Secretion of group 2 phospholipase A2 by lacrimal glands.

PURPOSE: Tears are known to have antimicrobial properties. The authors investigated the presence of the antibacterial enzyme phospholipase A2 (PLA2) in tears and lacrimal glands. METHODS: The catalytic activity of PLA2 and the amount of pancreatic group 1 PLA2 and nonpancreatic group 2 PLA2 were measured in homogenates of eight human lacrimal glands from autopsied subjects and in tears from four healthy volunteers. The localization of PLA2s in lacrimal gland sections was studied by immunohistochemistry. Skeletal muscle was used as a control. RESULTS: The catalytic activity of PLA2 was significantly higher in lacrimal glands than in skeletal muscle. Immunochemical analysis showed significantly higher amounts of group 2 PLA2 in lacrimal gland than in skeletal muscle homogenates. Group 1 PLA2 was present in trace amounts only. The concentration of group 2 PLA2 in tears was high (1451.3 micrograms/l) compared to that in the serum of healthy individuals (3.7 micrograms/l). By immunohistochemistry, a granular reaction of group 2 PLA2 was localized in the glandular cells of lacrimal glands. The apical cytoplasm of many duct cells also was labeled. CONCLUSIONS: The lacrimal glands secreted nonpancreatic group 2 PLA2, which most likely acts as an antiinfectious factor in tears.

Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands.

PURPOSE: The aim of this study was to demonstrate the synthesis and cellular distribution of group II phospholipase A2 and lysozyme in the main and accessory lacrimal glands. METHODS: The authors studied samples of normal main lacrimal glands of seven autopsied subjects and accessory lacrimal glands of eight patients who underwent ptosis surgery. The specimens were immunostained with a rabbit antiserum against group II phospholipase A2 and a monoclonal antibody against lysozyme. Expression of group II phospholipase A2 gene was shown using Northern hybridization and in situ hybridization. RESULTS: Lysozyme was present in the secretory granules of most acini, whereas group II phospholipase A2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands. Synthesis of group II phospholipase A2 in the glandular cells was confirmed by Northern hybridization and by in situ hybridization. CONCLUSIONS: There are two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II phospholipase A2 and the other synthesizing lysozyme. These enzymes are important nonspecific antibacterial factors in tears.

Severe degeneration of rabbit vastus intermedius muscle immobilized in shortened position.

The authors have previously shown that passive daily mobilization of the rabbit hind limb immobilized with the knee in extension leads to necrosis of the deep thigh muscles and myositis ossificans-like periosteal bone formation. In this study the effect of immobilization alone on the rabbit hind limb muscles was examined similarly to that of immobilized limbs. Serum creatine kinase activities increased significantly and intravenously administered Evans blue albumin showed increased vascular permeability in the deep vastus intermedius muscle even on day 1. Necrotic fibers were clearly present in the deep part of the vastus intermedius muscle on day 5 in light and electron microscopy and in enzyme histochemistry. Fibrosis and atrophy were found later. The superficial portion of the vastus intermedius and the deep contralateral nonimmobilized vastus intermedius showed degenerative changes. Bone formation was not noted. The conclusion was that the deep vastus intermedius muscle composed almost exclusively of type I fibers is exceptionally prone to damage when immobilized in a shortened position. Contact of the necrotic muscle with the underlying periosteum is not alone sufficient to induce heterotopic ossification. The additional trauma caused by daily mobilization is needed for the myositis ossificans-like bone formation.

Macrophages in trauma-induced myositis ossificans.

The basic cellular mechanisms of different forms of myositis ossificans are poorly known. In the current experiment the nature of the early (24-168 h) inflammatory cell reaction preceding trauma-induced myositis ossificans was studied. New bone formation was induced in the vastus intermedius region of the rabbit quadriceps muscle by means of immobilization and daily passive mobilization. Before the start of treatment, a cell harvesting device (viscose cellulose sponge in a silastic tube) was implanted in the region of interest. The opposite intermedius muscle and a standardized surgical skin wound served as the control sites. The results showed a significantly prolonged invasion of macrophages into the ossifying intermedius muscle as compared with the control intermedius muscle. It is hypothesized that microinjury and subsequent muscle necrosis cause the invasion of macrophages, and these cells respond to the conditions of the traumatized muscle under passive mobilization by releasing osteogenic growth factors.

Bone formation in experimental myositis ossificans. Light and electron microscopy study.

The development of ectopic ossification in experimental myositis ossificans of the rabbit thigh was studied. The right hind limb of 25 rabbits was immobilized with the knee in extension. Once a day the limb was passively mobilized for 2-3 minutes. The animals were killed 3, 5, 7, 14, 21, 28 and 35 days after the beginning of the experiment. Specimens for light and electron microscopy were obtained from both hind limbs. Extensive necrosis and fibrosis were observed in the right vastus intermedius muscle during the first week. Proliferation of chondrocytes and osteoblasts with newly formed woven bone and cartilage formation were found in the periosteum within 7 and 14 days after the beginning of the experiment. Intensive enchondral ossification and hard calcified tissue were observed later. It was evident that bone formation in this experimental model started in the periosteum after necrosis of the adjacent skeletal muscle. Therefore the temporal and spatial relationship of traumatic changes in the periosteum and muscle seems important for the development of myositis ossificans.

Magnetic resonance imaging of experimental renal hemorrhage.

Experimental renal hemorrhage was induced by injecting autologous blood into the left kidney of 13 rats. To investigate the magnetic resonance (MR) characteristics of acute renal hemorrhage and subsequent stages of resolution, repetitive MR images were obtained using a 0.35 Tesla imager during a period of 21 days postinduction. A dual spin-echo imaging (TR 500 and 2,000 msec, TE 28 and 56 msec) was used to calculate the relaxation times and record the intensities in the renal medulla and cortex. Histologic examination (n = 9) indicated that blood was dispersed intrarenally, and no encapsulated hematoma developed. The signal intensity on the T1- and T2-weighted images, as well as the relaxation times in the hemorrhagic renal parenchyma were unchanged during 21 days when compared with intact kidney values. Subcapsular fresh blood had a high signal intensity on T2-weighted images. A marked overlap of the relaxation parameters between intact kidney parenchyma and diffuse intrarenal hemorrhage was observed. Detection of dispersed intrarenal blood using spin echo MR imaging may be difficult.

Expression of the 43 kDa papain inhibitor during human fetal skin development.

The presence of the 43 kDa papain inhibitor protein in the skin of 40 fetuses with the gestational age varying from 9 to 40 weeks was studied. Immunohistochemistry showed the first evidence of the 43 kDa papain inhibitor in the acral parts, mostly in the nail bed, at the 14th week when the epidermis in all other sites was not stained. Staining of the follicular infundibulum as well as some parts of the uppermost epidermis and keratin layer was observed from 17 to 40 weeks. The staining was most intensive at 20-30 weeks gestational age, and later the intensity gradually decreased so that in the full-term newborn the 43 kDa papain inhibitor was hardly detectable or absent. It was concluded that the 43 kDa papain inhibitor is present in fetal skin at the stage of stratification (9-14 weeks) only in the nail region and its volar surroundings. It appears at the stage of interfollicular keratinization (14-24 weeks) in the uppermost epidermis, especially in the appendical openings and its amount seems the decrease during the stage of interfollicular keratinization (24 weeks--full-term). The expression of 43 kDa inhibitor protein is temporally related to that of filaggrin.

Collagens in neurofibromas and neurofibroma cell cultures.

Neurofibromas contain approximately 30-50% collagen of their lipid-free dry weight, which is about half of the value of skin but approximately twice that described for peripheral nerve endoneurium. Immunohistochemical stainings indicate that neurofibromas contain types I, III, IV, and V collagens and fibronectin. Most of the neurofibroma cells are type IV collagen and S-100 protein positive, which provides immunohistochemical evidence that neurofibromas are mostly composed of Schwann cell-like cells. The proteoglycan/collagen ratio is 4 to 10 times higher in the neurofibromas than in the surrounding dermal tissue. This would explain the typical soft consistency of the neurofibromas and may contribute to a favorable milieu for tumor growth. Pure fibroblastic cell cultures are obtained from neurofibromas after repeated passages. The cultured cells synthesized type I and III collagens and fibronectin, indicating that these cells are important in the production of the fibrous connective tissue proteins in neurofibromas.

Pancreatic phospholipase A2 in proximal tubules of rat kidney in experimental acute pancreatitis and after intravenous injection of the enzyme.

Kinetics and distribution of i.v. human pancreatic phospholipase A2 (h-PLA2) were determined in intact and nephrectomized rats, and tissue localization of rat pancreatic PLA2 (r-PLA2) was studied by immunohistochemistry in experimental acute pancreatitis. The concentration of h-PLA2 and the catalytic activity of phospholipase A2 in plasma decreased exponentially in intact and nephrectomized animals after the injection. The initial 15-min half-life was considerably longer in nephrectomized animals, and higher h-PLA2 concentrations and PLA2 catalytic activities were found in plasma. h-PLA2 was localized in endocytotic vesicles and apical cytoplasmic vacuoles in proximal tubule cells of the kidney. The intensity of the immunoreaction decreased considerably between 15 and 50 min in these cells. No signs of tubular damage were seen by light microscopy. Neither immunoreactive h-PLA2 nor PLA2 catalytic activity was found in urine. r-PLA2 was observed in proximal tubule cells 15 min after an injection of sodium taurocholate (necrotizing pancreatitis group) or saline (edematous pancreatitis group) into the pancreatic duct. Signs of tubular damage were present in necrotizing pancreatitis, but tubular morphology was normal in the animals with edematous pancreatitis. We conclude that the proximal tubule cells of the kidney participate in the metabolism of circulating pancreatic PLA2, and considerably higher PLA2 levels persist in plasma in nephrectomized animals. Endogenous pancreatic PLA2 is detected in kidneys in acute pancreatitis.

Ultrastructure of human osteosarcoma. Malignant transformation of a multipotential connective tissue cell.

Ten cases of osteosarcoma were studied by electron microscopy. The tumors consisted of six cell types: fibroblastic, myofibroblastic, chondroblastic, osteoblastic, unclassified and histiocytic cells. Disturbed structure of dilated endoplasmic reticulum was a common feature. The neoplastic character of myofibroblastic and histiocytic cells is controversial. Myofibroblastic differentiation was most abundant in parosteal osteosarcoma and in fibrosarcomatous intraosseal osteosarcoma. The malignant cells sometimes formed giant cells and many aggregates of these cells were seen. Osteoclasts and other reactive cells were encountered and this may indicate host reaction against the tumor cells. Formation of collagenous and cartilaginous ground substance was poor, and the capacity of collagen to mineralize was decreased. It is concluded that osteosarcoma is the malignancy of a multipotential connective tissue cell which forms callus in normal osteogenesis.

Group II phospholipase A2 in nasal fluid, mucosa and paranasal sinuses.

The aim of this study was to determine the type of phospholipase A2 (PLA2) in nasal fluid and to demonstrate its cellular origin. The concentration of group II PLA2 was high (591.5 micrograms/l) in nasal fluid compared with serum level (10.8 micrograms/l) and the fluid of paranasal sinuses (10.6 micrograms/l). Methacholine stimulated nasal fluid contained only small amounts (19.1 micrograms/l) of group II PLA2 when the flow of tear fluid through the nasolacrimal duct was obstructed. Occasional glands secreting group II PLA2 were found in nasal and paranasal mucosa by immunohistochemistry. Lysozyme was found in the majority of mucosal glands. It was concluded that nasal and paranasal mucosal glands contain group II PLA2. In nasal fluid, however, PLA2 is mainly derived from tear fluid.

Lacrimal bone thickness at the lacrimal sac fossa.

BACKGROUND AND OBJECTIVE: Because laser dacryocystorhinostomy techniques have become more popular during the past few years, interest has grown concerning the anatomic structures that need to be penetrated in these procedures. The authors therefore studied the thickness and the histologic type of the lacrimal bone at the lacrimal sac fossa. PATIENTS AND METHODS: The thickness of 69 lacrimal bones at the lacrimal sac fossa from 48 patients was measured. RESULTS: The mean thickness was 106 microns. In 67% of the patients the mean thickness of individual lacrimal bone was less than 100 microns and in 4% it was more than 300 microns. The thinnest measured cross section of the lacrimal bone sample was 11 microns and the thickest was 722 microns. The lacrimal bone was composed of a thin plate of lamellar bone. CONCLUSION: In most cases the lacrimal bone at the lacrimal sac fossa is so thin that it can be easily penetrated with most surgical instruments.

Solid facial edema as a complication of acne vulgaris: treatment with isotretinoin and clofazimine.

We present two patients, a 20-year-old female and an 18-year-old male, who suffered from persistent solid facial edema as a complication of acne vulgaris. They were treated with isotretinoin with moderate response and thereafter with lymph massage with further response. The female patient also received clofazimine with good response.

Experimental pancreatitis in the rat. Ultrastructure of sodium taurocholate-induced pancreatic lesions.

Necrotic lesions caused by the intraductal injection of sodium taurocholate in rat pancreas were studied by electron microscopy. The early (1 and 15 min after the injection) ductal cells were often necrotic. The acinar cell damage was characterized by mitochondrial swelling, vesiculation of the granular endoplasmic reticulum, and dissolution of cellular membranes. At later time intervals (1, 3, and 6 h) there were necrotic acinar cells with heterogeneous cytoplasmic inclusions representing degenerative cell organelles and their remnants. At 6, 12, and 24 h there were autophagic vacuoles in sublethally damaged acinar cells. The zymogen granules seemed relatively well preserved throughout the experiment. The vascular lesions consisted of endothelial detachment, extravasation of erythrocytes, thrombosis, and poikilocytosis. It was concluded that the initial cell injury was caused by the detergent action of the injected bile salt.

Standards of morphological evaluation and histological grading in experimental acute pancreatitis.

Acute pancreatitis is characterized morphologically by edema, hemorrhages, parenchymal necrosis and fat necrosis. The inflammation is accompanied by infiltration of polymorphonuclear leukocytes. According to the absence or presence of necrosis the disease can be divided into interstitial (or edematous) pancreatitis and hemorrhagic-necrotizing pancreatitis. The severity of disease can be graded in the histological sections either by giving scores to the different types of morphological alterations or by determining the proportion of necrotic tissue of the total lobular parenchyma. The former method is based on subjective assessment of histological slides and is suitable for the evaluation of both edematous and necrotizing pancreatitis. Histometric measurement of necrotic parenchyma can be used only in the necrotizing forms of experimental pancreatitis, e.g. in those induced by intraductal injection of bile, bile salts or digestive enzymes, and in the dietary ethionine-induced pancreatitis. Grading of the tissue damage is essential when the effects of different therapies are evaluated.

Annulo-aortic ectasia. Light and electron microscopic changes in aortic media.

The aortic wall of the human ascending aorta from 44 patients operated on for annulo-aortic ectasia (AAE) was studied. Light microscopy revealed significantly greater cystic change, elastic fragmentation, fibrosis and disappearance of smooth muscle cells in aortic media in AAE than in control specimens taken at autopsy. Occasional aortae, however, were morphologically almost normal. Eight of the patients had Marfan's syndrome. No significant differences were observed between them and the other 36 patients, except for a tendency to have less pronounced fibrosis. There were 9 patients who, in addition to the changes mentioned, had advanced atherosclerosis, and their aortae showed more extensive fibrosis and medial necrosis. Pooling of proteoglycan matrix, degeneration of elastic lamellae, increased amount of collagen and necrosis of smooth muscle cells characterized the electron microscopic findings of 13 patients. The collagen fibers seemed to be of normal shape. In conclusion, changes in annulo-aortic ectasia are characterized by severe cystic medial necrosis. The changes are basically similar in Marfan and non-Marfan patients.

Experimental pancreatitis in the rat. Light and electron microscopical observations on early pancreatic lesions induced by intraductal injection of trypsin, phospholipase A2, lysolecithin and non-ionic detergent.

Trypsin, phospholipase A2, lysolecithin or non-ionic detergent polyoxyethylene p-t-octyl phenol solutions were injected into the rat biliopancreatic duct. Histological and ultrastructural changes in the gland were studied 15 min and 3 h after the injections. The rough surfaced endoplasmic reticulum disintegrated in two ways: (1) the endoplasmic reticulum in the cell periphery was vesiculated but ribosomes were well preserved at 15 min, and (2) large, round membranous structures appeared in apical cytoplasm at 3 h. Zymogen granules disintegrated in the second type, which possibly represents autodigestion. Both types of injury lead ultimately to structureless necrosis. Lesions induced by phospholipase A2 and lysolecithin were identical. Trypsin-induced damage developed slowly and the two phases of endoplasmic reticulum disintegration were not sharply separable. Lesions caused by polyoxyethylene p-t-octyl phenol were variable at 15 min, but at 3 h the type 2 injury described above was observed. It was concluded that although the initial damage in pancreatic acinar cells may vary, necrotic changes are similar despite the injected material at the later time interval. During acute pancreatitis, the acinar cell necrosis is most probably due to the action of lysolecithin produced by the activation of phospholipase A2.

Experimental pancreatitis in the rat. Sodium taurocholate-induced acute haemorrhagic pancreatitis.

Sodium taurocholate injected into the pancreatic duct system of the rat caused acute haemorrhagic pancreatitis. The pancreatic lesions were immediate and characterized by interstitial oedema, extensive necrotic changes of the acinar cells, and haemorrhages during the first 24 h after the injection. In animals surviving 72 h there were marked acinar atrophy and pancreatic fibrosis. The mortality increased according to the amount of sodium taurocholate injected. Except for necrosis of occasional liver cells, other organs examined were histologically normal. This investigation created an experimental model for studying the pathogenesis of acute pancreatitis. The results support the hypothesis that bile can initiate acute haemorrhagic pancreatitis.

Cholinergic hypothesis of alcoholic pancreatitis.

Chronic alcohol intake interferes especially with the two main pathways regulating exocrine pancreatic secretion: the cholinergic and the pancreozymin pathway. Recently, a new theory of the pathogenesis of alcoholic pancreatitis was proposed emphasizing disordered agonist-receptor interaction at the level of pancreatic acinar cells. Accordingly, alcohol-induced alterations in the control of exocrine pancreatic secretion result in hyperstimulation of pancreatic acinar cells and their muscarinic receptors, mimicking the mechanism of acute pancreatitis caused by scorpion sting, intoxication with an anti-acetylcholinesterase-containing insecticide or supramaximal doses of secretagogues. The present review emphasizes the role of these alcohol-induced secretory alterations in the pathogenesis of alcoholic pancreatitis.