-glucosaminidase [NAG] and its isoform NAG-B, beta-d-galactosidase, and beta-glucouronidase) and brush border enzymes (alanyl aminopeptidase and gamma-glutamyl transpeptidase). Urine activities of nephrotoxicity markers were compared in CsA- and Tac-treated patients groups depending on the duration of treatment and allograft function as measured by serum creatinine concentration. Correlation studies between CsA and Tac levels and enzyme activities were performed in both groups and in the entire patient cohort. NAG and its isoform NAG-B seemed to be the most reliable markers of nephrotoxicity. Despite the significant correlation between NAG urine activity and serum creatinine concentration in the CsA group, there were no significant differences in NAG or NAG-B activities between CsA- and Tac-treated graft recipients.

Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo.

This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors expressing F134L and R273H grew slower than controls, and were associated with decreased necrosis and apoptosis, but not hypoxia. Interestingly, hypoxia levels were increased in tumors expressing M237L. There was less proliferation in F134L-bearing tumors compared to control, but this was not statistically significant. Angiogenesis was decreased in tumors expressing F134L and R273H compared with M237L, or controls. Conditioned medium from F134L tumors inhibited growth of normal human umbilical-vein endothelial cells but not telomerase-immortalized bone marrow endothelial cells. F134L tumor supernatants showed lower levels of VEGF and endostatin compared with supernatants from tumors expressing other mutants. Our results support the possibility that decreased angiogenesis might account for reduced growth rate of tumor cells expressing the F134L p53 mutation.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin.

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.

Structure of ovaries and oogenesis in Corydalidae and Chauliodidae (Insecta, Megaloptera). I. Architecture of adult ovarioles and previtellogenesis.

The results of histological and EM studies on the ovaries of three representatives of Megaloptera: Chauliodes pectinicornis, Nigronia fasciata (Chauliodidae), and Corydalus peruvianus Corydalidae) are presented. It is shown that the ovaries of all 3 investigated species are panoistic (secondary panoistic, = neopanoistic) and consist of numerous (more than a hundred) ovarioles that are differentiated into 3 well-defined regions: the terminal filament, the germarium, and the vitellarium. The germaria of adult females are apparently non-functional and contain germ and somatic cells in various stages of degeneration. The vitellaria are composed of 12-15 developing ovarian follicles (= oocytes surrounded by follicular cells) in a linear arrangement. In adult females these follicles can be classified into early previtellogenic, late previtellogenic, vitellogenic, and choriogenic. During early previtellogenesis oocyte nuclei (= germinal vesicles) contain single nucleolar masses. Histochemical analyses indicate that within the masses DNA as well as AgNOR proteins are present. During subsequent stages of the previtellogenic growth nucleolar masses gradually break down into smaller aggregations of coarse granular material, i.e. multiple nucleoli. In chauliodids the nucleoli are distributed evenly throughout the nucleoplasm while in the corydalid, C. peruvianus, they form a characteristic ring. The presented results are discussed in a phylogenetic context.

Evidence for gammadelta T cells with a restricted Vgamma6 junctional region in the normal mouse central nervous system.

In this study we present evidence that gammadelta T cells are present in the normal mouse central nervous system (CNS). Compared with matching spleen gammadelta T cells, CNS gammadelta T cells expressed only the CD45RBlow phenotype, suggesting that CNS gammadelta T cells belong to the memory cell population. Approximately 20% expressed exclusively the CD8alphabeta heterodimer, consistent with a thymic origin. Gammadelta T cells in both spleen and CNS expressed higher levels of the IL-2rbeta (CD122), as well as Fas and FasL, than alphabeta T cells, suggesting that these cells function as immunoregulatory T cells. RT-PCR analysis showed almost exclusive use of Vdelta6 in the CNS whereas more Vdelta genes were expressed in the spleen. Sequencing of Vdelta6 RT-PCR products demonstrated a polyclonal population of T cells in the spleen but a more clonal population within the CNS. The predominant CNS sequence was found in all animals studied and was also detected in the spleen. From these data we conclude that a selective component of circulating gammadelta T cells traffics through the CNS. Thus, all major populations of lymphocytes can be detected in the normal CNS and as such may play specific roles in the immunological surveillance of that organ.

[Venomous stings in the tropical world: bite and sting accident by a bumblebee swarm in a rainy Venezuelan forest]. / Picaduras venenosas en el mundo tropical: accidente por mordeduras y picaduras de un enjambre de abejorros en una selva lluviosa venezolana.

Two cases of poisoning produced by the unusual attack of colonies of insects from the family Bombidae, commonly known as bumblebees, are described. This type of poisoning may become severe toxicity and it is analyzed in the light of the latest findings. It is characterized by hemorrhagical symptoms, such as hematemesis and melaena.

Multiple sclerosis: the increased frequency of the ICAM-1 exon 6 gene point mutation genetic type K469.

Intracellular adhesion molecule-1 (ICAM-1) plays an important role in the cascade of adhesion events in the homing of inflammatory cells to the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Two single-base ICAM-1 polymorphisms have been described, in exons 4 and 6, changing codons 241 and 469 in the ICAM-1 gene, respectively. Both polymorphisms result in amino acid changes and can potentially lead to different interactions of ICAM-1 with its ligands. To detect ICAM-1 gene polymorphisms in MS, we have developed a highly sensitive and site-specific, two-stage, nested polymerase chain reaction. Genomic DNA was extracted from blood cells of 79 MS patients and 68 control subjects. The results were confirmed by direct dideoxy chain termination sequencing. The frequency of exon 6 allele T was found to be significantly higher in MS patients than in controls (68% vs 49%). Most interesting, the frequency of exon 6 homozygote K469 was significantly higher in MS patients than in controls (53% vs 34%). Higher frequency of the K469 genotype was found to be independent of possible linkage with the previously described MS susceptibility factor, the HLA class II DR2 allele. In the present study, we have shown for the first time the ICAM-1 gene polymorphisms in MS. The results indicate increased frequency of ICAM-1 exon 6 allele T in MS patients, which may contribute to the MS genetics background.

Multiple sclerosis: the frequency of allelic forms of tumor necrosis factor and lymphotoxin-alpha.

The cytokines LTa and TNF have been implicated as major mediators of tissue injury in multiple sclerosis (MS). In this study we have assessed the frequency of specific polymorphisms for these genes in MS (n = 53) and controls (n = 81) using a highly sensitive, two stage nested polymerase chain reaction (PCR), with the second stage using mutation-specific primers. Genomic DNA was extracted from blood cells and the results confirmed by direct dideoxy chain termination sequencing. The frequency of the -308 G to A mutation in the TNF promoter region in normal controls was 15% and in MS was 24%. For LTa gene the exon 3 polymorphism allele A was detected in 36% of controls and 34% of the MS patients. In MS, the combined genotype TNF G + A and LTa C + C was present 6 times more frequently (12%) than in controls (2%), and patients with this genotype showed the highest EDSS scores. We found the TNF and LTa polymorphisms to occur independently from the HLA class II DR2 allele distribution in MS. Whilst the G - A polymorphism in TNF gene promoter has been studied previously in MS, with conflicting results, this is the first study that has addressed the exon 3 polymorphism in LTa in MS. The results indicate that this polymorphism is not linked with the higher genetic predisposition for MS, but that combined TNF G + A and LTa C + C genotype might contribute to development of the disease.

Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g).

In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver cell line, (HEP G2ins) resulted in synthesis, storage and acute regulated release of insulin to analogues of cAMP, but not to the physiological stimulus glucose. In attempting to explain the lack of glucose responsiveness of the HEP G2ins cells we have stably transfected these cells with the human islet glucose transporter GLUT 2 (HEP, G2ins/g cells). The HEP G2ins/g cell clones exhibit glucose-stimulated insulin secretion and glucose potentiation of the secretory response to nonglucose secretagogues. While glucose responsiveness commenced at a lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated. Immunoelectron microscopy revealed the presence of insulin-containing granules, similar in size and appearance to those of the normal beta cell. These results demonstrate that while it is most likely that the HEP G2ins/g cell line predominantly secretes insulin via the constitutive pathway, significant acute regulated release was seen in response to glucose, and thus represents significant progress in the creation of a genetically engineered 'artificial beta cell' from a human hepatocyte cell line.

[The role of bronchoscopy and bronchoalveolar lavage in diagnosis of pulmonary tuberculosis in AIDS]. / Znaczenie bronchoskopii i plukania oskrzelowo-pecherzykowego w rozpoznawaniu gruzlicy pluc u chorych na AIDS.

Bronchoscopy was carried out in 32 HIV seropositive patients, most with AIDS during the period between January 1992 and August 1993. In 14 patients tuberculosis was diagnosed, in 13 it was bacteriologically confirmed. The mean age of the examined patients was 35.5 years (range 22-49 years). In 50% of the BAL samples bacterioscopy was positive. Bacteriological examination of the sputum and BAL fluid (bacterioscopy and culture) produced a confirmation of tuberculosis in 99.9% of the cases.