Subcellular localization of GFP-myosin-V in live mouse melanocytes.

Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.

Consistency achieved by 23 European pathologists from 12 countries in diagnosing breast disease and reporting prognostic features of carcinomas. European Commission Working Group on Breast Screening Pathology.

A detailed analysis of the consistency with which pathologists from 12 different European countries diagnose and classify breast disease was undertaken as part of the quality assurance programme of the European Breast Screening Pilot Network funded by the Europe against Cancer Programme. Altogether 107 cases were examined by 23 pathologists in 4 rounds. Kappa statistics for major diagnostic categories were: benign (not otherwise specified) 0.74, atypical ductal hyperplasia (ADH) 0.27, ductal carcinoma in situ (DCIS) 0.87 and invasive carcinoma 0.94. ADH was the majority diagnosis in only 2 cases but was diagnosed by at least 2 participants in another 14, in 9 of which the majority diagnosis was benign (explaining the relatively low kappa for this category). DCIS in 4 (all low nuclear grade) and invasive carcinoma (a solitary 1-mm focus) in 1. The histological features of these cases were extremely variable; although one feature that nearly all shared was the presence of cells with small, uniform, hyperchromatic nuclei and a high nucleo-cytoplasmic ratio. The majority diagnosis was DCIS in 33 cases; kappa for classifying by nuclear grade was 0.38 using three categories and 0.46 when only two (high and other) were used. When ADH was included with low nuclear grade DCIS there was only a slight improvement in kappa. Size measurement of DCIS was less consistent than that of invasive carcinoma. The majority diagnosis was invasive carcinoma in 57 cases, the size of the majority being 100% in 49. The remainder were either special subtypes (adenoid cystic, tubular, colloid, secretory, ductal/medullary) or possible microinvasive carcinomas. Subtyping was most consistent for mucinous (kappa, 0.92) and least consistent for medullary carcinomas (kappa, 0.56). Consistency of grading using the Nottingham method was moderate (kappa=0.53) and consistency of diagnosing vascular invasion, fair (kappa=0.38). There was no tendency for consistency to improve from one round to the next, suggesting that further improvements are unlikely without changes in guidelines or methodology.

Consistency achieved by 23 European pathologists in categorizing ductal carcinoma in situ of the breast using five classifications. European Commission Working Group on Breast Screening Pathology.

The increased detection of ductal carcinoma in situ (DCIS) by mammographic screening, the greater use of breast-conserving surgery, and the recognition that certain histological subtypes are associated with a greater risk of local recurrence has led to the formulation of several new classifications of DCIS in recent years. There are, however, no data concerning the degree of consistency with which these schemes can be applied by reasonable numbers of pathologists. Thirty-three cases of DCIS were thus examined by a working group of 23 European pathologists who categorized them using five recently published classifications: (1) that of the European Pathologists' Working Group based on differentiation (a combination of nuclear grade and cell polarization) with categories of poorly, intermediately, and well differentiated; (2) one based entirely on nuclear grade with categories of high, intermediate, and low, currently in use in the UK national and EC-funded breast screening programs; (3) the same classification in which only two categories, high nuclear grade and other, were used; (4) the Van Nuys system in which lesions are divided into high grade, non-high grade with necrosis and non-high grade without necrosis; and (5) a two-category classification based entirely on the presence or absence of comedo necrosis. Of the three systems with three categories, Van Nuys gave the highest overall kappa statistic of 0.42. Others gave similar values of 0.37 and 0.35 showing that assessing cell polarization in addition to nuclear grade neither improves nor worsens consistency. In all three systems, the middle category was associated with the lowest value for kappa. Of the two systems with two categories, that based on nuclear grade gave the highest overall kappa of 0.46 and that based on comedo necrosis the lowest of 0.34. The most robust histological features were thus high- and low-grade nuclei and necrosis as long as the latter did not involve the recognition of a comedo growth pattern. These values probably represent the maximum achievable, at least by reasonable numbers of pathologists in everyday practice. They are better than those previously reported for classification based entirely on architecture, but further improvement is needed.

Localization of myosin-V in the centrosome.

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.

Case report: sclerotic skeletal haemangiomatosis presenting with spinal cord compression--CT and MRI findings.

We report the case of a 59-year-old man with skeletal haemangiomatosis who presented with progressive bilateral lower extremity weakness. Computed tomography (CT) and magnetic resonance imaging (MRI) located the causative lesion in the neural arch of the T4 vertebra. CT demonstrated osseous expansion with a mixed lytic and sclerotic pattern. MRI of the lesion showed hypointensity on T1 weighted images, mixed signal intensity on T2 weighted images and moderate contrast enhancement. Similar but less extensive lesions were present in other vertebrae as well as ribs.

The anatomy of free paper sessions.

The interaction between speakers, audience, and chairmen was studied by 13 investigators during a medical meeting at which 356 free papers were given before a total audience of 2483 in 48 sessions. A protocol was used to score 21 questions relating to the presentation, 15 to the chairman of the session, and nine to the audience. Many speakers made technical faults in presentation and their use of slides. Most chairmen failed to comply with simple rules of procedure and with the expectations of speakers and audience. The interest of the audience was affected by the performance of speakers and chairman. Speakers should pay more attention to the technique of presentation, and organising committees of medical meetings should provide instructions to both speakers and chairmen.

Prognostic significance of lymph node histology in patients with squamous cell carcinoma of the larynx, pharynx, or oral cavity.

In a double-blind retrospective analysis, sections of lymph nodes regional to head and neck squamous cell carcinomas were microscopically examined to assess morphologically the immunologic pattern of response. Patients whose nodes showed evidence of immunologic stimulation had five-year survival rates significantly higher than those whose nodes showed no evidence of immunologic stimulation. None of the patients whose nodes showed the lymphocyte depletion pattern survived five years. The stage or histologic grade of the tumors did not influence these correlations. Metastases occurred much more frequently in patients whose nodes showed immunologic activity than in those whose did not. The data support the concept that immunologic capacities are important host defense mechanisms against malignancy. Histologic assessment of immunologic activity in regional nodes seems to be an important parameter for predicting survival.

Immunologic assessment of regional lymph node histology in relation to survival in head and neck carcinoma.

Histologic material from 84 cases of squamous cell carcinoma of head and neck regions was studied by double blind retrospective analysis. Sections of lymph nodes draining the tumors were examined microscopically to assess the morphologic pattern of response. Patients whose lymph nodes demonstrated active immunologic responses in the form of expanded inner cortices or increased numbers of germinal centers had 5-year survival rates significantly greater than those patients whose regional lymph nodes showed an unstimulated pattern. None of the patients whose lymph nodes showed the depleted pattern survived 5 years. These correlations were independent of the stage or grade of the tumor. Metastases occurred much more frequently in patients having regional nodes showing an unstimulated or depleted pattern than in those whose regional nodes showed evidence of immunologic activity. The data support the concept of a relationship between immunologic activity, progression of neoplasia, and survival. Morphologic assessment of immunologic activity in lymph nodes draining malignant tumors appear to be of significant value in the predicting survival.

In vitro studies of axillary lymph node cells in patients with breast cancer.

A total of 170 axillary lymph nodes were obtained from fresh mastectomy specimens from 81 women with breast cancer. Lymph node cells were tested in vitro for T and B cells by the rosette technique and immunofluorescence microscopy and for functional capacity by response to the mitogens phytohemagglutinin (PHA) and concanavalin A. T cells showed a wide range of relative values: 32-80 percent, with a mean of 63.5 percent. B cells defined by the presence of surface immunoglobulins ranged from 14 to 61 percent (mean, 35.8 percent); those defined by the presence of C3 receptors, from 8 to 54 percent (mean, 24.9 percent); and those defined by the presence of IgG-specific (Fc) receptors, from 10 to 45 percent (mean, 27.5 percent). Cells with the C3 and Fc receptors constituted approximately two-thirds of the cells not binding spontaneously to sheep red blood cells (non-SRBC-R), whereas virtually all non-SRBC-R stained for surface immunoglobulins. The proportion of T and B cells and the response to mitogens varied widely among nodes and among patients. Differences were significant between values observed in young and old patients, nodes with and those without metastatic disease, and lymph nodes with different morphology. Lymph nodes from patients over 60 years old showed a higher proportion of B cells and a lower proportion of T cells than did those from patients 45 years of age or younger. Lymph nodes with disease metastic to them also showed a higher percent of B cells and a lower percent of T cells than the nodes that did not have metastatic disease. Lymph nodes with lymphocyte predominance showed a relatively high proportion of T lymphocytes, a high PHA response, and a low content of B lymphocytes. By contrast, lymph nodes with germinal-center predominance showed a relatively low content of T cells, a low PHA response, and a relatively high proportion of B lymphocytes.

Prognostic evaluation of regional lymph node morphology colorectal cancer.

Histologic sections from 143 patients treated with surgery for infiltrating adenocarcinoma of the distal large bowel were studied with emphasis on the morphologic characteristics of the regional lymph nodes. Lymph nodes were classified into four groups designated lymphocyte predominance, germinal center predominance, lymphocyte depletion, and unstimulated. Results were correlated with the extent of disease and five year survival data. There was no significant association between the histologic pattern of the lymph nodes and the extent of the primary lesion. There was a higher survival rate in patients whose nodes showed germinal center predominance (71 per cent) compared with those whose nodes showed lymphocyte predominance or the unstimulated pattern (both 54 per cent), but these results were not statistically significant.

An autopsy study of human axillary lymph node histology.

This is a histologic study of axillary lymph nodes removed from 487 random autopsies. Histologic features such as germinal centers, deep cortical regions, sinus histiocytosis, hyalin deposition, fibrosis and overall cellularity were evaluated in each case. Results were correlated with the age of the deceased and cause of death. The results a) support the general opinion that germinal centers are more frequently found in children and young adults than in the old, b) showed that hyaline deposits increase with age, c) showed that lymphocyte depletion associated with fibrosis and hyaline deposits was found in patients dying of chronic disease, particularly cancer, d) pointed out a selective depletion of the deep cortical regions in patients dying of hemorrhage, and e) contributed to knowledge of the morphology of axillary lymph nodes in neonates. (Am J Pathol 78:7-22, 1975)