J. Chemnitz

STAUROSPORINE-INDUCED CELL DEATH IN TETRAHYMENA THERMOPHILA HAS MIXED CHARACTERISTICS OF BOTH APOPTOTIC AND AUTOPHAGIC DEGENERATION

Staurosporine blocks signal transduction associated with cell survival, proliferation and chemosensory behaviour in the ciliated protozoan, Tetrahymena thermophila. Staurosporine inhibits cell proliferation and in vivo protein phosphorylation induced by phorbol ester. It also reduces the in vitro phosphorylation of the PKC‐specific substrate, myelin basic protein fragment 4‐14. Our results show that cell death in the presence of staurosporine is associated with morphological and ultrastructural changes similar to both apoptosis and autophagic degeneration, but these in turn can be postponed or prevented by 8‐bromo‐cyclic GMP, protoporphyrin IX, hemin or actinomycin D, although phorbol ester and insulin were ineffective. The results support the notion that staurosporine‐induced cell death is an active process, associated with and/or requiring de novo RNA synthesis.

Collagen structural organization of healing colonic anastomoses and the effect of growth hormone treatment.

PURPOSE: This experimental study was designed to investigate the collagen fibrils of colonic anastomoses in rats and to compare normal healing with rats treated with biosynthetic growth hormone (bGH). METHODS: The healing zone of left colonic anastomoses was studied at days 2, 4, and 6 after surgery by means of scanning electron microscopy. RESULTS: After four days of healing a normal anastomosis was filled with loosely packed and unorganized collagen fibrils, which were organized into collagen fibers after six days. Compared with normal anastomoses, rats treated with bGH showed a more organized healing, characterized by a dense structure of a new-formed collagen framework of fibrils and immature collagen fibers after four days and with bundles of new collagen fibers after six days. CONCLUSIONS: Healing colonic anastomoses are characterized by new-formed collagen fibrils at postoperative day 4, and bGH seems to stimulate structural organization of the anastomotic collagen fibrils into fibers.

Two fetal antigens (FA-1 and FA-2) and endometrial proteins (PP12 and PP14) isolated from amniotic fluid: localisation in the fetus and adult female genital tract

Monospecific antisera against two fetal antigens (FA-1 and FA-2), alphafetoprotein (AFP) and two endometrial proteins (PP12 and PP14) were used to examine the distribution of these proteins and antigens in human trophoblast and gestational endometrium in first and third trimesters of pregnancy, normal human ovary and fetal tissues by indirect immunoperoxidase histochemical localisation techniques. Fetal liver stained exclusively for FA-1 and AFP which was used as a reference protein. Staining for FA-2 was seen in fetal connective tissue, in particular the basement membrane. FA-1 and FA-2 did not stain positively in decidua, trophoblast or ovarian tissue. Gestational endometrium stained positively for PP14 exclusively in the glandular epithelium, whilst staining for PP12 was seen only in the stromal cells. Trophoblast, both early and late, and ovarian tissue did not stain positively for any of the four substances tested.

Fetal antigen 1 (FA1) in the human pancreas: cell type expression, topological and quantitative variations during development

Monospecific rabbit anti-human fetal antigen 1 (FA1), was used to examine the distribution of FA1 during the development of the human fetal pancreas and liver using an indirect immunoperoxidase technique. FA1 was expressed by 94% of the glandular epithelial cells of the branching ducts in the pancreatic anlage at week 7 of gestation. This pattern changed during the development of the human pancreas, 64% of the glandular cells being FA1 positive at week 17 of gestation, decreasing to 11% in the infant (4 months after birth). In the infant and adults the FA1 expression was restricted to a subpopulation of β-cells within the islets of Langerhans. Insulin immunoreactive cells were scattered throughout the epithelium of primitive branching pancreatic ducts at week 7 of gestation, well before the formation of islets. From the 7th through to the 17th week of gestation, FA1 was found in the cytoplasm of fetal hepatocytes, whereas no staining was observed in the liver from a 4-month-old infant. No FA1 expression was found in the epithelium of the developing gut. The present findings indicate that the glandular epithelial cells in the developing pancreas may serve as stem cells, which, if appropriately induced, may differentiate into endocrine cells. Fetal antigen 1 (FA1) may take part in or be a result of this differentiation.

Injury and repair of smaller muscular and elastic arteries. Immunohistochemical demonstration of fibronectin and fibrinogen/fibrin and their degradation products in rabbit femoral and common carotid arteries following a dilatation injury.

Indirect immunoperoxidase staining for fibrinogen/fibrin and fibronectin was performed on normal and healing arterial tissue of muscular and smaller elastic arteries. Fibronectin was observed in the wall of the normal arteries, whereas fibrinogen/fibrin could not be demonstrated. Fibronectin was observed in the intima as well as the media deposited in a similar fashion in the femoral and carotid artery during repair. Apart from the early occurrence of fibrin/fibrinogen in the media of both arteries the distribution of fibrinogen/ fibrin and degradation products differed. In the femoral artery a progressively weakening positive reaction for fibrinogen/fibrin and degradation products towards the lumen was observed in the intima and the media 7 and 14 days after the lesion. By 28 days the reaction in the media was negative. No thrombus formation was observed. In contrast, all the specimens examined from the common carotid arteries were obliterated by luminal thrombi 28 days after the lesion. The thrombus as well as the damaged intimal thickening and the compressed media were loaded with fibrinogen/fibrin and degradation products. The deposition of fibronectin, fibrinogen, and degradation products in the carotid artery was similar to that previously reported in experimental aortic arteriosclerosis in rabbits as well as in giant cell arteritis.

Placental Proteins in Peripheral Blood and Tissues of Ectopic Pregnancies

Circulating human chorionic gonadotrophin (hCG), Schwangerschaftsprotein 1 (SP-1) and pregnancy-associated plasma protein A (PAPP-A) were examined in 10 women with ectopic gestation in relation to distribution and intensity of staining for these molecules using immunohistochemical techniques applied to matched trophoblastic and decidualized endometrial tissues. All 10 women revealed strong staining for hCG and SP-1 in the syncytiotrophoblast, which was apparently unrelated to maternal levels of hCG and SP-1, levels being less than the 10th centile of the normal range in 9/10 and 7/10 women, respectively. By contrast maternal PAPP-A levels seemed to correlate with the intensity and the distribution of staining for PAPP-A. Circulating PAPP-A was undetectable (less than 5 mIU/l) in 4 patients, and below the 10th centile in the remaining 6. Using immunohistochemical techniques hCG, SP-1 and PAPP-A could not be demonstrated in any of the decidualized endometrial tissues studied.

Localization of foetal antigen 2 (FA-2) in foetal and adult human skin.

Foetal antigen 2 (FA‐2) is a connective‐tissue‐associated antigen isolated from second trimester human amniotic fluid. FA‐2 has an α‐electrophoretic mobility and is a single‐chain molecule with a molecular weight of 26 kDa as determined by polyacrylamide gel electrophoresis (PAGE). Using indirect immunofluorescence and the immunoperoxidase technique, FA‐2 was found to be in the lamina densa/ sublamina densa region of the basement membrane zone (BMZ) in adult as well as in foetal skin. FA‐2 was found throughout the dermis in foetal skin, whereas in adult skin it was found to be associated with the BMZ and around the blood vessels, hair follicles and eccrine glands. Intracellular FA‐2 antigen was demonstrated in proliferating fibroblasts by the indirect immunoperoxidase technique and immunoelectron microscopy of the fibroblasts revealed staining of the antigen in the cisternae of the rough endoplasmatic reticulum at the trans‐side of the Golgi complex as well as in vesicles close to the plasma membranes. FA‐2, a hitherto undescribed antigen associated with human BMZ, is probably being synthesized by proliferating fibroblasts.

Fetal antigen 2 (FA2) in human fetal osteoblasts, cultured osteoblasts and osteogenic osteosarcoma cells.

SummaryImmunohistochemical staining techniques used on an 11-week-old fetus showed that fetal antigen 2 (FA2) was present intracellularly in endochondral and perichondral osteoblasts, and the immunoreaction was extended into the adjacent bone matrix. Osteoclasts and chondroblasts were found to be FA2 negative. A granular perinuclear intracytoplasmic FA2 immunoreaction was found in cultured osteoblasts and osteogenic osteosarcoma cells, and immunoelectron-microscopical examination revealed a granular immunoreaction product in the rough endoplasmic reticulum. These findings indicate that FA2 is synthesized by osteoblasts and osteogenic osteosarcoma cells. A reaction of immunological identity was found between FA2 purified from second trimester amniotic fluid and serum-free supernatants of cultured osteogenic osteosarcoma cells. This shows that an antigen recognized by the anti FA2 antibody is secreted by these malignant cells. Thus, FA2 may represent a marker for altered bone metabolism, and have a potential in the classification of osteogenic osteosarcoma/ chondrosarcoma.

Repair in arterial tissue 2 years after a severe single dilatation injury : the regenerative capacity of the rabbit aortic wall : the importance of endothelium and of the state of subendothelial connective tissue to reconstitution of the intimal barrier

The thoracic aortae from 11 rabbits that survived a single severe dilatation injury for 2 years were studied by vital staining with Evans blue, immunohistochemistry and transmission electron microscopy. Our results have shown almost total restitution of the thoracic aorta. Six of the 11 rabbits submitted to an injury had no blue-stained areas, indicating total reendothelialization. Five rabbits had a few blue areas often on the ventral side of the aorta. The reendothelialization from the first to the seventh pair of intercostal arteries ranged from 82% to 100%. There was intimal thickening inside the original internal elastic lamina in both white and blue areas. All blue areas had a surface composed of smooth muscle cells. Reendothelialized areas consisted of mature endothelium, reticular basal membrane, layered smooth muscle cells and an extracellular matrix consisting of pre-elastin, elastin, collagen and proteoglycans. An effective barrier had apparently been formed against penetration of macromolecules, judged from the absence of fibrinogen/fibrin and unmasked fibronectin. Intimal thickenings without endothelial cover were covered with smooth muscle cells without intercellular junctions. Our results indicate that an extracellular matrix of fibrin and fibronectin plays a role in forming an intimal thickening, and it is suggested that proteoglycans may modulate the biological role of the extracellular matrix in the healing process.

Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A

SummaryThe influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue.The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.