Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

Chromosomal changes in renal oncocytomas. Evidence that t(5;11)(q35;q13) may characterize a second subgroup of oncocytomas.

Many of the reported oncocytomas have different chromosome abnormalities, indicating that they comprise a cytogenetically heterogenous group of tumors consisting of potentially cytogenetic subgroups. We have performed cytogenetic studies on nine renal oncocytomas. Clonal abnormalities were present in eight tumors. The findings most observed were the loss of the Y chromosome, and abnormalities of chromosomes 1 and 22. We also observed telomeric associations (tas) in two tumors and structural aberrations of chromosomes 9p and 19q, as well as monosomy 10. In two cases we found a similar reciprocal t(5;11)(q35;q13) in two cases. Review of the literature disclosed one other oncocytoma with a t(5;11) (q35;q13). This suggests that t(5;11)(q35;q13) defines a (second) subset of oncocytomas apart from the subgroup specifically associated with the loss of chromosomes 1 and Y.

DNA ploidy analysis and cytologic examination of sorted cell populations from human breast tumors.

Two different flow cytometric procedures were applied on cell samples from human breast tumors. One procedure involved DNA ploidy analysis on suspensions of isolated nuclei. The mean ploidy ratios of 27 benign breast lesions to chicken erythrocytes and rainbow trout erythrocytes were found to be 2.66 +/- 0.03 and 1.25 +/- 0.02, respectively. From the 45 stemlines found in a series of 43 carcinomas, 12 were diploid, 13 hyperdiploid and 20 near-tetraploid. No association was found between the lymph node status and the DNA ploidy level. The second procedure involved sorting fixed cells from DNA "windows" for the preparation of permanent cytologic specimens. The sorted cells appeared to be shrunken, but the morphologic quality was similar to that of imprint specimens from the same tumors, permitting discrimination between various types of normal cells and tumor cells. The combined use of both flow cytometric procedures may lead to greater insight into the relationship between the cytologic and cytogenetic heterogeneity of breast carcinomas.

Presence of circulating macromolecular IgA in patients with hematuria due to primary IgA nephropathy.

The relation between renal histologic features and the presence of circulating immune complexes was studied in 50 patients with hematuria. Primary IgA nephropathy was found in 25 patients, and various other forms of glomerulopathy were seen in the remaining 25 patients. Circulating immune complexes were detected with the 125I-C1q-binding assay, the conglutinin-binding assay, and the anti-IgA inhibition binding assay, the latter detecting specifically IgA-containing immune complex-like material. The 125I-C1q-binding assay gave negative findings for all patients except one. With the conglutinin-binding assay, immune complexes were found in a similar frequency for patients with and without IgA nephropathy. However, the anti-IgA inhibition binding assay gave positive results only in patients with primary IgA nephropathy (68 percent) and in none of the other patients. Sucrose density ultracentrifugation, as well as experiments in which the anti-IgA inhibition binding assay was performed with and without pretreatment of serum with polyethylene glycol, showed the presumed IgA immune complexes to have intermediate sedimentation coefficients (11 to 21S). The presence and level of this macromolecular IgA in the circulation correlated significantly (p less than 0.001) with the presence of hematuria in patients who had this clinical manifestation intermittently. Furthermore, a significant correlation (r = 0.69, p less than 0.0001) was found between the degree of hematuria and the degree of positive findings of the anti-IgA inhibition binding assay. This study shows that in patients presenting with hematuria, a positive finding on the anti-IgA inhibition binding assay is restricted to patients with primary IgA nephropathy and therefore could be of diagnostic value.

Familial occurrence of the haemolytic uraemic syndrome.

A family is described in which the haemolytic uraemic syndrome (HUS) occurred in two generations. Both juvenile and adult onset of this syndrome were observed in this family. Those affected were all women, three developed HUS in the postpartum period, one towards the end of pregnancy and one as a five-year old child. Because five cases were observed over a period of 16 years, exposure to the same infectious agent is highly unlikely. Although the transmission of a "dormant" virus cannot be excluded, the occurrence of HUS in two generations of one and the same family seems compatible with the hypothesis that susceptibility to the disease is transmitted as an autosomal dominant characteristic. This observation suggests a genetic influence on the development of HUS, possibly in conjunction with other factors, such as infectious agents, pregnancy and/or delivery.

Antibodies directed against antigens on the endothelium of peritubular capillaries in patients with rejecting renal allografts.

This study was undertaken to examine the humoral immune response against endothelial antigens of the donor kidney in human renal allograft recipients. Sera from 61 transplant recipients who received 62 grafts were studied for the presence of circulating endothelial antibodies (CEAb) using an indirect immunofluorescence technique with a pretransplant biopsy of the graft as a substrate. IgG antibodies directed against the endothelium of peritubular capillaries were found in the sera of 6 of the 10 patients with graft rejection within 7 weeks after transplantation, whereas these antibodies were not found in the absence of rejection (P less than 0.001). Immunofluorescence studies of post-transplant biopsies showed IgG along the endothelium of peritubular capillaries only in the grafts of patients with CEAb. Eluates from these grafts contained IgG antibodies that bound to the endothelium of the donor as shown by the indirect immunofluorescence technique. Absorption of endothelial antibody (EAb)-positive sera with human platelets or Wistar strain rat erythrocytes showed that the EAb were not directed against serologically defined HLA antigens or against heterophile antigens on rat erythrocytes. We conclude from this study that the presence of antibodies directed against endothelial antigens is associated with poor graft prognosis and that these antibodies may be responsible for the rejection process.

Morphology and immunohistochemically-defined endocrine function of pancreatic islet cell tumours.

Thirty pancreatic islet cell tumours were histologically classified and analysed for their possible peptide hormone content using the immunohistoperoxidase method. Seven tumours contained insulin, six tumours contained gastrin and eight tumours contained glucagon. One tumour contained all three hormones. In the insulin and gastrin-containing tumours, the cells were usually arranged in solid nests of cells, with tubular and acinar formations in about half the cases. In the glucagon-containing tumours the cells were mainly arranged in anastomosing ribbons consisting of one of two layers of small cells. Most of the hormone-containing tumours were argyrophilic using Grimelius' silver reaction. All but one of the glucagon-containing tumours were incidental findings at autopsy. About half of the other tumours had metastasized. It is concluded that a relation exists between the histological pattern of growth and immunohistochemically defined endocrine function of pancreatic islet cell tumours.

Erythrocytosis and Wilms' tumour.

A Wilms' tumour was diagnosed in an 18-year-old male patient with erythrocytosis. After radical excision of the tumour and postoperative irradiation and chemotherapy, the erythrocytosis disappeared and did not recur during a 2-year observation period. The levels of erythropoiesis-stimulating activity in serum and in the renal mass suggest the tumour as the source of this activity. The rarity of the association is discussed and a review of the literature is given.

Unilateral renal vein thrombosis and nephrotic syndrome. Report of a case with protein selectivity and antithrombin III clearance studies.

A case of the nephrotic syndrome with unilateral renal vein thrombosis is reported. The patient, an 18 year old man, presented with a six month history of edema and the recent development of a left-sided varicocele. An enlarged left kidney and a thrombus in the left renal vein were demonstrated roentgenographically. A biopsy specimen of the right kidney was interpreted as membranous glomerulonephritis. Selective renal function studies showed nearly identical creatinine excretion, and similar total protein excretion and protein selectivity from each kidney. Thus, the thrombus in the left renal vein did not influence glomerular filtration rate or quantitative or qualitative protein excretion. A high urinary output and a decreased serum level of antithrombin III were measured. These findings suggest a mechanism to explain the increased thrombotic tendency seen in this and other patients with the nephrotic syndrome.

Preexisting glomerulonephritis in allografted kidneys. Occurrence in man.

Biopsy specimens were taken from 43 renal allografts after completion of the anastomoses. In five kidneys from four donors, mesangial lesions compatible with preexisting glomerulonephritis were found. Immunofluorescence study of three kidneys indicated immunecomplex pathogenesis. In one donor, congenital heart disease was present: the others were free of manifest (renal) disease. Clinical follow-up data and repeated renal biopsy specimens indicated an uneventful course of these allografts. Awareness of this kind of lesions is useful for interpretation of glomerular lesions in biopsy specimens from renal transplants.