[DNA flow cytometry. Comparison of the methods for nuclear extraction from frozen and paraffin-embedded sections]. / Citofluorimetria a flusso del DNA. Confronto delle tecniche di estrazione nucleare da campione congelato e campione incluso in paraffina.

Cellular DNA content of solid tumors can be determined either from fresh, frozen, or formalin fixed, paraffin-embedded tissues. However, discordant results have been obtained using the paraffin-embedded technique, and lack of abnormal DNA stemlines in the paraffin-embedded as compared to either fresh or frozen tissues has been reported. In this study we evaluated the validity of nuclear extraction method from paraffin-embedded tissues, using 75 breast carcinomas whose DNA content was previously analyzed from frozen tissue and resulted either normal (12 cases) or abnormal (63 cases). From representative paraffin blocks, nuclei were extracted following Hedley's technique. The results revealed excellent cell counting and good histogram resolution from all paraffin samples; the loss of G2M abnormal peak in eight histograms with abnormal stemline did not compromise the correct interpretation of DNA content. In addition, the comparison between DNA indices obtained from corresponding paraffin and frozen samples showed a good correlation in 69 cases (r = 94); discordance in six cases was demonstrated to be related to tumor heterogeneity. In conclusion the paraffin extraction method is a sensible, and reliable technique, which can be applied for DNA flow cytometric studies on archival cases, as well as whenever fresh sample from the tumor is not obtainable.

Usefulness of the scraping method for DNA flow cytometry in breast tumors.

Flow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0-G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed.

DNA index, cellular proliferative activity and nucleolar organizer regions in cancers of the larynx.

The DNA index, expression of cell-cycle-related proteins--proliferating cell nuclear antigen (PCNA, cyclin) and Ki-67--and the content of silver-binding nucleolar organizer regions (AgNORs) were evaluated in 30 unselected consecutive primary squamous cell carcinomas of the larynx. Results were compared and subsequently related to histological grading, lymph node status, pT category, and pathological stage. DNA content was non-diploid in 9 cases (30%). Mean AgNOR counts per tumor ranged from 2.52 to 8.76. PCNA and Ki-67 expressions were similar in 10 cases (33%). In the remaining cases, PCNA-positive cells usually outnumbered Ki-67-positive cells. No significant correlation was found among DNA index, PCNA and Ki-67 expressions, and AgNOR counts. Although there was a positive trend when Ki-67 was compared with histological grading, findings were not statistically significant. In contrast, a significant correlation was found between DNA index and lymph node status (P = 0.035), with a higher incidence of neck node metastases in non-diploid tumors. These data suggest that tumor ploidy can be correlated with lymph node spread in laryngeal squamous cell carcinoma and might be used as an additional prognostic factor when planning treatment.

Isoproterenol induces release of atrial natriuretic peptide from rat atrium in vitro.

To investigate the mechanism underlying the release of atrial natriuretic peptide (ANP) in in vitro condition, isolated, superfused rat atria were subjected to adrenergic, chronotropic, and mechanical stimulation. First administration of isoproterenol (Iso; either 10(-9) or 10(-6) M) caused a release of ANP, which was transient. Subsequent increments in concentration of Iso always resulted in a much lower release of ANP, despite the increased effects on the mechanical function of the atria. Stretching of the atria resulted in a transient release of ANP. Subsequent increments in stretching were followed by decreasing release of ANP. The total score of ANP in atrial tissue after Iso and stretching was not measurably depleted. Pacing the atria with increasing frequency did not induce release of ANP. Depolarization with 40 mM KCl abolished the release of ANP in response to Iso but not the release induced by stretch. In the presence of low external Ca2+, which abolished mechanical activity, both Iso and stretch could still induce release of ANP. Propranolol abolished the release of ANP by Iso but not that induced by stretching. Prazosin did not affect the release by either stretch or Iso. Stretching the atria 20 min after administration of Iso did not cause any further release of ANP. On the other hand, adding Iso 20 min after stretching induced a release of ANP. It is concluded that Iso and stretch cause a transient release from isolated strips of atria. The amount of ANP released is not related to the dose of Iso or to the load applied. Mechanisms involved in the release mediated by the two stimuli are different.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular and hormonal effects of calcitonin gene-related peptide in congestive heart failure.

The effects of infusing human alpha-calcitonin gene-related peptide were studied in eight patients with congestive heart failure, five normal rabbits and five rabbits with adriamycin-induced cardiomyopathy. In patients with heart failure, calcitonin gene-related peptide caused a dose-dependent increase in cardiac output and decrease in pulmonary and systemic vascular resistance and pulmonary artery pressure. The systemic blood pressure and right atrial and pulmonary wedge pressures decreased only at the highest infusion rate (16 ng/kg per min). Heart rate remained unchanged. Plasma epinephrine increased (p less than 0.05), whereas aldosterone, atrial natriuretic peptide and prolactin concentrations decreased (p less than 0.05). Plasma norepinephrine, renin activity, cortisol and growth hormone concentrations remained unchanged. In both groups of rabbits, the drug decreased blood pressure and increased cardiac output and heart rate. There was a significant increase in renal blood flow (p less than 0.05). The peptide did not affect the contraction amplitude of human and rabbit ventricular myocytes. These findings suggest that calcitonin gene-related peptide is a vasodilator in the rabbit and humans with little direct effect on ventricular myocardium. This peptide may be useful in some forms of heart failure.

Effect of congestive heart failure on rate of atrial natriuretic factor release in response to stretch and isoprenaline.

STUDY OBJECTIVE: The aim was to investigate the pattern of release of atrial natriuretic factor induced by mechanical and adrenergic stimulation from atria of rats with or without congestive heart failure. DESIGN: Monocrotaline, a pyrrolizidine alkaloid, was given to rats to cause severe pulmonary hypertension, leading to a marked degree of right ventricular hypertrophy and failure. Measurements of noradrenaline and atrial natriuretic factor were performed in each cardiac chamber and in plasma. Right and left atria of control rats and rats with congestive heart failure were isolated and subjected to mechanical or adrenergic stimulation to study the in vitro release of atrial natriuretic factor. MATERIALS: Studies were performed on plasma, ventricles, and isolated right and left atria of 276 male Wistar rats, 80-100 g weight, with or without congestive heart failure. MEASUREMENTS AND MAIN RESULTS: In monocrotaline rats right and left ventricular concentrations of noradrenaline were significantly reduced. In the same rats concentrations of atrial natriuretic factor fell to 15.2% in the right atria and to 65.5% in the left atria. Whole heart content of atrial natriuretic factor was diminished, while plasma concentrations were increased sevenfold. Isolated hypertrophied right atria of failing hearts did not release atrial natriuretic factor in response to stretch or to isoprenaline (10(-9)M) and they were insensitive to the inotropic action of isoprenaline. On the other hand, non-hypertrophied left atria from the same animals released increased amounts of atrial natriuretic factor under basal conditions and after both stimuli, despite reduced tissue stores of the peptide. CONCLUSIONS: Heart failure may deplete cardiac stores of noradrenaline and atrial natriuretic factor, especially in hypertrophied chambers, and can result in a decrease in the release of atrial natriuretic factor from atrial tissue in response to mechanical and adrenergic stimulation.

Effects of prolonged infusions of atrial natriuretic peptide and isoproterenol on the mechanical and endocrine function of isolated atria.

We have measured the rate of release of atrial natriuretic peptide in response to stretch and to isoproterenol from superfused atria isolated from rats after chronic in vivo infusion of either atrial natriuretic peptide or isoproterenol. The infusion lasted seven days, using minipumps filled with: 1) saline; 2) synthetic atrial natriuretic peptide to release 3 micrograms/kg/h; 3) l-isoproterenol HCl to release 400 micrograms/kg/h. Infusion of isoproterenol caused hypertrophy of the left chambers of the heart, a decrease of atrial content of atrial peptide with increased plasma levels. Infusion of atrial natriuretic peptide resulted only in elevated plasma levels of the peptide. Atria from animals infused with atrial natriuretic peptide responded to stretch and to isoproterenol (10(-9) M) with a prompt and transient release of peptide. Atria from animals infused with isoproterenol showed a severe reduction in sensitivity to the inotropic and chronotropic action of the drug. Their basal release of atrial peptide was extremely reduced and they were unable to respond to stretch or to isoproterenol. These data indicate that high circulating levels of atrial natriuretic peptide do not influence the release of the peptide from the atria, while high levels of isoproterenol drastically reduce it. Beta adrenoceptor desensitization, atrial hypertrophy and decrease of stores of atrial peptide are likely to account for this phenomenon.

Effects of temperature and osmolality on the release of atrial natriuretic peptide.

In isolated rat atria a 10 degrees C increase in temperature approximately doubled the output of atrial natriuretic peptide during relaxation and stretch. The effect was not due to the increased rate of contraction. Increasing the osmolality of the superfusate within the physiological range (290 to 320 m osmols) with sodium, potassium or glucose had no appreciable effect on the release of atrial natriuretic peptide.

Improved procedure for determining glutathione in plasma as an index of myocardial oxidative stress.

Glutathione plays an important role in the detoxification processes of electrophilic metabolites of xenobiotics and oxygen free radicals, such that release of reduced and oxidized glutathione into the plasma is considered a reliable index of oxidative stress. However, reduced glutathione in plasma undergoes spontaneous autoxidation, with mixed disulfide formation. We developed a new, simple, quick method to overcome this problem by treating the blood, immediately after collection, with thiol reagents. We add 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide to the blood before determination of total and oxidized glutathione, respectively. We find the proposed assay useful for investigating oxidative stress in clinical situations.