Human wound contraction: collagen organization, fibroblasts, and myofibroblasts

The closure of ungrafted sacrococcygeal pilonidal sinus excisional wounds was studied in 15 patients. Wound punch biopsies were taken on a regular basis, and histologic sections were made. To document changes, computer-assisted morphometric image analysis was employed. Initial average wound depth was 37.8 +/- 4.6 mm, and complete closure (0 wound depth) was reached by 68 days. Wound contraction contributed 88 percent to wound closure, whereas the deposition of scar only contributed 12 percent. Maximum cells density within granulation tissue was reached by day 18. Myofibroblasts, identified by alpha-smooth muscle actin immunostaining, first appeared on day 11. Unlike those observed in laboratory animals, myofibroblasts were a minor cell population of granulation tissue, never exceeding 10 percent of the cells. The pattern of collagen fiber organization was documented by polarized light microscopy of Sirius red-stained sections. Early granulation tissue collagen fibers demonstrated a fine greenish birefringence, whereas more mature granulation tissue collagen fibers were thicker, displaying orange-yellowish birefringence. Myofibroblasts were associated exclusively with thicker collagen fibers, whereas fibroblasts were associated with both fine and thick collagen fibers. It is proposed that human wound contraction involves a volume change whereby normal dermal and adipose tissues are pulled into the defect by forces generated within fibroblasts.     

Automated cell counting in tissue sections: a new approach by ‘multiple grey-level analysis‘

A system is described for the automated quantitation in tissue sections of the follicular and stromal cell populations of the thyroid gland, using a computer-linked TV image analyser. The paper illustrates how the problems of variable staining intensity and clustering of images (nuclei) may be overcome by a novel approach, in which multiple analyses of each field are made at increasing grey-level thresholds, and then ‘synthesized’ by computer to give a composite image. Discrimination between cell (nuclear) types was by a single minimum width criterion. The results were highly reproducible and correlated well with counts obtained by a comparable manual method.     

Immunohistological evidence for second or somatic mutations as the underlying cause of dystrophin expression by isolated fibres in Xp21 muscular dystrophy of Duchenne-type severity

Using five monoclonal antibodies against different parts of the dystrophin molecule, we have studied the dystrophin composition of 17 dystrophin-positive fibres in a muscle biopsy from a boy with Xp21 muscular dystrophy of Duchenne-type severity. The fibres showed five distinct, reproducible, immunoreactive dystrophin profiles. All the profiles included both the N-terminal and the C-terminal domains, but between these domains, different fibres were negative for different antibodies, suggesting the somatic loss of certain exons. We interpret this as the first in situ evidence of an individual having different patterns of missing exons leading to restoration of the reading frame in various ways in the original germline frame-shifting deletion of exons 35-43. It follows that various somatic mutations had taken place in different fibres.     

Simian virus 40 large T antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue

BACKGROUND: DNA sequences and immunoreactivity associated with Simian virus 40 transforming factors, large T and small t antigens (SV40LTAg), suggestive of an aetiopathogenetic link have been identified in fresh frozen tissue of a high proportion of recent cases of pleural mesotheliomas from the United States, Italy and Germany. SV40 is not normally infective in man though it can transform human cells in tissue culture. A large cohort of people in the western world was accidentally parenterally inoculated with live SV40 through contaminated polio vaccines given between 1959 and 1961, and this might be a factor in the current continuing rise in the incidence of mesothelioma in the United States, Britain and Europe. The present study investigated the presence of SV40LTAg DNA in recently diagnosed cases of mesothelioma in Britain and the feasibility of detecting the SV40 DNA in archival tissue for retrospective analysis of cases in the peri-vaccination period. METHODS: DNA was extracted from fresh frozen and/or rehydrated formalin fixed, paraffin embedded tissue sections from nine recently diagnosed cases of mesothelioma, nine cases of pulmonary adenocarcinoma, and three reactive pleurae, and amplified by the polymerase chain reaction (PCR) using the primer pairs used previously on fresh frozen tissues-namely, the SV primer set directed at the LTAg gene sequence unique to SV40 and the PYV primer set directed at a sequence shared by SV40 and papovavirus strains BK and JC, respectively. RESULTS: PCR positivity with the SV primer set was restricted to four of the nine cases of mesothelioma. In contrast, six of the nine mesotheliomas, two of the nine adenocarcinomas, and one of the three reactive pleurae showed positivity with the PYV primers. The fresh frozen and corresponding formalin fixed, paraffin embedded tissue results concorded well with each other. CONCLUSIONS: Our data provide evidence for the association of SV40LTAg primer specific DNA with human pulmonary mesothelioma in the British population.     

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function

The accumulation of functional neurotransmitter receptors by neurons during development is an essential part of synapse formation. Chick ciliary ganglion neurons express two kinds of nicotinic receptors. One is abundant, contains the alpha7 gene product, rapidly desensitizes, and binds alpha-bungarotoxin. The other is less abundant, contains multiple gene products (alpha3, beta4, alpha5, and beta2 subunits), slowly desensitizes, and binds the monoclonal antibody mAb 35. Rapid application of agonist to freshly dissociated neurons elicits responses from both classes of receptors. Between embryonic days 8 and 15, the whole cell response of alpha3-containing receptors increases fivefold in peak amplitude and, normalized for cell growth, 1.7-fold in current density. In addition, the response decays more slowly in older neurons, suggesting a developmental decrease in the rate of desensitization. The whole cell response of alpha7-containing receptors increases 10-fold in peak amplitude over the same period and 3-fold in current density. No change in the rate of desensitization was apparent for alpha7-containing receptors with developmental age, but analysis was limited by overlap in responses from the two kinds of receptors. Indirect immunofluorescence measurements on dissociated neurons showed that the relative levels of alpha7-containing receptors on the soma increased during development to the same extent as the whole cell response attributed to them. In contrast, the relative levels of alpha3-containing receptors increased more during the same time period than did the whole cell response they generated. The immunofluorescence analysis also showed that both classes of receptors become distributed in prominent clusters on the cell surface as a function of developmental age. The results indicate that during this period of synaptic consolidation on the neurons, the two major classes of functional nicotinic receptors undergo substantial upregulation; alpha3-containing receptors as a class may undergo changes in receptor properties as well.