Combined haematoxylin, eosin and fluorescent stain for paraffin sections.

Combined staining is achieved by adding Auramine O to 2 of the steps of the standard H & E staining procedure. Auramine O confers fluorescence to mucin, reticulin, elastin and protein aggregates without altering the H & E colouring pattern. The technique is designed as a substitute for some special stains.

An effect of actin on cell migration.

A specific inhibitory effect on mononuclear cell migration by smooth muscle actin and skeletal muscle actin is reported. Actin aged at 4 degrees C or heated at 56 degrees C for 10 min had little or no effect, but papain-denatured actin had a strong inhibitory action. Actin and/or its breakdown product(s) may interfere with mononuclear cell movement in vivo in pathological conditions where release of actin from damaged or dead cells occurs.

Immunological studies in chronic atrophic gastritis and chronic (superficial) gastritis.

Detection of autoantibodies, HLA typing and immunofluorescence studies on gastric biopsies were carried out in subjects with histologically proven chronic atrophic gastritis (CAG) and chronic superficial gastritis (CG). All were seronegative for parietal cell antibody and did not have pernicious anemia. Except for positive antismooth muscle and antimitochrondrial antibodies in one patient with CAG, autoantibodies (antinuclear, smooth muscle, mitochrondrial, parietal cell) were absent in patients with CAG and CG. Immunofluorescence studies showed that Ig-G and IgA were presented in the lamina propria of all cases with CAG or CG and of subjects with normal gastric histology. Ig-M was seen less often, in about half the cases. Complement C3 was an uncommon finding, being positive in only one case with CAG and one case with CG and in none of the cases with normal gastric histology. Fibrinogen was more commonly seen in patients with CG (5/5 cases) than in those with CAG (3/11 cases). Fibrinogen was found in one case with normal gastric histology. The most consistent fluorescence was obtained with antiparietal cell antiserum. All subjects with CAG showed negative or weak staining only. In contrast, subjects with CG and normal gastric histology had strong specific fluorescence. An increased frequency of HLA-A1 plus HLA-B8 was found in subjects with CAG (20.7% in controls; 40% in CAG).

Experimental smooth muscle antibodies.

Attempts were made to induce smooth muscle antibody (SMA) in rats by various procedures causing cell necrosis. Ligation of a liver lobe and cryosurgical damage to a liver lobe both resulted in subsequent appearance of SMA, provided the damage tissue was not removed. Transfer of the damaged liver tissue to the peritoneal cavity of a normal rat did not result in SMA production in the recipient. The SMA produced showed anti-actin and anti-heavy meromyosin specificity. Damage induced by hepatotoxic agents failed to give rise to SMA.

Antigenicity of janin, a new protein from smooth muscle.

The exact nature of smooth muscle autoantibodies is still unclear. The antigen(s) which they are directed against remain unknown. Janin, a so far unknown smooth muscle protein, was obtained from smooth muscle tissue extracts and purified by means of three different procedures. Its mol. w. was estimated at 60,000 daltons. It stimulated a specific anti-janin antibody in rabbits as shown by tube test, double diffusion in agar and indirect immunofluorescence. The new protein absorbed eighteen out of 104 human smooth muscle positive sera that were not absorbed with smooth muscle actin, myosin, heavy meromyosin, light meromyosin, tropomyosin and brain tubulin. This would vindicate a view that smooth muscle autoantibodies differ from patient to patient with regard to the autoantigen(s) involved.

The specificity of anti-actin serum.

The immunogenicity of smooth muscle actin is increased by ageing at 4 degrees for at least a week. Rabbits lacking natural smooth muscle antibodies were injected with 1 mg of aged purified actin in adjuvant. Fourteen out of thirty-six rabbits produced serum antibodies which precipitated with actin solution, but not with smooth muscle tropomyosin, myosin, light or heavy meromyosin or with other unidentified non-actin proteins in crude extracts. Analysis of crude actin extract before and after precipitation by antiserum (i) by Sephadex G-200 chromatography and (ii) for its stimulating effect on myosin ATPase activity showed that actin was selectively removed. The precipitate itself, analysed on SDS-polyacrylamide gel, showed one band in the actin position, and otherwise only bands representing immunoglobulins. The antiserum also inhibited the ability of actin to stimulate myosin ATPase activity, and prevented polymerization of G-actin to F-actin, as shown by viscosity and EM studies. On immunoflouresence with cryostat tissue sections or cell cultures, anti-actin serum stained smooth muscle fibres and many non-muscle cells, in the latter staining the microfilaments. The staining was prevented by absorbing the antiserum with actin (16 mug per 5 mul serum), and was abolished by pretreatment of the cells with cytochalasin B. No species specificity was demonstrated for these anti-actin antibodies.

Contractile proteins in human cancer cells. Immunofluorescent and electron microscopic study.

The presence of contractile proteins in human cancer cells has been studied by means of: a) immunofluorescent staining using specific antibodies, and b) electron microscopy in order to detect the presence of cytoplasmic filaments. The tissues examined were: normal human skin, basal cell carcinoma of the skin, squamous cell carcinomas (of skin, oral cavity, and larynx), normal nonlactating mammary gland, and infiltrating mammary carcinoma with or without fibrosis. Normal tissues were negative after immunnoflurosescent staining of contractile proteins and contained no or minimal amounts of microfilaments as judged by electron microscopy. Tumor cells were strongly positive after immunoflouorescent staining for actin, myosin, light and heavy meromyosin but were negative for tropomyosin. Moreover, they contained prominent microfilaments (40 to 80 A in diameter) with some filaments (100 to 120 A in diameter) scattered in between. It appears that malignant cells contain an increased amount of contractile proteins, organized in the form of a filamentous apparatus, when compared to their normal counterparts. The study of the presence of contractile proteins in tumor cells may be of potential importance in evaluating malignant growth.

Localization of smooth muscle-like contractile proteins in kidney by immunoelectron microscopy.

Contractile proteins of smooth muscle type were found in kidney cells by immunological methods. The reactions of rabbit anti-actin, anti-heavy meromyosin and anti-myosin antisera with rat kidney were investigated by immunoelectron microscopy. Anti-actin stained specifically the foot processes of epithelial cells in the glomerulus, the basal processes of tubular epithelial cells, and the cytoplasm of smooth muscle cells. Anti-heavy meromyosin stained the foot processes at their bases near the cell membrane and close to the basement membrane. Anti-myosin stained the cytoplasm of vascular smooth muscle cells. It is suggested that actin and heavy meromyosin-like proteins may act together to cause movement of foot process cytoplasm.

Increase of contractile proteins in human cancer cells.

Human cells from skin and mammary-gland carcinomas show strong immunofluorescent staining with specific antibodies against smooth-muscle actin, myosin heavy meromyosin and light meromyosin, and contain numerous microfilaments as seen by electron microscopy. This increase in the amount of contractile proteins distinguishes cancer cells from the cells of normal tissues.

Immunological studies of human placentae. Binding of complexed immunoglobulin by stromal endothelial cells.

Endothelial cells of foetal stem vessels in cryostat sections of normal, full-term human placentae bind fluorescein-conjugated heat-aggregated human IgG. Soluble immune complexes of sheep anti-human albumin also bind in a pattern that is similar to that of aggregated human IgG, but native human IgG is not bound by placental endothelial cells. Aggregated IgG binding, unlike soluble immune complexes, is blocked by pretreatment of the sections with antiserum to human IgM. Cross-blocking experiments with aggregated IgG and immune complexes suggest that they may be bound by different receptors.