An improved assay for nuclear estrogen receptor in experimental and human breast cancer.

We have validated a hydroxylapatite assay for measuring estrogen receptor in extracts from breast tumor nuclei. By adsorption of receptor of hydroxylapatite prior to addition of radioactive ligand and warming, receptor degradation can be avoided. Total binding sites are measured at 30 degrees by exchange, and receptor sites unoccupied by steroid are measured at 4 degrees. A single saturating dose of 5 nM tritiated estradiol (with or without a 100-fold excess of nonradioactive diethylstilbestrol to estimate nonspecific binding) yields results similar to a six-dose Scatchard plot. Following in vivo injection of estradiol into rats bearing mammary tumors, receptor translocation in the tumors can be accurately quantitated with this assay. Applying the assay to human breast cancer, we find that tumor biopsies may contain cytoplasmic receptor alone or may also have appreciable nuclear receptor. The latter may be bound to estradiol or may be found in "free" form. The finding of free receptor in the nuclei in certain cases raises the possibility that unoccupied receptor might be able to stimulate cell replication in these cases, even in the absence of estrogen.

Estrogen receptor and proteolytic activity in human breast tumor nuclei.

We have previously found that human breast cancer cells in tissue culture contain estrogen receptor in their nuclei despite the absence of estrogen. We have now investigated solid human breast cancer biopsies and find that proteolytic activities in extracts from the nuclear pellets of these biopsies interfere with or prevent the measurement of nuclear estrogen receptor when the protamine assay is used. However, the problems of receptor degradation can be avoided by the use of a hydroxylapatite assay.

A hydroxylapatite micromethod for measuring estrogen receptor in human breast cancer.

The concentration of estrogen receptor (ER) in a human breast tumor is a critical variable predicting the response to endocrine therapy and the course of the disease. Since many tumor specimens are quite small, a reliable and simple ER assay requiring a minimum of tissue is desirable. We here describe a hydroxylapatite assay for ER that (a) requires only a single, saturating concentration of [3H]estradiol, (b) agrees with more complex multiple-concentration assays and with the standard dextran-coated charcoal assay at normal protein concentrations, (c) is far more reliable than the latter at low protein concentrations, and (d) can be adapted to an accurate and reliable ER microassay requiring less than 50 mg of tissue.

Measurement of progesterone receptor in human breast cancer biopsies.

PIP: An improved sucrose gradient procedure for measuring progesterone receptor (PR) status was compared with other methods in an attempt to improve the assay of PR, a possibly important determinant in predicting endocrine therapy responses. Tritiated 3H-labeled R 5020 PR assay was modified to determine the actual nature of the competitive binding detected using sucrose density gradient centrifugation. An underestimation of 8S receptor was the result when using the swinging bucket technique, perhaps due to the prolonged run time (16 hours) involved. Hence, a vertical tube rotor was used which eliminated the problem. The 4S component was shown not to be PR, but probably an aberration due to failure to adsorb to hydroxylapatite. Dextran-coated charcoal and hydroxylapatite Scatchard plots confirmed this aberrant finding in that values derived from these methods correlated well with the 8S component of the gradient profile and not with the sum (8S + 4S) in a series of 27 human breast cancer specimens. In the same series the use of dextran-coated charcoal or hydroxylapatite single-saturating-dose assays correlated well with the 8S component of sucrose gradients. However, it is cautioned that until more experiments with these types of correlations are compiled, the 8S component of sucrose density gradient profiles as obtained from vertical tube rotor centrifugation yields the most reliable data on PR concentrations in human breast tumors.^ieng

X-linked Alport syndrome in females.

Alport syndrome (AS) is in the differential diagnosis of hematuria. Variability in clinical presentation and in the ultrastructural changes of the glomerulus can make the diagnosis of AS a challenge in female patients. The purpose of this report is to present immunostaining for glomerular basement membrane (GBM) expression of alpha5(IV) as an adjunctive diagnostic method. Renal biopsy specimens from eight female patients with clinical presentation suggestive of AS were studied. The patients were between 7 and 36 years of age; six were between 12 and 15 years. Light microscopy and immunohistochemistry using a monoclonal antibody to alpha5(IV) were performed. Controls showed a continuous linear pattern along the GBM in normal kidneys and absence in renal biopsy specimens from male X-linked AS patients. To express the variability of the ultrastructural GBM changes among the patients in the series, we developed a semi-quantitative Alport Index, obtained by quantification of severity and extent of ultrastructural GBM changes. With immunohistochemistry, we showed an interrupted, discontinuous linear pattern for alpha5(IV) in glomeruli from the eight patients in the series, confirming the diagnosis of X-linked AS. The ultrastructural Alport Index varied between 6 and 47, showing the heterogeneity in the severity of the GBM changes, even among the six patients aged between 12 and 15 years. In three of the eight biopsy specimens, the predominant change was thin GBM, and the Alport Index was below 20. Immunohistochemistry for alpha5(IV) in renal biopsy specimens can identify female patients heterozygous for X-linked AS. In this series, the method led to the diagnosis of AS in female patients in whom the predominant ultrastructural change was thin basement membrane.

Hormones in breast cancer: update 1978.

The role of cytoplasmic estrogen receptor (ER) assays in determining therapeutic strategies for advanced breast cancer is certainly well established. The use of ER assays in the primary breast tumor specimen to predict for early recurrence and ultimate survival is a new finding, however, and will probably be employed in future trials of adjuvant therapy. The prevalence and significance of nuclear-bound ER still requires additional clarification. Our previous suggestion that progesterone receptor measurements might be a useful marker for hormone dependence in advanced breast cancer is gaining support and may soon have a place in routine therapeutic decision-making. The emphasis on early adjuvant therapy has hastened the search for a safe endocrine therapy that would have good patient compliance and achieve remission rates comparable to previous agents and procedures. Antiestrogens show promise of meeting these requirements. We are now beginning an era in which primary and secondary systemic therapies for breast cancer can be based on sound biologic principles. The empirical approach is outdated.

Endocrine characterization of a human ovarian carcinoma (BG-1) established in nude mice.

The steroid receptor-positive human ovarian cancer (BG-1) was evaluated to determine its usefulness as a tumor model. This tumor grows in intact male and female nude mice without hormone supplements. Moreover, its growth was significantly accelerated in ovariectomized mice, and the increased growth rate could be reversed by estradiol administration. Evaluation of tumor growth following endocrine therapy revealed that, while antiandrogens did not affect the tumor growth, both an aromatase inhibitor and a luteinizing hormone-releasing hormone agonist significantly impaired growth of this human ovarian tumor. Estradiol was also shown to up-regulate both estrogen and progesterone receptors in tumors grown in ovariectomized mice. Therefore, the BG-1 human ovarian carcinoma grows without hormonal supplements and yet responds to specific forms of endocrine therapy. Moreover, the steroid receptors present in this tumor respond to exogenous steroids. In conclusion, this tumor may serve as an ideal model for the study of hormonal regulation of ovarian tumor growth.

Pancreatoduodenectomy for neonatal myofibromatosis of the pancreas.

Myofibromatosis is a rare congenital disorder consisting of one or more fibrous nodules in the skin, soft tissues, bones, and internal organs. The authors report the unique case of a newborn who presented with obstructive jaundice caused by a single myofibroma in the head of the pancreas that was treated successfully by pancreatoduodenectomy on the eighth day of life.

Cell-cycle regulatory proteins in the podocyte in collapsing glomerulopathy in children.

Podocyte is a terminally committed cell in G1 arrest of cell cycle, and is unable to overcome G1/S transition phase in children with minimal change disease (MCD) and classic focal segmental glomerulosclerosis (FSGS), in contrast to dysregulated proliferative phenotype of idiopathic collapsing glomerulopathy (CGN) in adults. Forty-two kidney biopsies, MCD (14), FSGS (12), CGN (4), and normal (CON) (12), were evaluated by immunohistochemistry using dual staining for expression of p27, p21, and p57, and cyclins D and A, in podocytes of children with CGN. On light microscopy, all podocytes expressed p27, whereas p21 and p57 expression was seen in a portion of podocytes in normal kidney biopsies. Cyclin D was expressed in a small percentage of podocytes. Cyclin A expression was absent in normal biopsies. The staining for p27 decreased significantly, in order, from normal (100%) to MCD (45.8%) to CGN (24.2%) to FSGS (16.6%). p21 staining was significantly decreased from normal (69.8%) to CGN (15.5%) to MCD (2.2%) to FSGS (0.6%), and the difference between CGN and MCD and FSGS was also significant. There was no significant difference in staining of p57. Cyclin D staining was significantly increased in CGN (26.8%) compared to normal (7.2%), MCD (1.6%), and FSGS (0.0%), and the difference between CGN and MCD and FSGS was also significant. De novo cyclin A staining was only observed in children with CGN. Thus, p27 and p21 but not p57 was decreased in CGN, as in FSGS when compared to normal. Both cyclins D and A staining were increased in CGN. The staining pattern in CGN would suggest that podocyte is able to overcome G1/S transition phase, and has a proliferative phenotype. We propose, based on the significant contrast observed in podocytes injury response between CGN (proliferative) and classic FSGS (non-proliferative), that CGN not be considered as a morphological variant of FSGS.

Alveolar capillary dysplasia, with and without misalignment of pulmonary veins: an association of congenital anomalies.

Two new cases of alveolar capillary dysplasia (ACD), one without misalignment of the pulmonary vessels (MLV), are reported. They are unique for their association with complex cardiac malformations and asplenia. By reporting these cases we want to stress that ACD is associated with multiple malformations, and that the absence of MLV does not rule out the diagnosis of ACD.

Nephrocalcinosis and nephrolithiasis in infants with congestive heart failure treated with furosemide.

Nephrocalcinosis and nephrolithiasis developed in five children after furosemide therapy for congestive heart failure. In four children renal calcifications were detected by ultrasonography and in one by autopsy. Discontinuation of the loop diuretic in three children resulted in resolution of the calcifications in two of the patients. Residual renal morbidity included reduced creatinine clearance, microscopic hematuria, and hypercalciuria. The phenomenon of renal calcifications associated with furosemide treatment is more frequent than previously recognized.

Autosomal dominant inheritance of multicystic dysplastic kidney.

Unilateral multicystic dysplastic kidney (MCDK) in a normal infant is believed to be a sporadic disorder, with an incidence of about 1 in 4,300 live births. Isolated unilateral MCDK occurring in a family without other genitourinary abnormalities has not been described. We report a family in which isolated unilateral MCDK occurred in a woman and her two children. The mother presented with a palpable abdominal mass during infancy, which on excision was found to be a MCDK. Both the children were found to have MCDK on prenatal ultrasonography, which was later confirmed on postnatal evaluation. The MCDK in the children continues to involute on follow-up urinary tract ultrasonography. The inheritance of MCDK appears to be autosomal dominant in this family.

Monocyte chemoattractant protein-1 and its receptor CCR-2 in piglet lungs exposed to inhaled nitric oxide and hyperoxia.

Monocyte chemoattractant protein-1 (MCP-1), acting through its C-C chemokine receptor 2 (CCR-2), has important roles in inflammation, angiogenesis, and wound repair. The individual and combined effects of inhaled nitric oxide (NO) and hyperoxia on lung MCP-1 and CCR-2 in relation to lung leukocyte dynamics are unknown. Because MCP-1 gene is up-regulated by oxidants, we hypothesized that inhaled NO with hyperoxia will increase MCP-1 production and CCR-2 expression more than either gas alone. We randomly assigned young piglets to breathe room air (RA), RA+50 ppm NO (RA+NO), O(2), or O(2)+NO for 1 or 5 d before sacrifice. Lungs were lavaged and tissues preserved for hybridization studies, Western blotting, histology, and immunohistochemistry. The results show that lung MCP-1 production and alveolar macrophage count were significantly elevated in the 5-d O(2) and O(2)+NO groups relative to the RA group (p < or = 0.05). In contrast, lung CCR-2 abundance was diminished in the O(2) group (p

Synaptopodin expression in idiopathic nephrotic syndrome of childhood.

BACKGROUND: Synaptopodin is a proline-rich protein intimately associated with actin microfilaments present in the podocytes' foot processes. We investigated for synaptopodin expression in children with idiopathic nephrotic syndrome (INS), including minimal change disease (MCD), diffuse mesangial hypercellularity (DMH), and focal segmental glomerulosclerosis (FSGS); in children with congenital nephrotic syndrome of the Finnish type (CNF); and in normal kidney tissue. In particular, we examined whether an association exists between synaptopodin expression in podocyte cells and the response to steroids in INS, and whether synaptopodin expression can predict FSGS upon the initial kidney biopsy in children who progress from MCD or DMH to FSGS. METHODS: Immunohistochemistry was performed for synaptopodin expression on renal tissues from MCD (N = 18), DMH (N = 7), FSGS (N = 13), CNF (N = 9), and normal children (N = 7). Synaptopodin expression in nonsclerosed glomeruli was quantitated by computerized image analysis on the Optimastrade mark software for both luminance (L) and percentage of glomerular area (A). RESULTS: Synaptopodin expression was absent in areas of sclerosis. In nonsclerosed glomeruli, synaptopodin was significantly less expressed in all groups of INS and in CNF compared with normal (P < 0.0001 for both L and A, in each MCD, DMH, FSGS, and CNF). In INS, synaptopodin expression decreased in order from MCD to DMH to FSGS, reaching statistical significance between MCD and FSGS (P = 0.001 for L and P = 0.05 for A). Greater synaptopodin expression in podocytes was associated with a significantly better response to steroid therapy (P < 0.05 for both L and A). On the other hand, the expression of synaptopodin did not predict progression of MCD or DMH to FSGS. CONCLUSION: We conclude that measurement of synaptopodin has the potential to be used as a marker to study the alteration in podocyte cell and response to therapy in INS.

Ketorolac-induced irreversible renal failure in sickle cell disease: a case report.

Nonsteroidal anti-inflammatory drugs are often used in the management of those with acute pain secondary to sickle cell disease due to potent analgesic effects along with a lack of addictive potential, respiratory depression, and central nervous system effects, as may occur with narcotics. Caution should be observed in the use of nonsteroidal anti-inflammatory drugs in patients with compromised renal function. We present a case of a 17-year-old sickle cell disease patient with an acute painful episode and normal renal function indices who subsequently developed irreversible renal failure and a perirenal hematoma following the administration of ketorolac, despite adequate hydration. Due to its inhibitory effect on prostaglandin-mediated vasodilation, we advise caution in the use of ketorolac for the pain management of sickle cell painful episodes. We recommend following the administration guidelines for ketorolac for renal-compromised patients in those with painful episodes of sickle cell disease, and if used in this patient population, renal function must be very closely monitored.

Podocyte proteins in Galloway-Mowat syndrome.

Galloway-Mowat syndrome is an autosomal recessive disorder characterized by early onset nephrotic syndrome and central nervous system anomalies. Mutations in podocyte proteins, such as nephrin, alpha-actinin 4, and podocin, are associated with proteinuria and nephrotic syndrome. The genetic defect in Galloway-Mowat syndrome is as yet unknown. We postulated that in Galloway-Mowat syndrome the mutation would be in a protein that is expressed both in podocytes and neurons, such as synaptopodin, GLEPP1, or nephrin. We therefore analyzed kidney tissue from normal children (n=3), children with congenital nephrotic syndrome of the Finnish type (CNF, n=3), minimal change disease (MCD, n=3), focal segmental glomerulosclerosis (FSGS, n=3), and Galloway-Mowat syndrome (n=4) by immunohistochemistry for expression of synaptopodin, GLEPP1, intracellular domain of nephrin (nephrin-I), and extracellular domain of nephrin (nephrin-E). Synaptopodin, GLEPP1, and nephrin were strongly expressed in normal kidney tissue. Nephrin was absent, and synaptopodin and GLEPP1 expression were decreased in CNF. The expression of all three proteins was reduced in MCD and FSGS; the decrease in expression being more marked in FSGS. Synaptopodin, GLEPP1, and nephrin expression was present, although reduced in Galloway-Mowat syndrome. We conclude that the reduced expression of synaptopodin, GLEPP1, and nephrin in Galloway- Mowat syndrome is a secondary phenomenon related to the proteinuria, and hence synaptopodin, GLEPP1, and nephrin are probably not the proteins mutated in Galloway-Mowat syndrome.