Ultrastructural studies of interactions of B16 melanoma cells and normal mouse skin fibroblasts in the three-dimensional collagen gels in vitro.

In the in vivo system, cancer metastasis is a complex multi-step process. We have utilized an in vitro three-dimensional system to study the initial stages of tumour cell invasion. In this communication, the sequence of events in interactions between B16 melanoma cells and normal mouse skin fibroblasts are described. It is proposed that tumour cells produce a "retractive factor" which induces the underlying fibroblasts to retract so as to facilitate invasion and metastasis.

Ultrastructural study on the invasion of the basal lamina of retinal inner limiting membrane by esophageal carcinoma cells.

Esophageal carcinoma cells EC/CUHK1 were allowed to interact with the basal lamina of the inner limiting membrane of the retina in vitro. Both scanning and transmission electron microscopy revealed that tumour cells first attached to the basal lamina with filopodia and subsequently invaded into the ganglion cell layer of the retina. In addition, transmission electron microscopic studies demonstrated that tumour cells induced mechanical distortion as well as dissolution of the basal lamina.

Scanning electron microscopic study of invasiveness between normal and neoplastic cells and fibroblasts in monolayer culture.

Normal cells are non-invasive except for a few cell types such as leukocytes and macrophages, while neoplastic cells are active in both migration and invasion. In the present investigation, the confrontations of normal rat hepatocytes, lung macrophages and B16 melanoma cells with monolayers of fibroblasts grown on collagen gel have been studied. When monolayers of fibroblasts were confronted with melanoma cells, there was obvious retraction of the fibroblasts. When hepatocytes or macrophages were seeded onto monolayers of fibroblasts, infiltration always occurred at intercellular contacts and no retraction of fibroblasts was seen. Therefore it would appear that normal and neoplastic cells penetrate into monolayers of fibroblasts by different mechanisms.

Biological characteristics of a newly established human oesophageal carcinoma cell line.

An epithelial cell line, EC/CUHKI was established from a human squamous cell carcinoma of the oesophagus. The cells were polygonal in shape with numerous microvilli. Transmission electron microscopy (TEM) revealed the presence of desmosomes and tonofilaments. Karyotypic analysis of cells from the cell line demonstrated an aneuploid human type with a modal chromosome number of 85. Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or subcultures. The tumour cells form colonies in agar and have a plating efficiency of 20%. The cells are highly invasive in vitro and are tumorigenic in nude mouse.

Cryopreservation of cultured dermal fibroblast impregnated collagen gels.

The survival of cultured dermal fibroblasts was evaluated following manufacture, freezing and disaggregation of fibroblast-impregnated collagen gels. The concentration which gave optimal cell survival was determined for three cryoprotectants (glycerol, dimethyl, sulphoxide (DMSO) and ethanediol) and their efficacy compared. DMSO led to the highest cell viability after freezing and thawing. The effect of rate of freezing was also compared and 0.5 degree C/min (within the range 20 degrees C to -70 degrees C) was found to result in a significant enhancement of cell viability in comparison with freezing at 1.0 degree C/min or rapid freezing.

Cytotoxicity of wound dressing materials assessed using cultured skin equivalents.

An in vitro system, based on the Bell model of cultured composite skin equivalents, was used to assess the effect of a number of wound dressing materials on DNA synthesis. DNA synthesis was quantified using immunocytohistochemical identification of incorporated bromodeoxy-uridine and the percentage of labelled cells measured, following 7 days' exposure to the dressing material. Differences in labelling index were observed from replica gels covered by different dressing materials and between dressings of the same type of material, but made by different manufacturers.

The long head of the biceps femoris: anatomic basis for its possible use in the construction of an electrically stimulated neoanal sphincter.

Electrical stimulation of the nerve to the gracilis muscle following its transposition around the anal canal creates an artificial sphincter capable of actively opposing intrarectal pressure. Not all patients have an available or suitable gracilis. This paper describes the anatomic basis for the use of the long head of the biceps femoris as a potential electrically stimulated neoanal sphincter. The muscle was found to have an adequate length and a suitable arc of rotation for transposition around the anal canal. In 75 percent of thighs studied the neurovascular anatomy of the long head of the biceps femoris was compatible with its utilization in this manner as an alternative to the gracilis.

Private medical education: provider's perspective.

Issues related to the provision of private education are discussed in relation to the need, clinical teaching, professional standards and financial implications. The advantages and disadvantages are summarised.

Ultrastructural localisation of anionic sites at the dermo-epidermal junction in normal human skin.

Glycosaminoglycans have been demonstrated throughout the cutaneous BMZ at the ultrastructural level. Colloidal iron and cationised ferritin proved of limited value, whilst staining with Alcian blue and application of the critical electrolyte concentration principle has provided evidence for the presence of sulphated GAGs at the lamina lucida and lamina reticularis. Digestions with chondroitin ABC lyase and heparin lyase have confirmed the existence of chondroitin and/or dermatan sulphates and heparan sulphates, although the results obtained with hyaluronate lyase have indicated that hyaluronates are also present.

Periodate-labile structures at the normal human cutaneous basement membrane zone.

The distribution of carbohydrates containing 1,2-glycol groups at the basement membrane zones of the dermo-epidermal junction and dermal blood vessels was investigated using the periodic acid-thiosemicarbazide-silver proteinate technique. Controls included pre-incubation with dimedone, dimedone treatment both before and after periodate oxidation, and dimedone treatment followed by thiosemicarbazide and silver proteinate. At the dermo-epidermal junction the anchoring fibrils were stained selectively after both short (45 min) and long (71 h) thiosemicarbazide incubation periods, there being no staining of the lamina densa; however, the lamina densa surrounding blood vessels was labelled. After prolonged treatment with thiosemicarbazide, fibrils were observed traversing the lamina lucida at the dermo-epidermal junction, and collagen fibres were stained faintly. Pre-incubation with dimedone to block aldehyde groups did not affect the staining. However, when periodic acid treatment was omitted, or when sections were incubated with dimedone both before and after periodate oxidation, only melanin granules and the cytoplasm of fibroblasts were stained. Thus, anchoring fibrils are rich in carbohydrates containing 1,2-glycol groups susceptible to periodate oxidation, and correspond to the periodic acid Schiff-positive layer seen at the dermo-epidermal junction. Furthermore, the basement membrane zones of dermal blood vessels and the dermo-epidermal junction differ in their carbohydrate distribution.

The healing of suction blisters in pig skin.

The healing of intact suction blisters in pig skin was studied using fluorescein-labelled lectins as well as conventional histological techniques. After blistering, initially overt separation was observed at the dermo-epidermal junction, but the separated epidermis appeared to re-attach within 9 h. Massive infiltration of the dermis and the epidermis by inflammatory cells, including eosinophils, was observed. By day 4 focal detachment of the epidermis occurred spontaneously, and towards day 9 the appearance of the blistered areas had almost returned to normal, apart from isolated patches of thin epidermis devoid of rete ridges. It is proposed that the sloughing the of the epidermis on day 4 is related to the presence of inflammatory cells and that the restoration of normal dermo-epidermal relations is dependent on the basement membrane zone remaining intact. The secondary blistering seen in day 4 specimens and the inflammatory cell involvement are in many ways similar to the blistering processes observed in certain human cutaneous disorders, such as dermatitis herpetiformis and bullous pemphigoid, and therefore suction blisters in pig skin might provide a suitable animal model for these diseases.

The fine structure of haemopoiesis in the human fetal liver. II. Origin and differentiation of the megakaryocyte.

The differentiation of the megakaryocyte was studied at the ultrastructural level in the liver of human fetuses of between 49 and 134 mm crown-rump length. The development of the cells was traced from lymphoid elements with the features of haemopoietic stem cells and was divided on the basis of nuclear morphology into three stages. Granule formation commenced during the first stage and demarcation membrances could be demonstrated in the perinuclear cytoplasm early in the second stage. Late stage 2 cells often contained more than one nucleus, and the possibility that this was due to cellular fusion is discussed. The third stage was characterized by the appearance of cytoplasmic zoning and by the gradual extension of the demarcation system throughout the cytoplasm. There was evidence that the demarcation membrances were initially formed directly from the Golgi apparatus, but that their further development was due to the incorporation of elements of the agranular endoplasmic reticulum. The surface projections associated with platelet release were observed only in fully developed cells, and the formation of a zone of clear cytoplasm at the periphery was related to events occurring during the later stages of platelet release.

Toxicity testing of wound dressing materials in vitro.

There is a bewildering array of dressing materials available for wound coverage. The choice of dressing is often by local custom or practical experience. We wished to investigate if different dressings varied in their ability to either stimulate or inhibit proliferative activity and differentiation in an in vitro test system. We have used a number of test systems for this study. Human dermal fibroblast and keratinocyte cultures were used to screen for proliferative and cytotoxic effects. A more complex "organotypic" method involving fibroblast-impregnated collagen gels overlaid with epidermal keratinocytes was used to investigate effects on differentiation. Dressings were selected from each of the major types available, from simple gauze to hydrocolloids. Of the dressings tested, some reduced cell growth rates but the majority showed no major effect on proliferation or differentiation. Of those displaying an effect, only one could be attributed to cytotoxicity.

A fine structural study of microwave fixation of tissues.

Microwave fixed liver and kidney tissues were examined by electron microscopy. It was found that the preservation of fine structure of these tissues by this method is equal to that processed by routine methods. No difficulty was encountered in sectioning microwave fixed tissue blocks. It is obvious that microwave fixation is a faster and more efficient method.

The ultrastructural changes in the skin in dermatitis herpetiformis after withdrawal of dapsone.

The development of skin lesions in patients with dermatitis herpetiformis after the withdrawal of their dapsone therapy was studied with the electron microscope. In control biopsies from patients prior to cessation of treatment, membrane-bound vacuoles were found beneath the basal lamina of the epidermis as previously described. After dapsone withdrawal, there was an apparent increase in the number of vacuoles and occasionally several vacuoles appeared to have coalesced forming an early blister. At this stage, the basal lamina and associated hemidesmosomes were normal although in places there were small discontinuities in the basal lamina. Where the reaction was more intense, vacuoles and cells, mainly eosinophils, were embedded in fibrin de posits. Above this, the basal lamina was usually disrupted with involvement of the basal epidermal cells. These results suggest that the vacuoles do play a part in the formation of the pathological lesion in dermatitis herpetiformis. In addition, the basal lamina is shown to be only secondarily involved. The nature of the vacuoles has still to be elucidated.

Surface morphological study of Ehrlich ascites tumor cells exposed to microwave irradiation and heat.

Microwave irradiation of EAT cells caused an increase in length and number of surface microvilli. The tumor cells tend to form large aggregates by means of extensive interdigitation of surface microvilli. On the other hand, heat hyperthermia caused a decrease of surface microvilli but an increase of surface blebs. Hence the surface morphology of EAT cells after in vitro exposure to microwave irradiation differs markedly from that after heat hyperthermia.