[Urinary MicroRNAs as Potential Biomarkers of Bladder Cancer]. / Mocové mikroRNA jako potenciální biomarkery karcinomu mocového mechýre.

BACKGROUND: Currently, there are no urinary-based tumour markers with sufficient sensitivity and specificity to replace cystoscopy in the detection of bladder cancer (BCA). Urinary microRNAs are emerging as clinically useful class of biomarkers for early and non-invasive detection of urologic malignancies. PATIENTS AND METHODS: In this study, 155 patients with BCA and 83 healthy controls were enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays and candidate miRNAs further validated in independent cohort using specific TaqMan assays and quantitative real-time polymerase chain reaction method. RESULTS: Whole-genome profiling identified miRNA signature with significantly different concentrations in urine of BCA compared to controls (p < 0.01). In the independent validation phase of the study, three miRNAs were confirmed to have significantly higher levels in urine of patients with BCA in comparison with control groups (p < 0.0001). In addition, we observed significant decrease in two miRNAs (p < 0.01) concentrations in the urinary samples collected 3 months after surgery compared to pre-operative samples. CONCLUSION: We identified and validated miRNAs to have significantly higher concentrations in urine of patients with BCA in comparison with controls. Our data have shown that urinary miRNAs could serve as sensitive and specific biomarkers enabling non-invasive detection of BCA.Key words: urinary microRNAs - biomarkers - bladder cancer The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This study was supported by Ministry of Health of the Czech Republic, grant No. 15-31071A. All rights reserved.Submitted: 19. 3. 2018Accepted: 20. 3. 2018.

Comparison of different regimens of short-term antibiotic prophylaxis in transrectal prostate biopsy.

BACKGROUND: Prostate cancer is the most common malignant solid tumour in men aged >70 years and is the second most common cause of death from oncological circumstances. AIM: To evaluate the effect of different short-term prophylactic antibiotic regimens in transrectal prostate biopsy (PB) on the incidence of infectious complications. METHODS: Patients who underwent transrectal ultrasound-guided PB between January 2021 and December 2022 were included in the prospective randomized study. According to the regimen of prophylaxis, patients were randomized into three groups: (1) fosfomycin trometamol 3 g, 3 h before the procedure + ciprofloxacin 500 mg, 2 h before the procedure; (2) fosfomycin trometamol 3 g, 3 h before and 24 h after the procedure; (3) ciprofloxacin 500 mg 12, 2 h before the procedure, and 12 h after the procedure. A rectal swab was performed 1-2 weeks before PB to evaluate the culture findings. Complications were evaluated during follow-up visits within one month after PB. FINDINGS: In the monitored period, 605 PBs were performed, and 544 patients met the inclusion criteria (184, 161, and 199 in groups 1, 2, and 3). Infectious complications occurred in 10 cases (1.83%), namely 3, 4, and 3 according to patient groups. There was no statistically significant difference between the individual groups. None of the patients required hospitalization and all were free of symptoms of sepsis. CONCLUSION: Short-term antibiotic prophylaxis in PB using fosfomycin trometamol, ciprofloxacin, or their combination appears to be effective. Fosfomycin trometamol is a suitable alternative to fluoroquinolone antibiotics.

Urinary tract infections in patients with multiple sclerosis and different methods of bladder evacuation.

INTRODUCTION AND OBJECTIVES: To evaluate the incidence and course of urinary tract infections (UTI) in patients with multiple sclerosis (MS) and their relationship to the method of bladder evacuation. MATERIALS AND METHODS: Patients with neurogenic bladder dysfunction due to MS (n=111) were enrolled in the study. During one-year follow-up, clinical examination with urine culture was performed every 4 months or whenever symptoms occurred. The control group included patients with symptomatic UTI, without neurological or autoimmune disease. Incidence of symptomatic and asymptomatic bacteriuria, the effect of urine drainage on UTI incidence, and the effect of antibiotics were statistically evaluated. RESULTS: 54 MS patients completed the protocol. The mean incidence of symptomatic and asymptomatic bacteriuria in the MS group was 12.5% and 29.6%, respectively. A decreasing trend in the incidence of symptomatic, and an increasing trend in the incidence of asymptomatic bacteriuria was observed. Eradication of UTI in symptomatic MS patients was significantly lower than in controls (37.75% vs. 92.93%, P<0.05). Causative agents significantly differed in both groups (P=0.0005). The hypothesis that the incidence of UTIs in MS patients is independent of the method of bladder evacuation was not rejected (P>0.99 at visit 0, 1 and 3, P=0.078 at visit 2). CONCLUSIONS: There is a significant difference between the causative agents of UTI in both groups. Eradication of bacteriuria in symptomatic MS patients is difficult when compared to the normal population. We have insufficient evidence to confirm the relationship between the incidence of UTI and the method of bladder evacuation.

Morphology and peroxidase cytochemistry of mouse promonocytes, monocytes, and macrophages.

Mouse promonocytes have been identified and studied in cultures of bone marrow cells. These cells have a diameter of 14-20 micro, and in stained preparations reveal a large, indented or folded nucleus, and basophilic, finely granular cytoplasm. The living promonocyte viewed by phase contrast shows additional features: nucleoli, small dense bodies, and vesicles in the cytoplasm adjacent to the nuclear hilus, and slight membrane ruffling. Prominent ultrastructural components of promonocytes include a well developed Golgi apparatus, small numbers of centrosomal granules and vacuoles, extensive ribosomal aggregates, and finger-like projections of the cell surface. Promonocytes engage in pinocytosis and phagocytosis, but they are less active in these functions than are peripheral blood monocytes of peritoneal macrophages. Promonocytes are positive for peroxidase, the reaction product being localized to granules most of which are centrally situated in the cell. Monocytes in blood or in inflammatory peritoneal exudates display much smaller numbers of peroxidase-positive granules, and various types of mature mouse macrophages are peroxidase negative.

The in vitro differentiation of mononuclear phagocytes. IV. The ultrastructure of macrophage differentiation in the peritoneal cavity and in culture.

The structure of unstimulated mouse peritoneal phagocytes has been examined by electron microscopy and compared to cells obtained from the inflamed peritoneum and from cultures maintained in vitro. The unstimulated cell resembles the blood monocyte and contains a moderate amount of rough surfaced endoplasmic reticulum, a small but well defined Golgi apparatus and a few, small, electron-opaque granules in the cytoplasm. During in vitro cultivation there are marked changes in cell ultrastructure. Most prominent is the formation of large electron-opaque granules, some of which have a complex matrix containing both electron-opaque and lucent vesicles. In addition, there is an increase in size of the Golgi apparatus with the appearance of new lamellae and tiny, smooth surfaced vesicles. With continued cultivation, large lipid droplets are found in apposition to the rough endoplasmic reticulum. The formation and size of electron-opaque granules as well as the enlargement of the Golgi region is stimulated by high concentrations of serum in the medium. Cells obtained from the peritoneal cavity of lipopolysaccharide stimulated animals demonstrated changes in ultrastructure similar to those seen in cells cultured in vitro.

The in vitro differentiation of mononuclear phagocytes. V. The formation of macrophage lysosomes.

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles. These electron-lucent vesicles subsequently fused with and discharged their contents into larger pinocytic vacuoles. Colloidal gold was homogeneously distributed in the large pinosomes. In contrast, gold was initially deposited in the periphery of preformed dense granules indicating that these structures were also in constant interaction with the external environment. Colloidal gold was not observed within the cisternae of the endoplasmic reticulum nor within the saccules or vesicles of the Golgi apparatus. There were, however, many small, gold-free vesicles, indistinguishable from Golgi vesicles, which were preferentially aligned about and appeared to fuse with the large pinosomes. The intracellular flow of leucine-H(3)-labeled protein was followed by electron microscopic autoradiography. After a 15 min pulse of labeled amino acid there was initial labeling of the rough endoplasmic reticulum. Subsequently, much of the label appeared in the Golgi complex. At still later time periods the cytoplasmic dense granules contained the majority of the isotope. Acid phosphatase activity was localized to the dense granules and in the majority of cells to the Golgi apparatus. It is suggested that hydrolytic enzymes are initially synthesized in the endoplasmic reticulum and are then transferred to the Golgi apparatus. Here they are packaged into small Golgi vesicles which represent the primary lysosome of macrophages. The Golgi vesicles subsequently fuse with pinosomes, thereby discharging their hydrolases and forming digestive granules or secondary lysosomes.

Loss of iron from mouse peritoneal macrophages in vitro after uptake of (55fe)ferritin and (55fe)ferritin rabbit antiferritin complexes.

Mouse peritoneal macrophages in culture for 24 h were exposed to horse [(55)Fe]ferritin and rabbit antihorse [(55)Fe]ferritin antibody complex and the amount of (55)Fe in the medium was assayed up to 2 days after the pulse uptake. Cell survival was assayed by photographing the same areas of the tissue culture Petri dish on successive days and by counting cell numbers per unit area. In experiments in which quantitative assay for cell death is negligible, about 10-20% of the iron ingested by pinocytosis or phagocytosis is released to iron-free medium containing either freshly dialyzed or deironized newborn calf serum (10%). Over the 2-day postpulse period, iron loss is linear. This loss of iron to the medium is significantly reduced by adding iron-saturated newborn calf serum in the postpulse recovery period. A significant portion of the iron released to the medium is bound to transferrin. When human serum is used in the tissue culture system, similar quantities (10-25%) of the ingested iron are lost to the medium 2 days after the pulse.

Cytoplasmic granule formation in myelocytes. An electron microscope radioautographic study on the mechanism of formation of cytoplasmic granules in rabbit heterophilic myelocytes.

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.

Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and "postfixation" in uranyl acetate.

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.

Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique.

Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.

Autophagic vacuoles produced in vitro. I. Studies on cultured macrophages exposed to chloroquine.

Mouse macrophages exposed to 30 microg/ml of chloroquine in vitro develop autophagic vacuoles containing various cytoplasmic components and acid phosphatase. The early toxic vacuoles appear in the perinuclear region within 15 min; on electron microscopy, they show irregular shape, amorphous moderately dense content, apparent double membranes, and in some instances curved thin tubular extensions with a central, dark linear element. Cytoplasmic structures are probably transported into the vacuoles by invagination of the vacuolar membrane. After exposure to chloroquine for 1-4 hr, macrophages display large vacuoles containing degraded cytoplasmic structures, membranous whorls, and amorphous material. When chloroquine is removed by changing the culture medium after 4 hr, the cells survive and 24 hr later they exhibit no abnormality except for large cytoplasmic dense bodies packed with membrane lamellae. During recovery chloroquine disappears from the cells. 24 hr after exposure to chloroquine the macrophages have accumulated less hydrolases than control cells.

Autophagic vacuoles produced in vitro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine.

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1-2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.

Appearance and distribution of ferritin in mouse peritoneal macrophages in vitro after uptake of heterologous erythrocytes.

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.

Effect of chloroquine on morphology of cytoplasmic granules in maturing human leukocytes--an ultrastructural study.

Bone marrow and peripheral blood from patients who had received chloroquine phosphate were studied to determine the effect of this drug on the ultrastructure of cytoplasmic granules in leukocytes. Neutrophils from approximately one-half of the patients who were treated developed abnormal cytoplasmic granules. Vacuolar, lamellar, and particulate components within abnormal, large granules were present in myelocytes from certain patients who received chloroquine therapy. Mature neutrophils and lymphocytes from these patients showed variable numbers of large, membrane-bounded structures containing myelin figures. Cytoplasmic granules in eosinophilic myelocytes from patients treated with chloroquine did not contain the usual crystalloid structure, but instead contained small whorls of osmiophilic material. The granules in abnormal mature eosinophils were replaced by large vacuoles which contained amorphous material. The abnormal granules seen in these various white cells after chloroquine therapy may either reflect defective granule formation or autophagy.

Tannic acid effect on membrane of cell surface origin in guinea pig megakaryocytes and platelets.

Plasma membranes in isolated guinea pig megakaryocytes and washed platelets are poorly stained with the usual methods used to outline cell membranes. The addition of tannic acid and calcium to the initial fixative is useful to enhance electron density of all surface-derived membrane systems in these cells. The method described here shows that the increased electron denisty of membrane after fixation in the presence of tannic acid occurs both at the cell surface and along the invaginated membrane systems.

Secretion of cytokines and modulation of function of immunologically competent cells.

Cytokines and their effects are widely studied in immunology, but the cellular mechanisms responsible for producing these substances are not yet described or proven. Antigen presenting cells (APCs), macrophages and dendritic cells are suitable subjects for such a study. One of the many papers illustrating the interplay between phagocytosis, cytokine secretion, and cell adhesion is one by Kitajima et al. The intracellular processes involve both cytokine secretion and mobilization of the membrane system of the cell. Five pieces of evidence are presented to propose that the Golgi complex is the source both of the membrane translocated and cytokine production. Morphologic evidence is cited that reverse pinicytosis does not play a significant role in the mouse macrophage system.

Infecciones del tracto urinario en pacientes con esclerosis múltiple y los diferentes sistemas de vaciado vesical / Urinary tract infections in patients with multiple sclerosis and different methods of bladder evacuation

Introducción y objetivos: Evaluar la incidencia y la evolución de las infecciones del tracto urinario (ITU) en pacientes con esclerosis múltiple (EM) y su relación con el sistema de vaciado vesical. Materiales y métodos: Se incluyeron en el estudio pacientes con disfunciones miccionales neurógenas debido a la EM (n=111). Durante un año de seguimiento, se realizó una evaluación clínica con cultivo de orina cada 4 meses o ante la presencia de síntomas. El grupo de control incluyó a pacientes con ITU sintomática sin enfermedad neurológica o autoinmune. Se evaluó estadísticamente la incidencia de bacteriuria sintomática y asintomática, el efecto del drenaje urinario en la incidencia de ITU y el efecto del tratamiento antibiótico. Resultados: Cincuenta y cuatro pacientes con EM completaron el protocolo. La incidencia media de bacteriuria sintomática y asintomática en el grupo de EM fue del 12,5% y del 29,6%, respectivamente. Se observó una tendencia decreciente en la incidencia de la bacteriuria sintomática y una tendencia creciente en la incidencia de la asintomática. La erradicación de la ITU en los pacientes sintomáticos con EM fue significativamente menor que en los controles (37,75% frente a 92,93%, p<0,05). Los agentes causales fueron significativamente diferentes en ambos grupos (p=0,0005). No se rechazó la hipótesis de que la incidencia de ITU en los pacientes con EM es independiente del sistema de evacuación vesical (p>0,99 en las visitas 0, 1 y 3; p=0,078 en la visita 2). Conclusiones: Existe una diferencia significativa entre los agentes causales de la ITU en ambos grupos. La erradicación de la bacteriuria en los pacientes sintomáticos con EM es difícil en comparación con la población normal. No disponemos de pruebas suficientes para confirmar la relación entre la incidencia de ITU y el sistema de evacuación vesical. (AU)

Introduction and objectives: To evaluate the incidence and course of urinary tract infections (UTI) in patients with multiple sclerosis (MS) and their relationship to the method of bladder evacuation. Materials and methods: Patients with neurogenic bladder dysfunction due to MS (n=111) were enrolled in the study. During one-year follow-up, clinical examination with urine culture was performed every 4 months or whenever symptoms occurred. The control group included patients with symptomatic UTI, without neurological or autoimmune disease. Incidence of symptomatic and asymptomatic bacteriuria, the effect of urine drainage on UTI incidence, and the effect of antibiotics were statistically evaluated. Results: Fifty-four MS patients completed the protocol. The mean incidence of symptomatic and asymptomatic bacteriuria in the MS group was 12.5% and 29.6%, respectively. A decreasing trend in the incidence of symptomatic, and an increasing trend in the incidence of asymptomatic bacteriuria was observed. Eradication of UTI in symptomatic MS patients was significantly lower than in controls (37.75% vs. 92.93%, P<.05). Causative agents significantly differed in both groups (P=.0005). The hypothesis that the incidence of UTIs in MS patients is independent of the method of bladder evacuation was not rejected (P>.99 at visit 0, 1 and 3, P=.078 at visit 2). Conclusions: There is a significant difference between the causative agents of UTI in both groups. Eradication of bacteriuria in symptomatic MS patients is difficult when compared to the normal population. We have insufficient evidence to confirm the relationship between the incidence of UTI and the method of bladder evacuation. (AU)