Cell proliferation rate and nuclear morphometry in Roberts syndrome

Roberts syndrome (RS) is a rare autosomal recessive disorder characterized primarily by symmetric reduction anomalies of all limbs, growth retardation and craniofacial abnormalities. Most RS patients are reported to present a typical abnormality of their constitutive heterochromatin, accompanied by abnormal cytological growth characteristics. We present an extremely severe case of an RS fetus, karyotypically documented, with a clinical presentation including growth deficiency, tetraphocomelia, frontal meningocele, craniofacial abnormalities and penile enlargement with hypospadias. Nuclear morphometrical analysis in tissues of various organs revealed a reduced nuclear size in RS as compared to normal controls, and statistically significant differences in morphometric parameters related to the nuclear shape. Immunohistochemical study of the same organs showed a reduced expression of proliferating cell nuclear antigen in the presented case, thus indicating a decreased cell proliferation rate in RS. Our results reconfirm previously reported findings in cultured fibroblasts of RS cases, thereby reinforcing on a histologic level, the hypothesis that reduced cell proliferation may be involved in the growth retardation and the reduction abnormalities observed in RS.     

Enhanced polymer one-step staining (EPOS) for proliferating cell nuclear antigen (PCNA) and Ki-67 antigen: Application to intra-operative frozen diagnosis

Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technlque was applied to intra-operattve frozen diagnosis. The markers of choice were proliferating cell nuclear anmen (PCNA) and Ki-67 antigen. These cell prollferation markers were both identifiable in fresh frozen see tions of the human tonsil In approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyitimethoxysilane-cpated glass slides are immediately fixed, without air drying, for 15s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalln for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PSS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H₂O₂ solution containing 10mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxi-dase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.     

Transfection of Lepidopteran insect cells with Baculovirus DNA

Summary Several protocols are presented for preparation and transfection of Baculovirus and plasmid DNAs into Lepidopteran insect cells using the calcium-phosphate co-precipitation technique. Important parameters for optimum efficiency include the inherent susceptibility of the recipient cell line for transfection, and the method of preparation of viral and plasmid DNAs. The protocols presented provide reproducible high efficiencies for transfection of several Lepidopteran cell lines.     

Argyrophilic nucleolar organizer regions in colorectal malignancy: application of typing by their density

Colorectal neoplasms obtained at colonoscopy were examined by argyrophilic nucleolar organizer region (AgNOR) staining to evaluate the usefulness of AgNOR as a discriminant for malignancy. AgNOR dots were divided into two kinds: 'structures' (larger and less-densely stained) corresponding to the nucleolus, and 'units' (smaller and densely stained) presumed to be true AgNOR within the structure. The number of structures per nucleus did not differ between the adenoma and carcinoma groups, whereas the number of units per nucleus showed a significant difference. However there were several cases showing an overlap between the adenoma and carcinoma groups, leading to a difficulty in deciding whether any given case was benign or malignant. Three types of AgNOR patterns were categorized based on the ratio of units to structure. Type I was defined as the unit being indistinguishable from the structure, Type II as each structure having one to five units, and Type III as at least one structure having six or more units, irrespective of total number of units per nucleus. The colorectal lesions in which more than half of the neoplastic cells showed Type III coincided well with carcinomas histologically diagnosed, with the exception of adenomas with severe atypia. Labeling index of proliferating cell nuclear antigen (PCNA LI) differed between the adenoma and carcinoma groups with a considerable extent of overlap, and correlated to some extent with the AgNOR values. These results showed that the AgNOR staining was useful for determining malignancy and its usefulness appeared superior to PCNA LI.     

Proliferating cell nuclear antigen in malignant and pre-malignant lesions of epithelial origin in the oral cavity and the skin: an immunohistochemical study

Summary Proliferating cell nuclear antigen (PCNA) is a nuclear protein synthesized in the late G₁ and S phase of the cell cycle and immunohistochemical detection of the protein represents a useful marker for the proliferating fraction of cells in tissue specimens. A series of malignant and pre-malignant lesions of the oral cavity and skin were evaluated by the streptavidin biotin immunoperoxidase method for detection of this protein. Monoclonal anti-PCNA antibody (PC 10) labelled proliferating cells in all cases with varying intensity of nuclear staining. In squamous cell carcinoma (n=48), PCNA positivity correlated with the differentiation and atypia of the tumour cells; however, in poorly differentiated tumours, the relationship between PCNA expression and proliferation was lost. Basal cell carcinoma showed an increased growth fraction in tiny epithelial nests (mean 43.8, SD 6.0,n=20) than in neoplastic basal cells (mean 30.1, SD 6.9,n=8). The growth fractions were significantly higher in the pre-malignant lesions (leukoplakia, mean 22.3, SD 7.7,n=14; Bowen's disease, mean 45.2, SD 11.7,n=12; senile keratosis, mean 41.2, SD 7.0,n=12) than in the normal mucosa (mean 9.8, SD 4.9,n=10), suggesting that cellular growth fractions correlate with the degree of dysplasia in pre-malignant lesions.     

The juxtaglomerular apparatus in malignant hypertension of man

Summary Investigation of the behaviour of the renal Juxtaglomerular apparatus in 19 patients with malignant hypertension has shown that in kidneys fixed immediately after operation the Juxtaglomerular granulation index is twice as high as in autopsy kidneys. The formation of renin by the epitheloid cells begins with the appearance of osmiophilic substances in the region of the endoplasmic reticulum. The first stages of granule formation are small rhomboid particles in the Golgi cisternes, which aggregate to form bigger round or polymorphic granules in the Golgi area.In pathological conditions the substances synthesized may be set free and become active locally as a result of fibrinoid necrosis of the vascular wall. The rate of production is increased firstly by forcing rhe production of active agents in the preexistent epitheloid cells, secondly by transformation of the so-called bivalent cells and finally, by cell division.In accelerated hypertension the production of renin also takes place in nephrons whose glomeruli, tubules and macula densa, are damaged. There is a correlation between blood pressure elevation and the Juxtaglomerular granulation index.

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Zusammenfassung Untersuchungen über das Verhalten des juxtaglomerulären Zellkomplexes der Niere bei 19 Patienten mit maligner Hypertonie haben folgendes ergeben: In operativ gewonnenen und sofort fixierten Nieren ist der juxtaglomeruläre Granulationsindex doppelt so hoch wie in Nieren aus dem Sektionsgut. Die Reninbildung der epitheloiden Zellen beginnt mit dem Auftreten osmiophiler Substanzen im Bereich des endoplasmatischen Reticulums. Kleine rhomboide Gebilde in Golgizisternen sind Vorstufen von reifen Sekretgranula, die sich im Golgifeld zu größeren runden oder vielgestaltigen Sekrettropfen zusammenlagern.Unter pathologischen Bedingungen können infolge fibrinoider Gefäß- wandnekrosen Sekretsubstanzen lokal frei und lokal wirksam werden. Vermehrte Arbeitsleistung erfolgt zunächst durch Forcierung der Sekretproduktion in den präexistenten epitheloiden Zellen, sodann durch Transformierung sogenannter bivalenter Zellen und schließlich durch Zellneubildungen.Reninproduktion findet bei akzelerierter Hypertonie auch in solchen Nephronen statt, deren Glomerula und Tubuli samt Macula densa strukturell geschädigt sind. Es besteht eine Korrelation zwischen Höhe des Blutdruckes und Höhe des Granulationsindex.

     

Two Autopsy Cases of Primary Leiomyosarcoma of the Liver Superiority of Muscle-specific Actin Immunoreactivity in Diagnosis

Two autopsy cases of leiomyosarcoma of the liver in a 49-year old female and 63-year-old male are reported. Both of the liver tumors showed electron microscopically dense patches in the cytoplasm and intermediate junctions between the tumor cells, suggesting a smooth muscle cell origin, irrespective of their different histological features. The nature of both tumors was confirmed by positive immunoreactivity for muscle-specific actin in the tumor cells, whereas desmin immunoreactivity was labile in both cases, showing a higher diagnostic value of the former compared with the latter in these leiomyosarcomas. Both cases, showed extensive distant metastases in spite of an evident difference in their mitotic indices, indicating that this index is not reliable for judging the metastatic potential of these tumors. Acta Pathol Jpn 41: 461–465, 1991.