RNA quality in fresh frozen prostate tissue from patients operated with radical prostatectomy.

BACKGROUND: High-throughput technologies such as microarray have enhanced the discovery of new biomarkers in prostate cancer. However, the reliability of transcriptome analyses is limited by the RNA quality. OBJECTIVE: Identification of variables influencing the RNA quality in radical prostatectomy specimens. MATERIAL AND METHODS: RNA was extracted using an automatic extraction method for 354 samples from 38 fresh frozen prostate slices, and by manual extraction for 28 samples from 5 slices. RNA quality was measured using the RIN method (RNA Integrity Number). Evaluation of tissue composition was performed by light-microscopy for each sample. Age, total operative time, estimated blood loss, prostate volume, prostate specific antigen (s-PSA) and postoperative Gleason score were registered. The independent variables were correlated to the RIN score in a multiple linear regression model, taking p < 0.05 as the significance limit. RESULTS: The amount of blood loss during prostatectomy and the amount of stroma in the tissue sample both correlated negatively with the RIN score (p = 0.03 and 0.02). Automatically extracted samples which were exposed to heat according to the RNA extraction protocol, had lower mean RNA quality (5.5, 1.46 SD) than manually extracted samples, not exposed to heat (8.7, 0.86 SD), suggesting degradation by temperature sensitive RNases, mainly residing in the stroma. CONCLUSION: The highest RNA quality isolated by an automatic method from fresh frozen prostate tissue is obtained from patients with low peroperative blood loss and from samples with a low stromal fraction.

Uptake of IgG in osteosarcoma correlates inversely with interstitial fluid pressure, but not with interstitial constituents.

The uptake of therapeutic macromolecules in solid tumours is assumed to be hindered by the heterogeneous vascular network, the high interstitial fluid pressure, and the extracellular matrix. To study the impact of these factors, we measured the uptake of fluorochrome-labelled IgG using confocal laser scanning microscopy, interstitial fluid pressure by the 'wick-in-needle' technique, vascular structure by stereological analysis, and the content of the extracellular matrix constituents collagen, sulfated glycosaminoglycans and hyaluronan by colourimetric assays. The impact of the microenvironment on these factors was studied using osteosarcomas implanted either subcutaneously or orthotopically around the femur in athymic mice. The uptake of IgG was found to correlate inversely with the interstitial fluid pressure and the tumour volume in orthotopic, but not subcutaneous tumours. No correlation was found between IgG uptake and the level of any of the extracellular matrix constituents. The content of both collagen and glycosaminoglycans depended on the site of tumour growth. The orthotopic tumours had a higher vascular density than the subcutaneous tumours, as the vascular surface and length were 2-3-fold higher. The data indicate that the interstitial fluid pressure is a dominant factor in controlling the uptake of macromolecules in solid tumours; and the site of tumour growth is important for the uptake of macromolecules in small tumours, extracellular matrix content and vascularization.

TNF, IL-1, IL-6, IL-8 and soluble TNF receptors in relation to chorioamnionitis and premature labor.

Inflammatory cytokines seem to play a key role in mechanisms initiating labor. Since cytokine levels are higher in preterm than in term labor, it has been hypothesized that labor-inducing effects of cytokines are inhibited by an upregulated production of cytokine antagonists, such as soluble cytokine receptors, at early stages of gestation. In this study, TNF, IL-1, IL-6, IL-8 and soluble TNF receptors (sTNFRs) were measured in amniotic fluid samples from a) 39 women in premature labor, b) 25 women who where not in labor but delivered prematurely, and c) 33 women in term labor. Fifty-four of the placentas from premature deliveries were evaluated for presence of histological chorioamnionitis. Chorioamnionitis was associated with increased levels of TNF, IL-1 and IL-6, whereas elevated IL-1, IL-6 and IL-8 concentrations were found in premature parturition with no signs of infection. Concentrations of sTNFR were lower in preterm than in term deliveries. The present study confirms the participation of inflammatory cytokines in parturition. Multivariate analysis suggests a dominant, role of IL-1 in the presence of chorioamnionitis, whereas IL-6 seems to be more important during idiopathic premature labor. TNFR data do not support the hypothesis that production of cytokine antagonists is upregulated prematurely to prevent partirution.

Mast cells in human coronary arteries; is there any correlation with luminal narrowing?

Histamine in the human coronary arteries is harboured in mast cells and possibly in a non-mast cell store. Upon histamine exposure atherosclerotic coronary arteries react differently than normal coronary arteries, and histamine has been proposed to provoke vasospasms, ischaemia and sudden cardiac death. Previous reports have indicated a correlation between the mast cell density and the histamine content on the one hand and the degree of atherosclerosis on the other hand. Since previous reports applied visual assessment of the luminal narrowing only, we reexamined these results utilising planimetric methods. The study was accomplished by examining coronary arteries from autopsy cases (50-70 years, N = 29) with a diagnosis of sudden death, myocardial infarction or a non-cardiac cause. The mast cell density was calculated as the ratio between the mast cell count and the adventitial area as seen by light microscopy, and was compared to the degree of luminal narrowing. We were not able to find any correlation between the mast cell density and the luminal narrowing. The myocardial infarction group had the most severe degree of luminal narrowing, but there was no significant difference in mast cell density between the groups. We therefore question the previous notion that there is a correlation between the adventitial mast cell density in the coronary arteries and the luminal narrowing. Earlier reports may have been biased by visual assessment and by erroneously treating several measurements from the same person as independent samples.

Cytokine levels in amniotic fluid and inflammatory changes in the placenta from normal deliveries at term.

Cytokine levels in amniotic fluid have been shown to increase towards term in normal pregnancies, and may play a regulatory role in parturition by stimulating the local production of prostaglandins. The work reported in the present paper was conducted in order to test the hypothesis that the increased cytokine levels may be induced by a subclinical inflammatory reaction in intrauterine tissues. The concentrations of tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 2 (IL-2) and interleukin 6 (IL-6) were determined in samples of amniotic fluid from 38 women in delivery at term, after a clinically normal pregnancy. In 33 of the cases, tissue material was available for histological examination. In these, the extent of inflammatory cell infiltration was assessed in the fetal membranes, placenta and umbilical cord. A close interrelation was observed between the levels of the mediators typically released during inflammatory processes (TNF, IL-1, IL-6). Frank chorioamnionitis was not found in any of the histological specimens, although most placentae showed varying degrees of granulocyte infiltration in the fibrin layer under the chorion, sometimes also in the chorionic membrane. The degree of such leukocytic infiltration correlated positively with the levels of TNF, IL-1 and IL-6. These findings lend support to the hypothesis that a low-level inflammatory process may be a normal occurrence in the term placenta, and that this process may induce the production of cytokines, which, in turn, may play a role in the regulation of parturition. Such inflammation could be due to exposure of the fetal membranes to microbial material from the vagina, as the cervix dilates towards term.

Microcurettage sampling of the endometrium for histopathological examination--simpler but not safe? Comparison of endometrial histopathology in samples obtained by a disposable mechanical curette and by traditional curettage.

The routine use of dilatation and curettage (D&C) is questioned as a standard procedure for endometrial sampling. Instruments have been developed in order to simplify the procedure. The aim of the present study was to estimate the sensitivity to detect malignancy/premalignancy by histological examination of endometrial samples obtained by microcurettage using a disposable plastic curette, Gynoscann. The histological examination of the combined microcurettage and D&C material was defined as the true diagnosis. All patients undergoing diagnostic D&C at the two participating hospitals were included in the study. The endometrial samples were evaluated at the Department of Pathology as part of their routine work. The microcurettage samples were re-examined independently by two pathologists (observer 1 and observer 2) who did not have access to the rest of the tissue material nor the diagnosis given previously. A total of 902 patients were enrolled in the study. Endometrial cancer was found in four women below 50 years of age. None of these were detected by histological examination of the Gynoscann obtained sample. Among women aged 50 and above, samples from 16 women turned out to have endometrial cancer. The microcurettage samples were correctly classified as malignant/premalignant by observer 1 in 13 cases and by observer 2 in 14 cases. We conclude that diagnostic D&C among women 50 years and below cannot be replaced by histological examination of Gynoscann samples without the risk of overlooking significant pathology.

[Normal labor. Is it induced as a result of subtle intrauterine inflammatory reactions?]. / Den normale fødsel. Settes den i gang som resultat av subtile inflammatoriske reaksjoner intrauterint?

The present study was conducted in order to test the hypothesis the elevated cytokine levels in amniotic fluid from normal deliveries may be generated by a subclinical inflammatory response in intrauterine tissue. Specimens were collected from 38 women with normal deliveries. The levels of tumour necrosis factor, IL1 and IL6 in amniotic fluid samples correlated with the extent of inflammatory cell infiltration in specimens of the foetal membranes, placenta and umbilical cord. Tumour necrosis factor, IL1 and IL6 are generally produced as part of the inflammatory reaction and stimulate synthesis of prostaglandin. Thus a subclinical, intrauterine inflammatory response may play a role in initiation of normal parturition at term.

Cellular differentiation in prostatic explant cultures: assessed by electron microscopy and X-ray microanalysis.

A method developed for X-ray microanalysis (XRMA) of cell monolayers cultured on Formvar film has been shown to be well suited for the study of explant cultures of human prostate. Adherence and epithelial cell outgrowth occur as readily on the formvar as on ordinary tissue culture plastic, giving rise to cultures with the same morphological characteristics. Conventional transmission electron microscopy revealed intracytoplasmic granules with the appearance of secretory vesicles. XRMA of electron-dense intracytoplasmic granules in freeze-dried cryosections showed significant local sequestration of calcium, but not of magnesium or zinc, elements that have previously been shown to colocalize with calcium in secretory granules in vivo. It is concluded that some aspects of the secretory phenotype are supported in this in vitro model. The factors regulating the expression of a differentiated phenotype in prostatic epithelium await further elucidation. XRMA may be useful in assessing the effects on secretory differentiation induced by variations in the culture conditions.

Cytokine-associated tissue injury and lethality in mice: a comparative study.

A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.

[Examination of uterine hemorrhagic disorders using fractionated abrasion]. / Utredning av uterine blødningsforstyrrelser ved hjelp av fraksjonert abrasio.

The aim of the study was to describe the use of uterine curettage in the diagnosis of vaginal bleeding disorders. 301 pathological specimens were identified where uterine curettage was performed because of vaginal bleeding disorders. These specimens represented 23% of all recorded examinations in the files of Department of Pathology in 1987. Based on the above data the overall yearly rate of uterine curettage was estimated to be 8.7 per 1,000 women aged 20 years and over. The highest age-specific rate was found among women under 50 years of age. Most examinations (222), were carried out on tissue from women younger than 50. In only one of them (= 0.5%) was the diagnosis endometrial carcinoma. Most cases of carcinoma were found in women older than 50 years, with a total of seven out of 79 examinations. Questions should be raised about the routine use of uterine curettage, especially among women under 45 years of age.

Complement activation and pulmonary dysfunction in experimental Escherichia coli septicaemia.

Septicaemia was induced in anaesthetized and artificially ventilated rabbits. Three groups receiving different doses of Escherichia coli 055:B5 and a control group were studied. The animals were followed for 240 min after the start of the bacterial infusion. One animal in the high-dose and one in the medium-dose group died. Septic shock developed in the high-dose group. The infusion of E. coli led to a severe, sustained neutropenia and a dose-dependent thrombocytopenia. A severe arterial hypoxaemia developed during the infusion of bacteria in three of four animals, and in one of three animals in the high- and medium-dose groups, respectively. Both the levels of C5a in plasma and the accumulation of granulocytes in the lung tissue, as assessed by a morphometric method, were dependent on the dose of bacteria. A strong positive correlation was found between levels of C5a and endotoxin. The levels of both C5a and endotoxin correlated positively to the degree of accumulation of granulocytes in the lung tissue. The results from the present study suggest that the magnitude of bacteraemia and the degree of complement activation are probably important factors determining the course and the severity of septicaemia.

Cystitis cystica treated with the neodymium-YAG laser. Case report.

We report on two patients with severe urgency and pollakisuria where further investigations revealed cystitis cystica (glandularis). Conservative treatment and TUR of the lesions had no effect on the symptoms. After treatment with Neodymium-YAG laser, however, both have been asymptomatic for more than 18 months and urethrocystoscopic examination demonstrated normalization of the bladder mucosa.

Intracellular penetration and accumulation of radiographic contrast media in the rat kidney.

Radiographic iodine-containing contrast media (meglumine calcium metrizoate, iohexol and meglumine sodium ioxaglate) were injected intravenously in rats. At various intervals after exposure, in situ cryofixation of kidneys was performed. Thin, freeze-dried cryosections were examined by electron microscopy and X-ray microanalysis. In endothelial cells, erythrocytes and tubular cells high dry weight concentrations of iodine were found. Twenty-four hours after iohexol was injected, no trace of iodine was found in the plasma, microvilli or the nuclei of the tubular cells. Small organelle-like compartments in the cytoplasm of the proximal tubular cells contained high concentrations of iodine, whereas no iodine was found in the surrounding cytoplasm. Since no metabolism of contrast medium has been demonstrated, the iodine signals must be emitted from contrast medium molecules. Other elements were also measured, with the concentrations being always within the ranges found in tubular cells of control animals. The detection of intracellular contrast thus does not seem to be an artifact due to cell injury, but rather represents a physiological event in healthy cells in the rat kidney. Our results are in contradiction to the prevailing opinion that contrast media do not enter healthy cells. However, previous conclusions have been based on the use of conventional preparation methods, and the highly water soluble contrast molecules may have been lost during the different steps of fixation and processing.

[Students of Trondheim--10 years after. Former students' evaluation of the curriculum in Trondheim]. / Trondheimsstudenter--ti år etter. Tidligere studenters evaluering av det medisinske studium i Trondheim.

Teaching of clinical medicine started in Trondheim in 1975, and in 1978 the first students graduated. As part of a curriculum reform programme, a questionnaire was distributed to the doctors who graduated in 1978, asking their opinion about various aspects of the kind of education they had received ten years previously. We present some of the answers.

[Gynecomastia. Investigation and surgical treatment]. / Gynekomasti. Utredning og kirurgisk behandling.

Enlargement of the male breast, gynecomastia, commonly occurs in otherwise healthy men. If the gynecomastia is marked, surgical treatment may be necessary. Underlying disease must be considered. History, physical examination, laboratory tests and in some patients ultrasonography of the testes should be conducted prior to surgery. If the gynecomastia is due mainly due to fat, suction lipectomy alone is sufficient treatment. However, liposuction will not remove breast parenchyma, and some patients also require local excision of the parenchyma. In our experience, suction lipectomy provides a new means of acquiring better contour, with reduced morbidity and complications.

Effects on the ATP content of cultured cells after radiographic contrast media exposure. Evidence for accumulation of contrast media in cultured cells.

The ATP content of cultured cells after exposure to meglumine-calcium metrizoate, sodium metrizoate, iohexol, iopamidol and saline was studied. Initially, the ATP content diminished rapidly for a short period and thereafter slowly during the incubation. After incubation with contrast media or saline, the ATP content slowly increased to normal when the cells were reincubated with fresh nutrient medium. Different contrast media and saline with the same final osmolality produced a similar effect on the ATP content of the cultured cells. Cellular association of meglumine-sodium diatrizoate, sodium metrizoate, sodium-iothalamate, iohexol and iopamidol was also examined. The established cell line NHIK 3025 as well as primary cultures of human umbilical endothelium were found to accumulate contrast media in a time- and concentration-dependent manner. When the incubation was carried out at 4 degrees C, the cellular accumulation of contrast medium was less than 35 per cent of that seen at 37 degrees C. It therefore seems that energy-dependent processes are involved to some degree.

Incorporation of contrast media in cultured cells.

Monolayer cultures of human prostatic (PC-3) and cervical (NHIK 3025) carcinoma cells were grown on formvar film and exposed to moderate concentrations of contrast agents for 30 minutes to 4 hours. After the exposure period, the monolayers were quickly frozen, and cryosections were examined by electron microscopy and X-ray microanalysis. Iodine was not detected in control cells, but was found in the cells that had been exposed to iodine-containing contrast media. The amount of intracellular iodine increased with increasing exposure dose and time. Because the cells mostly presented no sign of membrane damage, our findings support the view that contrast media have the ability to enter intact cells.

Rapid freezing of small biopsies and standard for cryosectioning and X-ray microanalysis.

A method for clamp-freezing of small biopsies for ultrathin cryosectioning is described. A formvar film is used to support the specimen before freezing with a pair of pliers, precooled in liquid nitrogen. This method makes it possible to align the piece of tissue close to a drop of standard solution and freeze the two simultaneously. After clamping, the flat, frozen sample contains both the piece of tissue and the standard in one solid block, which may thus be sectioned as a whole. The method has been used in the surgical theater, yielding good results with needle biopsies of the prostate and cystoscopically obtained biopsies of bladder tumors. The time lapse between excision and freezing may be less than 30 seconds, which represents a great improvement compared with the far more time-consuming mincing of tissue and mounting of the pieces on metal pins. From the frozen specimen, a large number of sections with good morphologic characteristics may be produced. The method was evaluated with the use of rat liver and lactating mammary tissue. Directly frozen tissue with intact blood supply was compared with biopsies prepared as described. Redistribution of Ca could not be demonstrated in either specimen type. However, the results indicate that the monovalent ions are far more mobile, and a significant increase of the Na:K ratios was observed in the biopsies compared with directly frozen tissue.

Intracellular distribution of calcium and zinc in normal, hyperplastic, and neoplastic human prostate: X-ray microanalysis of freeze-dried cryosections.

X-ray microanalysis has been performed on freeze-dried cryosections of normal, hyperplastic, and neoplastic human prostates. Needle biopsies from prostates suspected to be malignant were collected from ten patients. Seven of these patients had fully developed nodular hyperplasia, whereas the remaining three specimens were histologically classified as moderately differentiated adenocarcinomas. Prostates with normal light microscopic appearance were obtained from two brain-dead kidney donors. The concentration of calcium in secretory vesicles was found to be several orders of magnitude higher than the concentration of magnesium and zinc in all of the glands studied, which is consistent with calcium being the major cation secreted by the prostatic acinar cells. Some elderly prostates and neoplastic prostates revealed even lower mean zinc:calcium ratios in secretory vesicles. Even though both elements varied considerably, the wide variation of the zinc:calcium ratios was due mainly to variations in the concentration of zinc. Large, electron-dense bodies occasionally were found in the cytoplasm of prostatic acinar cells. These bodies contained high concentrations of sulphur which by far was the dominating element.