Type 2M von Willebrand disease - more often misidentified than correctly identified.

INTRODUCTION: Appropriate diagnosis of von Willebrand disease (VWD), including differential identification of qualitative vs. quantitative von Willebrand factor (VWF) defects has important management implications, but remains problematic. AIM: The aim of the study was to assess whether 2M VWD, defining qualitative defects not associated with loss of high molecular weight (HMW) VWF, is often misidentified, given highly variable reported frequency ranging from 0 to ~60% of all type 2 VWD. METHODS: A comparative evaluation of laboratory ability to appropriately identify 2M VWD (n = 4) vs. HMW VWF reduction (n = 4), as sent to participants of an international external quality assessment programme. RESULTS: Laboratories had considerably greater difficulty identifying type 2M VWD, correctly identifying these on average only 29.4% of occasions, with the 70.6% error rate representing use of insufficient test panels (41.7%), misinterpretation of test results (10.0%) and analytical errors (13.3%). One type 2M case, giving a median of 49 U dL(-1) VWF:Ag, was more often misidentified as type 2A/2B VWD (46.7%) than 2M (34.8%). Another 2M case, giving a median of 189 U dL(-1) VWF:Ag, was instead often misidentified as being normal (non-VWD) (36.4%), with identifications of type 2A/2B VWD (13.6%) also represented. In comparison, errors in identification of HMW VWF reduced samples only averaged 11.5%, primarily driven by use of insufficient test panels (6.3%) or misinterpretation of results (4.2%) and infrequently analytical errors (1.0%). CONCLUSION: Type 2M VWD is more often misidentified (70.6%) than correctly identified as 2M VWD (29.4%), and potentially explaining the relative under-reported incidence of 2M VWD in the literature.

Establishment and characterization of a cell line (SS78) from a human renal cell carcinoma.

A new cell line, SS78, was established from a primary renal cell carcinoma of a Caucasian male. The tissue was dispersed with collagenase, and viable cells were separated by flotation on a Ficoll-Hypaque gradient. In culture, the SS78 cells retained a distinct epithelial morphology, and no fibroblast like cells were seen. The cultured cells were aneuploid with a modal chromosome number of 80 and had several marker chromosomes. Inoculation of the cultured cells into athymic nude mice caused tumors at the sites of inoculation.

In vitro chemotherapeutic testing of urologic tumors.

We studied 20 transitional cell tumors of the bladder and 25 adenocarcinomas of the kidney in vitro to determine their chemotherapeutic sensitivity. The different sensitivity patterns among the individual tumors were demonstrated. Identical drug sensitivity patterns could be identified in the primary and metastatic sites, and in tumor tissue removed from the primary and metastatic deposits in the same patient. Human renal adenocarcinoma maintained in the athymic mouse demonstrated identical chemotherapeutic sensitivity patterns in vitro and in vivo. Our data would support that these in vitro chemotherapy studies may assist in the selection of agents to use in human tumor-bearing hosts.

An in vitro assay procedure to test chemotherapeutic drugs on cells from human solid tumors.

An in vitro assay was developed to measure the chemotherapeutic drug susceptibility of cells from human tumors. The assay utilized live cells, freshly isolated from tumor tissue, which were incubated for a short period in vitro. The drug-induced inhibition of incorporation of radiolabeled precursor into DNA, RNA, and protein was measured. The assay is sensitive to concentrations of chemotherapeutic drugs in the therapeutic range and is reproducible when tested with replicates of the same tumor cell population.

Activation of cyclophosphamide for in vitro testing of cell sensitivity.

Two methods are described for the activation of cyclophosphamide by a liver microsome preparation. These procedures were applicable to an assay in vitro which tests the sensitivity of tumor cells to the drug. Satisfactory results were obtained either by pretreatment of the cyclophosphamide and removal of the microsomes before testing or by the somewhat simpler procedure of mixing drug, microsomes, and test cells for the assay. Microsome treatment of bleomycin gave a smaller increase in activity, and much smaller effects were seen on some other drugs.

Clastogenic effect of 4-nitroquinoline-1-oxide on swine urothelial cells in culture.

Swine bladder epithelial cells in culture were treated with 4-nitroquinoline-1-oxide (4NQO) for 4 hr with three different concentrations to examine dose-dependent effects. To study time course effects, they were treated with one concentration for 4 hr and then incubated in 4NQO-free medium for three different periods before harvesting. Three different effects were recorded: (i) a suppression of mitotic activity that was dose-dependent and which continued for more than 30 hr posttreatment; (ii) no marked changes in the proportions of different ploidy classes; and (iii) chromatid gaps, breaks, and exchanges that were dose-dependent. This clastogenic effect decreased with increasing time after treatment.

Polyploidy in mammalian urothelial cells.

The mitotic indices and the extent of polyploidy in urothelial cells of baboons, dogs and swine were studied. All three species had very low mitotic activity in vivo but short-term culturing of these cells in vitro stimulated mitosis thus enabling chromosome counts. Tetraploid cells were found in the urothelium of all three species, and higher ploidies also in dog and swine. There were substantial differences in the proportions of diploidy and higher ploidies among the three species and among individuals within each species. Dog urothelial cells were predominantly tetraploid (70%) while more swine cells were diploid (68%). Baboon urothelial cells had only two ploidy classes and 92% were diploid.

Development and application of basic research techniques in bladder cancer research.

The growth of transitional epithelial cells with different growth media and growth supports was examined. Sephadex G-10, Bio-Gel P-20, Bio-Glas-1000, DEAE-Sephadex A-50, DEAE-cellulose, CM-Sephadex C-50, acid-soluble collagen, and immobilized collagen fibers were used to enhance plating efficiency. Acid-soluble collagen layers optimally increased the plating efficiency of primary cultures of bladder carcinoma. Media alterations with serial combinations of fetal calf, newborn calf, calf, bovine, and bull serum with minimum essential medium, Roswell Park Memorial Institute Tissue Culture Medium 1640, Connaught Medical Research Laboratories Medium 1066, Medium 199, Grand Island Biological, National Cancer Tissue Culture 135, 1415, McCoy's 5A, and National Cancer Institute medium were established. No promotion of cell division was noted with any one of these basic medium formulations.

Canine urinary bladder epithelial cells: preparation for cell culture by enzyme dispersion.

In a qualitative and quantitative study of enzymic dispersion of cells from the mucosal layer stripped from canine urinary bladder, trypsin was found to be equal or superior to the other enzymes tested for dispersal of urothelial cells specifically. Collagenase or collagenase plus trypsin served to disperse the whole tissue. A procedure for recovering the urothelial cells as a single-cell suspension and establishing them in culture is presented. The morphology, culture behaviour, and chromosome complement of these cells is described.

Acute tubular necrosis. An experimental model detailing the biochemical events accompanying renal injury and recovery.

Male Charles River mice, divided into control or experimental groups, received on Day 0 either sterile 0.3 MNaHCO3 in 0.9 per cent saline (pH7.4) intraperitoneal injection or pteroylglutamic acid (200 mug per body weight), similarly buffered to pH7.6, and were sacrificed on Days 0, 1/4, 1/2, 1,2,3,4,7, and 14. The experimental kidneys demonstrated intratubular deposits of pteroylglutamic acid with edema between Days 1 and 4 with cortical scarring by Day 14. The experimental kidneys reached maximal increases in weight (+90 per cent) on Day 2, RNA (+61 per cent, protein (+67 per cent) on Day 3, and DNA (+25 per cent) on Day 4 before falling to below control levels on Day 14. The control kidneys demonstrated the gradual incremental increases of normal renal growth throughout this period. No change in renal size, protein, RNA, or DNA could be detected in those animals who failed to demonstrate renal tubular damage. It is postulated that the response of the kidney to folic acid administration is a reparative response and not a response directed toward accelerated renal growth.

Effect of transient hydronephrosis on subsequent compensatory renal growth.

Apparent augmentation of renal growth occurs in kidneys made temporarily ischemic, or partially obstructed, before contralateral nephrectomy. The study herein was undertaken to investigate the effect of acute complete ureteral occlusion on a subsequent course of renoprival hypertrophy and hypoplasia. Three groups of animals were established. Animals in Group 1 underwent high ligation of the right ureter. Animals in Groups 2 and 3 underwent exposure and manipulation of the right ureter. Forty-eight hours later, animals in Group 1 underwent deligation and contralateral nephrectomy, animals in Group 2 underwent contralateral nephrectomy, and animals in Group 3 underwent sham operation. Animals were then selected 6 and 17 days after their second operative procedure and decapitated; the right kidneys were removed and underwent analysis with respect to wet and dry weight, total RNA, DNA, and protein content. At 6 days and at 17 days, animals in Groups 1 and 2 demonstrated no difference between these groups, although the remaining kidneys from animals in Group 1 and Group 2 were significantly larger than Group 3 animals. When compared to Group 3 animals, wet renal weight at 17 days had increased by 41 per cent, total bulk RNA had increased by 26 per cent, and total bulk DNA had increased by 33 per cent. The data support the clinical impression that transient, complete ureteral obstruction is well tolerated by the normal kidney, and that the metabolic response to obstruction does not hinder recovery after release of obstruction.

Properties of prostatic cultures transformed by SV40.

SV-40-transformed hamster prostatic tissue has been previously evaluated as a model for human prostatic carcinoma. Because the original cell line was lost, Syrian golden hamster prostatic tissue has been established in explant culture and infected with a 10-6-cell tissue culture infectious dose (50 percent effective) of SV40. After in vitro transformation, the cells were produced in quantity and 60 times 10-6 cells were injected into adult male Syrian golden hamsters 24 hours after 400 rads of whole-body radiation. After 60-90 days, a small palpable tumor developed. These tumors could be serially transplanted in adult male animals without immunosuppression. The tumor cells were established in tissue culture and the cells were returned to adult animals without immunosuppression where they rapidly produced fast-growing tumors. The solid tumors were composed of sheets of pleomorphic polygonal cells with large nuclei and many nucleoli; they resembled undifferentiated human prostatic carcinoma. In vitro, the cultures contained small, rapidly growing cells with a population doubling time of about 1.3 days. The cells carried the SV 40-specific antigen. The modal chromosome number was 66-68 with a distribution of 47-120. Cells exposed to 2-bromo-5'-deoxyuridine in culture did not release particles with RNA-dependent DNA polymerase activity. Endocrine sensitivity in vivo and in vitro is undertermined to date.

Immunoelectrophoretic analysis of avian ribonucleic acid tumor virus group-specific antigens.

Tumors induced in pigeons by inoculation with the Schmidt-Ruppin strain of Rous sarcoma virus regressed after about 6 weeks. Sera from these pigeons, taken 8 weeks after inoculation, had complement-fixing group-specific antibody titers of 1:2 to 1:256. In immunoelectrophoresis with the pigeon serum, disrupted BAI strain A (myeloblastosis) avian tumor virus showed at least five precipitin arcs. The pattern of precipitin lines was dependent in part on the means used for virus disruption, and ethyl ether and nonionic detergents appeared to be both effective and relatively mild reagents. Immunoelectrophoretic comparison of pigeon serum with serum from a tumor-bearing hamster and that from virus-inoculated rabbits yielded similar, though not identical, results.