Accumulation of 4- and 5-ene steroid sulfates in human breast cyst fluids

Gross cystic disease of the breast is one of the most common diseases of adult females. Breast cyst fluid contains various steroid hormones. In order to obtain more information about the concentrations of 4- and 5-ene steroids in human breast cyst fluids, levels of pregnenolone sulfate (PREGS), pregnenolone (PREG), dehydroepiandrosterone sulfate (DHEAS) and dehydroepiandrosterone (DHEA) were determined by high-performance liquid chromatography (HPLC). A total of 35 human breast cyst fluid samples, obtained from 35 patients (28-54 years old) were analyzed. Cyst fluid electrolytes were simultaneously determined. Levels of PREGS (mean+/-S.D.) were 26.9+/-20.0 micromol/l (N=35) and of PREG were <0.1 micromol/l. Levels of DHEAS and DHEA were 89.1+/-111.7 micromol/l (N=35) and 0.3+/-0.2 micromol/l (N=35), respectively. Cyst fluids were divided into two groups (types I and II) according to their electrolyte ratio (K(+)/Na(+)). The cysts of the type I group (K(+)/Na(+) >1.5) contained significantly higher levels of PREGS (39.9+/-21.1 micromol/l) and DHEAS (133.2+/-87.9 micromol/l) than those of the type II group (K(+)/Na(+) <1.5), the mean levels of which were 19.8+/-16.2 micromol/dl for PREGS, and 36.3+/-29.0 micromol/dl for DHEAS (P<0.05). PREGS and DHEAS levels in the cysts were significantly correlated (r=0.49; P<0.01). Human breast cyst fluids contain high concentration of DHEAS and PREGS, especially in the cyst fluids containing high K(+)/Na(+) ratios.     

Correlation of concentrations of estriol-3-sulfate with those of potassium and sodium in human breast cyst fluid

Estriol-3-sulfate (E3-3S) was assayed in 92 specimens of human breast cyst fluid (BCF) obtained by needle aspiration from women with fibrocystic disease. The concentrations of K+ and Na+ were determined in the same samples. The median concentration of E3-3S in the fluids from premenopausal women under 51 years of age (69 cases) was 4.4 ng/mL. Based on the K+ levels the samples were divided into two groups, above 50 mM (Type I) and below 50 mM (Type II). Correlations were made between the concentrations of the estrogen conjugate and the univalent ions. In the premenopausal women, Type I cysts were associated with above median E3-3S and Type II cysts with below median E3-3S (P less than 0.01). A K+/Na+ ratio of more than one was also related to elevated E3-3S (P less than 0.025). The BCF obtained from postmenopausal women and women older than 50 years tended to be low in E3-3S (median 1.64 ng/mL) and high in K+ but there were too few cases to permit statistical comparisons to be made. Since fibrocystic disease constitutes a risk factor for the development of breast cancer, it will be of interest to determine retrospectively whether any of the above subsets of BCF may be useful in identifying a patient at such risk.     

Mechanism of dissociation of cortisol and adrenal androgen secretion after removal of adrenocortical adenoma in patients with Cushing's syndrome

We investigated the mechanism of dissociation of cortisol and dehydroepiandrosterone sulfate (DHEA-S) secretion by the adrenal glands after the removal of an adrenal gland containing an adrenocortical adenoma in a patient with Cushing's syndrome. After removal of the adrenocortical adenoma, the serum cortisol rapidly decreased from 24.6 +/- 6.4 micrograms/dl (mean +/- SD, n = 6) to 0.7 +/- 0.5 micrograms/dl. Serum DHEA-S levels were 15 +/- 14 micrograms/dl and 6 +/- 9 micrograms/dl before and after surgery, respectively, and significantly lower than the control values. Serum cortisol levels reverted to normal levels 1.5 to 3 years after the surgery. On the other hand, DHEA-S levels reverted to normal 5 to 7 years after the serum cortisol levels had normalized. Monolayer cultures of normal human adrenal cells obtained at adrenalectomy in patients with advanced breast cancer and atrophic adrenal cells adjacent to the adrenocortical adenoma in patients with Cushing's syndrome were used to study the mechanism of the dissociation of cortisol and DHEA-S secretion. ACTH caused significant increases in the productions of pregnenolone (P5), progesterone (P4), 17-hydroxypregnenolone (17-OH-P5), 17-hydroxyprogesterone (17-OH-P4), DHEA, DHEA-S, androstenedione (delta 4-A), and cortisol. The amounts of 17-OH-P5 and 17-OH-P4 produced by ACTH in atrophic adrenal cells were significantly greater than those in normal adrenal cells. The amounts of DHEA, DHEA-S and delta 4-A produced by ACTH in atrophic adrenal cells were significantly smaller than those of normal adrenal cells. The conversion rate of 17-OH-[3H]P5 to 17-OH-[3H]P4 and 11-deoxy-[3H] cortisol was higher in atrophic adrenal cells than in normal adrenal cells, but the conversion rate to [3H]DHEA, [3H]DHEA-S and [3H]delta 4-A was significantly lower in atrophic adrenal cells than in normal adrenal cells. These results suggest that the dissociation of cortisol from DHEA-S after the removal of adrenocortical adenoma is a probably due to diminished C17,20-lyase activity in the remaining atrophic adrenal gland.     

A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).     

Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guinea-pig heart.

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

High molecular weight basic and acidic immunosuppressive protein components in uterine secretions of pregnant cows

Immunosuppressive proteinaceous components were determined in bovine uterine milk (UTM) collected during late pregnancy. Crude UTM was separated by ion-exchange (carboxymethylcellulose-CMC) and gel filtration (Sephacryl S-200 and Sepharose CL-6B) chromatography. Basic (CMC+) and acidic (CMC-) protein molecular weight (Mr) components were tested for immunosuppressive activity in an in vitro mitogen (phytohemagglutinin-PHA)-treated lymphocyte blastogenesis assay. For most experiments, cultures containing 1 x 10(5) lymphocytes were incubated with 0.08 micrograms PHA and varying concentrations of test protein in RPMI-1640 with supplements. At 48 +/- 1 h, 0.1 microCi of [3H]thymidine was added to cultures and [3H]DNA was quantified at 60 +/- 1 h of culture. Results were expressed as percentage of control values. Crude UTM, CMC+, and CMC- components exhibited immunosuppressive activity. For immunosuppressive Sephacryl S-200 fractions, activity was greater (p less than 0.05-0.01) for CMC+, S-200 fraction I (greater than or equal to 250,000 Mr, void volume [Vo]) than for CMC-, S-200 fractions I (Vo) and III combined for protein concentrations of 20, 40, and 50 micrograms/ml. For the high Mr Sepharose CL-6B protein components, CMC+, CL-6B fraction I (greater than or equal to 4 x 10(6) Mr, Vo) exhibited greater (p less than 0.05-0.005) activity than CMC-, CL-6B fractions I (greater than or equal to 4 x 10(6) Mr, Vo) and II (2.8 x 10(6) Mr) combined at protein concentrations ranging from 20 to 80 micrograms/ml. In summary, bovine UTM contains basic and acidic immunosuppressive protein components, with the greatest activity being associated with a high Mr, basic component.     

Levels of androgen conjugates and oestrone sulphate in patients with breast cysts

Plasma and cyst fluid were obtained from patients with palpable breast cysts and analysed for androgen conjugates and oestrone sulphate content by radioimmunoassay. Concentrations of androgen conjugates in cyst fluids varied from 15.6 to 475.5 mumols/l. These levels were much greater than those in plasma (1.3-5.2 mumols/l) and there was no association between values in cyst aspirates and plasmas obtained from the same individuals. Levels of oestrone sulphate in breast cyst fluids (1.5-744.0 nmol/l) were also generally in excess of those in plasma (2.0-59.9 nmol/l) and again no relationship was evident between concentrations in cyst fluid and the circulation. Neither was there a relationship between levels of androgen conjugate and oestrone sulphate in plasma. In contrast, a highly significant correlation (P less than 0.001) was identified between the androgen conjugate and oestrone sulphate content of cyst fluids. Levels of both androgen conjugates and oestrone sulphate were also significantly different in groups of cysts subdivided according to electrolyte classification, cysts with low Na+:K+ ratios having higher steroid concentrations than those with high Na+:K+ ratios. The biological significance of the relationship between the two conjugates in cyst fluids remains unclear but it is suggested that the accumulation of these steroids involves a common mechanism.     

Determination of human thrombin-antithrombin III complex by enzyme immunoassay

We have developed a specific and sensitive ELISA for the measurement of the TAT in human plasma. The assay follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The anti-thrombin antibody population used for coating was purified by immunoadsorption on immobilized prothrombin and thrombin, respectively. Antithrombin III antibodies were conjugated with peroxidase. Plasma samples containing TAT were incubated in polystyrene tubes coated with anti-thrombin antibodies; after washing, peroxidase-conjugated antithrombin III antibodies were added and bound enzyme activity was subsequently measured using o-phenylenediamine. The assay was calibrated with definite concentrations (2.0 to 60 micrograms/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance at 492 nm against TAT concentrations revealed a linear correlation (r = 0.98). A reference range from 0.85 to 3.0 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT was found up to 7-13 micrograms/l. Patients with septicemia associated with a consumption coagulopathy (n = 10) showed markedly increased TAT values (greater than or equal to 10 micrograms/l). From these data it can be concluded that measurement of TAT might be a parameter for detection of a latent clotting pathway activation.     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of toxic pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) in ovine ruminal fluid under anaerobic conditions.

The ability of ovine ruminal fluid to metabolize pyrrolizidine alkaloid (PA) from Senecio jacobaea under anaerobic conditions was evaluated. Four fistulated sheep fed PA served as individual sources of ruminal fluid, which was incubated in a defined minimal salts medium under two different anaerobic conditions, denitrifying and methanogenic. Anaerobic cultures amended with ovine ruminal fluids (20%), PA (100 micrograms/ml), and a defined minimal salts medium were monitored for a period of several days. These cultures revealed that while PA was not depleted in sterile, autoclaved controls or under denitrifying conditions, it was metabolized during periods of active methanogenesis under methanogenic conditions. In addition, samples of ruminal fluid were separated by differential centrifugation under anaerobic conditions, and the resultant supernatants were tested for their ability to metabolize PA as compared with those of the respective uncentrifuged control fluids. Uncentrifuged controls exhibited a PA depletion rate of -4.04 +/- 0.17 micrograms of PA per ml per h. Supernatants 1 (centrifuged at 41 x g for 2 min), 2 (centrifuged at 166 x g for 5 min), and 3 (centrifuged at 1,500 x g for 10 min) exhibited significantly slower depletion rates, with slopes of data representing -1.64 +/- 0.16, -1.44 +/- 0.16, and -1.48 +/- 0.16 micrograms of PA metabolized per ml per h, respectively, demonstrating no statistically significant difference among the supernatant cultures. Microscopic evaluations revealed that protozoa were present in the control whole ruminal fluid and to a lesser extent in supernatant 1, while supernatants 2 and 3 contained only bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.     

Tyrosine kinase inhibitors and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle.

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, less than 7.4 microM (2 micrograms/mL); tyrphostin, less than 20 microM (4 micrograms/mL)) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1 microM (0.30 micrograms/mL; mean +/- SEM) and 3.6 +/- 0.5 microM (0.74 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS: TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3 microM (0.81 micrograms/mL) for genistein and 2.4 +/- 0.2 microM (0.49 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40 microM) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of a panel of monoclonal antibodies in the evaluation of cytologic specimens from the central nervous system

A panel of monoclonal antibodies was tested immunohistochemically to determine the utility of such reagents in distinguishing among metastatic carcinoma, lymphoma, leukemia and primary brain tumors. The monoclonal antibodies used were: (1) a cocktail comprised of three anti-glial fibrillary acidic protein antibodies (alpha-GFAP); (2) UJ13A, a panneuroectodermal antibody; (3) B72.3, which recognizes a carcinoma-distinctive tumor-associated glycoprotein complex; and (4) 2D1, a pan-leukocyte antibody. Fifty-three specimens (21 cerebrospinal fluids, 1 ventricular fluid, 2 brain cyst fluids, 12 needle washings, 15 imprints, 1 subdural fluid and 1 post-shunt fluid) were obtained from 21 gliomas, 2 meningiomas, 1 pineoblastoma, 11 metastatic tumors, 3 lymphomas, 1 leukemia and 14 cases without tumor. alpha-GFAP stained all 21 gliomas and 5 of 5 cases containing reactive brain fragments. UJ13A had a reactivity pattern similar to that of alpha-GFAP, but also stained the meningiomas, pineoblastoma, oat-cell carcinoma and embryonal rhabdomyosarcoma. B72.3 stained all adenocarcinomas and the large-cell carcinoma. 2D1 stained lymphoma and leukemia, all inflammatory cells and 4 of 12 glioblastomas. Although no single antibody was diagnostic of a specific tumor type, this panel accurately differentiated among most primary brain tumors, metastases, leukemias and lymphomas.     

The concentration of 16x-hydroxy androgens in serum and cyst fluid of women with gross cystic disease of the breast

The concentrations of 16 alpha-hydroxydehydroepi-androsterone sulfate (16 alpha-OHDHAS) and androst-5-ene-3 beta, 16 alpha-, 17 beta-triol-3-sulfate (A-TriolS) were measured in the plasma and breast cyst fluid (BCF) of women with gross cystic disease of the breast. In the 19 BCF samples analyzed, the 16 alpha-OHDHAS and A-TriolS concentrations ranged from 15 to 1130 ng/mL, and 12 to 871 ng/mL, respectively. However the concentrations of these steroids in the sera of these women were lower (15-179 ng/mL, 8-80 ng/mL, respectively). Estriol-3-sulfate (E3-3S) concentrations in the BCF samples ranged from barely detectable (0.2 ng/mL) to 3 micrograms/mL. In BCF or serum a positive linear correlation was observed in the concentration of 16 alpha-OHDHAS and A-TriolS (p less than 0.001 and 0.05, respectively). However, in the same patients no statistical significance was observed in the BCF vs serum concentrations of these two steroids. When the specimens from this and previous studies were combined, positive correlation was found between potassium ion concentration and E3-3S or 16 alpha-OHDHAS. The origin of the high concentration of E3-3S is still obscure. Although no linear correlation between 16 alpha-OHDHAS and E3-3S was observed, the possibility of a precursor-product relationship between the two is not elimnated.     

The value of routine cytologic examination of breast cyst fluids

The results of the cytologic examination of 6,782 consecutive breast cyst fluids aspirated from 4,105 women during the period 1976 to 1983 were reviewed to assess the value of the routine cytologic study of such specimens. Cases in which cancer had been suspected by physical examination and/or mammography before aspiration of the cyst were excluded from the evaluation. Five clinically and radiologically inapparent intracystic papillomas were detected overall (0.1%). All cases of intracystic papilloma produced a blood-stained fluid and showed an intracystic mass at pneumocystography. Cytology was negative in two of these cases and falsely positive in one case while correctly identifying the papilloma in two cases. One incidental case of occult in situ lobular carcinoma was also detected. Routine cytologic examination of all breast fluids is thus not recommended as a cost-effective practice. Cytology should be used only when a blood-stained fluid is obtained (2% in the present series) since its indiscriminate application to all cyst fluids does not affect the rate of detection of intracystic lesions, most of which are suspected before the aspiration.     

Quantification of the sulfates of 16α-hydroxy androgens that are possible precursors of estriol-3-sulfate in human breast cyst fluid

The concentration of 16 alpha-hydroxydehydroepiandrosterone-3-sulfate (16 alpha-OHDHAS) was determined in 29 samples of human breast cyst fluid (BCF) and in 15 of these, androst-5-ene-3 beta,16 alpha,17 beta-triol-3-sulfate (A-TriolS) was also assayed. The median value of both was about 100 ng/mL and the ranges were from 1.4 to about 1800 ng/mL. There was a significant association in the values for the two sulfates (p less than 0.05). These concentrations are consistent with a role for 16 alpha-hydroxy androgens as possible precursors for estriol-3-sulfate. The latter is highly elevated relative to other body fluids in BCF. The androgens also correlated directly with the concentrations of K+, an indicator of apocrine proliferation of breast cysts.     

Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.     

Cytokines in human breast cyst fluid

Gross cystic breast disease is a common benign disorder in which palpable cysts occur in the breast and are normally treated by aspiration of the contents. The cysts are classified as either Type 1, containing a high level of potassium ions and a low level of sodium ions, or as Type 2, with low potassium and high sodium ion concentrations. Steroid sulphatase activity in MDA-MB-231 and MCF-7 cell lines is regulated by exogenous breast cyst fluid (BCF), possibly because of cytokines in the BCF. A screening method was used to determine the range of cytokines in eight BCFs, four of each type. This was an array system, which uses antibodies immobilised on a membrane to qualitatively detect 79 different cytokines or growth factors. Nine cytokines were detected well above background levels: all were found in both types of BCF, but only epidermal growth factor (EGF) was higher in Type 1. All the other factors were higher in Type 2 BCF. Two of these cytokines, IL-6 and EGF, have previously been suggested to affect steroid sulphatase expression and several (MIP-1β, IL-8, NAP-2) are known to affect MCF-7 cell chemotaxis. In addition two cytokines were measured by ELISA in 57 BCFs, and both IL-1β and IL-13 were found in BCF, with significantly higher amounts of IL-1β in Type 1 than Type 2 BCF (35.5 ± 4.4 pg/ml versus 9.9 ± 2.9 pg/ml).     

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.