High serum levels of YKL-40 in patients with systemic sclerosis are associated with pulmonary involvement.

OBJECTIVES: YKL-40, a growth factor of connective tissue cells, is elevated in sera from patients with diseases characterized by inflammation, tissue remodelling, or fibrosis. The aim of the study was to determine serum YKL-40 levels in patients with systemic sclerosis (SSc) and to explore any possible clinical and prognostic associations. METHODS: YKL-40 was measured in sera from 88 patients with SSc (26 with diffuse and 62 with limited skin involvement) and in sera from 88 matched healthy controls. Immunohistochemical staining for YKL-40 antigen was performed in a biopsy from a patient with pulmonary SSc. RESULTS: Serum YKL-40 levels of the SSc patients were significantly higher than those of the controls (p<0.00001). Patients with pulmonary fibrosis by chest X-ray, obstructive ventilatory pattern, reduced diffusing capacity (DLco), and digital joint deformity due to skin retraction had significantly higher serum YKL-40 compared with patients without these findings. Patients with elevated serum YKL-40 had shorter survival times than patients with normal serum YKL-40 (p = 0.0005), although this was not independent of age and pulmonary function. YKL-40 protein expression was found in inflammatory cells in fibrosing pulmonary tissue from a patient with SSc. CONCLUSIONS: Serum YKL-40 is elevated in patients with SSc with pulmonary involvement.

Histochemical studies of the masseter, the temporal and small zygomaticomandibular, and the temporomandibular masticatory muscles from aged male and female humans. Fiber types and myosin isoforms.

The purpose of this study was to investigate the histology of two small masticatory muscles from females and males of more than 70 years of age. By using immuno- and enzyme histochemistry the muscles were characterized by their fiber types and myosin heavy chain pattern. The observations were compared with similar studies of the masseter and temporalis muscles. Previously the two small muscles have been described based solely upon their gross anatomy. One muscle originates from the anterior, deep surface of the temporal fascia and inserts in the temporal tendon: the temporo-mandibular muscle (TM). The other muscle originates from the upper part of the temporal surface of the frontal process of the zygomatic bone and the adjacent part of the frontal bone and inserts in the temporal tendon: the zygomaticomandibular muscle (ZM). In the masseter, TM, and ZM, most of the autopsy samples contained an abundant number of fibers containing neonatal myosin heavy chains while in the temporal muscle specimens, such fibers were sparse and scattered. Electrophoresis followed by immuno-staining of Western blots supported the histochemical findings. There was no obvious correspondence between fiber typing based upon ATPase activity and the neonatal myosin heavy chain content in the muscle fibers. Neither did the fibers show accordance in their content of adult slow and fast myosin heavy chains and in their content of neonatal myosin heavy chain.

Serum YKL-40 is increased in patients with hepatic fibrosis.

BACKGROUND/AIMS: YKL-40, a mammalian member of the chitinase family, is a lectin that binds heparin and chitin. The function of YKL-40 is unknown, but it may function in tissue remodelling. The aims of this study were to assess the level of circulating YKL-40 in patients with various kinds and degree of chronic liver disease and its possible relation to liver fibrosis. METHODS: Serum YKL-40 levels were determined by radioimmunoassay in 129 patients with suspected liver disease and related to histological findings and immunohistochemical staining of YKL-40 in a liver biopsy taken simultaneously with the blood sample. RESULTS: The median serum YKL-40 was highest in patients with alcoholic cirrhosis (532 microg/l), in particular in patients with additional alcoholic hepatitis (740 microg/l). Patients with alcoholic cirrhosis, post-hepatitic cirrhosis (425 microg/l) and non-cirrhotic fibrosis (330 microg/l) had significantly higher serum YKL-40 than normal subjects (102 microg/l), patients with fatty liver (195 microg/l) or patients with viral hepatitis without fibrosis (174 microg/l). Serum YKL-40 was significantly (p<0.001) related to the degree of liver fibrosis with the highest levels in patients with moderate (466 microg/l) to severe (676 microg/l) fibrosis. Serum YKL-40 was also increased (p=0.018) in patients with slight fibrosis (270 microg/l) compared to patients without fibrosis. Immunohistochemical analysis demonstrated positive staining for YKL-40 antigen in areas with fibrosis, particularly areas with active fibrogenesis. YKL-40 staining was never found in hepatocytes. CONCLUSIONS: Our study indicates that the increased serum YKL-40 in patients with liver disease of various degree and aetiology seems to reflect fibrosis and fibrogenesis.

Aging affects different human muscles in various ways. An image analysis of the histomorphometric characteristics of fiber types in human masseter and vastus lateralis muscles from young adults and the very old.

This study is an attempt to objectively evaluate age-related changes in human muscles by use of histomorphometric methods. Aging in humans induces dramatic transformations in the skeletal muscles but little is known as to whether or not the aging processes per se may affect all muscles equally. In this study aging of two human muscles with different functions, origin and nerve supply is compared. Sections were cut from masseter and vastus lateralis muscles obtained from young adults aged 18-24 years and from the very old aged 90-102 years. Muscle fiber types were classified with the traditional myofibrillar ATPase staining. Various histomorphometric parameters of the different fiber types in human masseter and vastus lateralis muscle sections were obtained by image analyses to evaluate the age-related changes in the muscle fibers. The following variables were calculated: the number of each fiber type per photographed area; the area of each fiber and two indicators for the shape of the muscle fibers. In the aging muscles there was no relative preferential loss of a fiber type. High numbers of intermediate ATPase-stained fibers (IM fibers) were found in some old vastus muscles but were only sporadic in young vastus muscles. However, there was no change in the percentage distribution of intermediate ATPase-stained fibers when young and very old human masseter muscles were compared. Incubation of the sections with antimyosin antibodies showed that the IM fibers in old masseter and old vastus contained different myosin heavy chains. Thus ATPase activity and anti-myosin staining displayed a somewhat different pattern of fiber type distribution. The main changes in the shape and area indicated that type I fibers in the masseter became more circular while in the vastus they decreased significantly in size. The type II fibers in the vastus became very small and deviated significantly from circularity whereas the type II fibers in the masseter only exhibited a decrease in the size of the fibers. Histomorphometric measurements show that aging affects different human muscles in various ways.

The distribution of YKL-40 in osteoarthritic and normal human articular cartilage.

YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found in chondrocytes of osteoarthritic cartilage mainly in the superficial and middle zone of the cartilage rather than the deep zone. There was a tendency for high number of YKL-40 positive chondrocytes in areas of the femoral head with a considerable biomechanical load. The number of chondrocytes with a positive staining for YKL-40 was in general low in normal cartilage. The present findings, together with previous observations, suggests that YKL-40 may be of importance in cartilage remodelling/degradation of osteoarthritic joints.

YKL-40 in giant cells and macrophages from patients with giant cell arteritis.

OBJECTIVE: YKL-40, a mammalian member of the family 18 glycosyl hydrolases, is secreted by activated macrophages at a late stage of differentiation. Macrophages are present in inflammation of the arterial wall and are thought to participate in the pathogenesis of giant cell arteritis (GCA). The aim of this study was to evaluate whether macrophages and giant cells of patients with GCA produce YKL-40, and whether serum YKL-40 concentrations are elevated in these patients. METHODS: Serum YKL-40 was determined by radioimmunoassay in 19 patients with GCA and 8 patients with polymyalgia rheumatica (PMR) who were followed up prospectively during 1 year of treatment with prednisolone. Immunohistochemical staining for YKL-40 was performed in temporal artery biopsy samples that were obtained before treatment. RESULTS: In the arteritic vessels of patients with GCA, positive staining for the YKL-40 antigen was found in CD68+ giant cells and mononuclear cells located in the media. Macrophages located in the adventitia and intima were negative for YKL-40. At the time of diagnosis, patients with GCA had an increased median serum level of YKL-40 (256 microg/liter; P<0.01) compared with healthy age-matched controls (median 118 microg/liter), and the serum level of YKL-40 decreased to normal levels during prednisolone treatment (-38% after 1 month; P<0.001). Most patients with PMR had normal serum YKL-40 levels (median 158 microg/liter) and had no changes in the serum YKL-40 levels during prednisolone treatment. The observed changes in serum YKL-40 did not always parallel the changes in serum C-reactive protein levels and erythrocyte sedimentation rate during the 1-year study period. CONCLUSION: YKL-40 is found in CD68+ giant cells and mononuclear cells in the media of arteritic vessels of patients with GCA, and the concentration of serum YKL-40 may reflect the local activity of these cells in the inflamed artery.

Sirius red and acid fuchsin staining mechanisms.

The purpose of the study was to investigate the staining mechanism of acid fuchsin and Sirius red. Acid (poly-glutamic acid), neutral (poly-hydroxyproline) and basic (poly-arginine, poly-histidine, poly-lysine) poly-amino acids, collagen types I, II and III, and arginine- and lysine-containing histones were used as test substances applied to nitrocellulose membranes as dot blots. Five micrometer sections of granulation tissue on slides were tested in parallel. Some dots and sections were treated with chloramine-T before staining with acid fuchsin and Sirius red and some with 1 M NaOH after staining. The acid and neutral poly-amino acids were not stained, but the basic amino acids polylysine and poly-arginine, poly-amino acids containing these basic amino acids and the histones and the collagens exhibited intense staining. Oxidative deamination by chloramine-T abolished the staining and 1 M NaOH removed the staining except in the case of poly-arginine. Tissue sections treated in the same way showed a considerable decrease in staining after oxidative deamination with chloramine-T; in particular, the staining of the smaller fibers was abolished. The staining was totally removed by destaining with 1 M NaOH. Therefore, acid fuchsin and Sirius red are not selectively bound to collagen; they are also bound to other proteins containing basic amino acids, and staining to a large extent is influenced by electrostatic forces. The staining seems not to be selective for collagen, and one must account for this when quantitative conclusions are drawn from collagen methods using these stains.

Immunohistochemical quantification of heparan sulfate proteoglycan and collagen IV in skeletal muscle capillary basement membranes of patients with diabetic nephropathy.

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteoglycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30-300 mg/24 h) (D1), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means +/- SD): 0.477 +/- 0.082 (C), 0.627 +/- 0.031 (D0), 0.542 +/- 0.098 (D1), and 0.371 +/- 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means +/- SD): 0.836 +/- 0.111 (C), 0.838 +/- 0.300 (D0), 0.970 +/- 0.173 (D1), and 0.512 +/- 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albuminuria (r = -0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = -0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.

Plasma YKL-40: a new potential marker of fibrosis in patients with alcoholic cirrhosis?

BACKGROUND: YKL-40 (human cartilage glycoprotein-39, or 38-kDa heparin-binding glycoprotein) is a mammalian member of a protein family that includes bacterial chitinases. YKL-40 mRNA is expressed by human liver and may play a role in tissue remodelling. The aims were to assess whether circulating YKL-40 is released or extracted in the hepatosplanchnic system and to localize YKL-40 in liver tissue. METHODS: Plasma YKL-40 was determined by radioimmunoassay in 25 patients with liver diseases (alcoholic cirrhosis (n = 20), chronic active hepatitis (n = 2), cirrhosis of unknown aetiology (n = 2), and fatty liver (n = 1) and in 18 subjects with normal liver function during a haemodynamic investigation with catheterization of liver vein and the femoral artery. Immunohistochemical studies of the localization of YKL-40 in cryostal liver biopsy specimens were obtained from eight other patients with alcoholic liver disease. RESULTS: Plasma YKL-40 was significantly increased in patients with alcoholic cirrhosis (median, 523 micrograms/l; P < 0.001) compared with controls (106 micrograms/l), and plasma YKL-40 in the hepatic vein was higher (P < 0.01) than that of the artery in both the patients and controls, showing release of YKL-40 from the hepatosplanchnic area. The release rate of YKL-40 from the hepatosplanchnic area was higher in patients with liver disease than in controls (11.0 versus 2.1 micrograms/min, P < 0.05). Furthermore, the highest plasma YKL-40 levels were found in patients with a moderate or severe degree of liver fibrosis, and immunohistochemical studies showed positive staining for YKL-40 antigen in areas of the liver biopsy with fibrosis. CONCLUSIONS: The increased plasma YKL-40 in patients with alcoholic cirrhosis may reflect the remodelling of liver fibrosis.

Hyaluronan in human deciduous tooth germs in the bell stage. Histochemistry and immunohistochemistry.

The primary aim of the present study was a localization of hyaluronan (HA) in human deciduous tooth germs in the bell stage. HA was compared to the content of chondroitin sulfates (CSs). HA was detected with a biotin-labeled HA-binding protein (HABP) and CS with a monoclonal antibody. As controls, enzyme digestions were carried out. Furthermore, the glycosaminoglycans were investigated histochemically with enzyme digestions followed by alcian blue staining. The investigation showed a considerable content of HA in the stellate reticulum, although CS was also found, primarily when treatment with protease was omitted. The dental papilla contained both HA and CS, while the predentin and the dentin contained only CS. The enamel did not contain any CS, but some staining with HABP was observed along the borderline between the ameloblasts and the enamel. The significance of HA in the stellate reticulum is discussed. The importance of carrying out investigations with and without protease digestions is stressed.

Dose response profiles of human growth hormone in subcutaneous wound chambers in rats.

OBJECTIVE: To investigate dose response profiles of human growth hormone in soft connective tissue healing when it is given locally in subcutaneous wound chambers. DESIGN: Placebo controlled parallel study. SETTING: Institute of Medical Anatomy, Denmark. MATERIAL: 36 male Sprague Dawley rats, in three group of 12. INTERVENTIONS: Stainless steel wire mesh cylinders 7 mm in diameter and 20 mm long were implanted subcutaneously in pairs in the upper and lower back on either side of the midline in three groups of male Sprague Dawley rats. Two groups were each given two different doses of growth hormone (group 1, 0.2 and 0.7 IU; and group 2, 0.02 and 2 IU) in two cylinders and vehicle alone in the two cylinders on the opposite side. Group 3 were given vehicle alone in two cylinders and needle puncture (sham) on the opposite side. Injections of growth hormone or vehicle (placebo) were given every three days for 16 days. MAIN OUTCOME MEASURES: Body weight, weight of granulation tissue, and concentrations of hydroxyproline and aminoterminal propeptide of procollagen type III. RESULTS: The dose response curves for weight of granulation tissue and deposition of collagen were upward convex (ANOVA p < 0.001 and 0.001, respectively). Growth hormone in doses of 0.2 and 0.7 IU stimulated formation of granulation tissue to means of 180% (95% confidence interval (Cl) 149% to 210%) and 174% (95% Cl 148% to 200%) more than in the placebo treated cylinders (group 3) (p < 0.05 and < 0.01, respectively). Doses of 0.2 and 2 IU, however, had less effect. The placebo cylinders in animals in groups 1 and 2 contained a mean of 157% (95% Cl 137% to 177%) more granulation tissue than the cylinders in group 3, indicating that locally applied growth hormone also had a systemic effect. CONCLUSION: The clinical use of topical growth hormone in wound healing may be complicated by the relatively narrow therapeutic interval.

Effect of craniotomy and cerebrospinal fluid loss on the inner ear. An experimental study.

Craniotomy with cerebrospinal fluid (CSF) suction was performed on 18 guinea pigs to determine the effects on the inner ear morphology. Six control animals received anaesthesia only and 12 were operated on with a postoperative survival time of 1 or 24 h. The histologic examinations showed no signs of endolymphatic hydrops or injury to other structures in any of the animals. In 11 of the operated animals, red blood corpuscles were demonstrated in the perilymphatic space of the cochlea, the subarachnoid space, and the cochlear aqueduct (CA). After 1 h survival time blood had entered primarily the basal part of the scala tympani, but in the animals of 24 h survival time the blood was more abundant in both the scala tympani and the scala vestibuli indicating flow within the inner ear. The CA thus provides a pathway between the CSF and the whole of the perilymph through which noxious effects could take place.

The protein effect on determination of DNA with Hoechst 33258.

The present study was designed to afford a critical review of the effect of proteins on the Hoechst 33258 method for determination of DNA of crude homogenates. A considerable effect of proteins on the fluorescence was observed when the concentration exceeded 100 micrograms BSA equivalent protein. Below that value, practically no effect of proteins was noted. We used proteinase K to remove the proteins, but dilution of homogenates could be used as well. Moreover, we found that the concentration of the fluorochrome should be between 1 microgram and 2 micrograms when microgram levels DNA are to be determined.

Immunohistochemistry of the intercellular matrix components and the epithelio-mesenchymal junction of the human tooth germ.

The immunohistochemical localization of heparan sulphate, collagen type I, III and IV, laminin, tenascin, plasma- and cellular fibronectin was studied in tooth germs from human fetuses. The lamina basalis ameloblastica or membrana preformativa, which separates the pre-ameloblasts from the pre-dentin and dentin, contained heparan sulphate, collagen type IV, laminin and fibronectin. Enamel reacted with antifibronectin, but the reaction varied depending on the type of fibronectin and the source of antibody. In early pre-dentin, collagen type I, laminin, tenascin and fibronectin were present. In late pre-dentin and dentin collagen type I was found in intertubular dentin and in the zone between enamel and dentin. The close relationship between collagen type I in dentin and fibronectin in immature enamel is interesting, as it may contribute to the stabilization of the amelodentinal interface. In dental pulp, collagen type IV and laminin were found in the endothelial basement membranes. Collagen type I and III, tenascin and fibronectin were localized to the mesenchymal intercellular matrix. The results of this study have supported the assumption that the lamina basalis ameloblastica is a basement membrane, and have lead to the suggestion that ameloblasts are producers of fibronectin or a fibronectin-like substance.

Influence of human growth hormone on granulation tissue formation, collagen deposition, and the aminoterminal propeptide of collagen type III in wound chambers in rats.

The influence of growth hormone on granulation tissue formation was investigated in wire mesh cylinders implanted subcutaneously in rats. Two groups of 10 rats (study 1) and 1 group of 12 rats (study 2) were used for the investigation. Growth hormone, 0.02 and 0.2 IU (study 1), 0.05 and 0.2 IU (study 2), or vehicle only, was injected into the cylinders every third day for 16 days. In study 2, wound fluid was aspirated before injection of growth hormone and saved for later analysis of the aminoterminal propeptide of collagen type III. In both studies, growth hormone significantly increased the formation of granulation tissue and of total collagen content dose-dependently, whereas the relative amount of collagen was unaffected by growth hormone treatment. Wound fluid aminopropeptide increased significantly after implantation of the cylinders until day 7, before declining slightly, with no difference between the groups. We conclude that growth hormone stimulated granulation tissue formation and collagen deposition dose-dependently in the wound cylinders when injected every third day. The results suggest that growth hormone treatment does not cause excessive collagen deposition in newly formed granulation tissue.

Collagen metabolism during wound healing in rats. The aminoterminal propeptide of type III procollagen in serum and wound fluid in relation to formation of granulation tissue.

The aminoterminal propeptide of type III procollagen (PIIINP) in serum has been shown to correlate with fibrillogenesis, and thus to be a potential direct marker of type III collagen deposition. The aim of the study was to investigate the correlation between changes in serum PIIINP and formation of granulation tissue during pharmacological suppression. Granulation tissue was induced in rats by the implantation of viscose cellulose sponges. Pharmacological suppression was achieved by cyclophosphamide treatment. To distinguish between the isolated effect of cyclophosphamide and the influence of the weight loss caused by treatment, weight loss caused by starvation was investigated. In untreated rats, serum PIIINP and wound fluid PIIINP were related to formation of granulation tissue (serum: r = 0.58, p < 0.05; wound fluid: r = 0.56, p < 0.05). In rats treated with cyclophosphamide, collagen deposition and formation of granulation tissue were markedly reduced, as compared within the untreated rats (6% vs 33%, p = 0.01). Wound fluid PIIINP reflected the sparse collagen deposition (r = 0.48, p < 0.05), whereas serum PIIINP decreased (-35%, p < 0.01) and was not correlated with the formation of granulation tissue. In starved rats, with a weight loss of 8%, formation of granulation tissue, vascular density, and collagen deposition were not reduced. Wound fluid PIIINP reflected the formation of granulation tissue (r = 0.52, p < 0.05), whereas serum PIIINP remained unchanged despite normal formation of granulation tissue. Starvation of rats without implants caused a decrease in serum PIIINP (-33%(-)-48%, p < 0.01). We conclude that during cyclophosphamide treatment and after a moderate weight loss, serum PIIINP is not a valid marker of fibrillogenesis. However, in normal rats with free access to food, changes in serum PIIINP mirror fibrillogenesis. Furthermore, our study provides experimental evidence consistent with the hypothesis that wound fluid PIIINP directly mirrors the local formation of granulation tissue, independent of weight loss and cyclophosphamide treatment.

Quantitative PAS assay of some carbohydrate compounds and detergents.

A spectrophotometric method for determination of color development of glycocompounds subjected to PAS reaction was investigated with various carbohydrate compounds and related chemicals. The conditions of the oxidation with periodic acid was found to influence the amount of the colored Schiff dye produced. Mono- and di-saccharides (mannose, glucose and maltose) were PAS-negative. Glycogen was more reactive than dextran. When glycogen was hydrolyzed by amylase the intensity of the PAS product dropped until a certain limit probably reflecting the limit dextrin. The presence of proteins (albumin) or electrolytes (NaCl) did not influence the PAS reaction. Many non-ionic detergents commonly used in membrane biology such as alkyl glycosides and gluco-methyl alkanamides were strongly PAS-positive and so was the anionic detergent SDS while the zwitterionic detergents tested, such as CHAPS and CHAPSO, were PAS-negative. The color development of the spectrophotometric PAS reaction showed linearity with the concentration of a simple glycoprotein solution (peroxidase) and a complex solution (bovine serum). By the PAS reaction it was also possible to measure the content of soluble and membrane bound carbohydrate compounds in a pellet of liver cell membranes. We find that the PAS reaction is sensitive and reliable for quantitative estimations of complex carbohydrates as well as soluble and membrane-bound carbohydrate compounds. The latter should be treated with PAS-unreactive zwitterionic detergents.

Changes of soluble glycoproteins in dystrophic (dy/dy) mouse muscle shown by lectin binding.

Lectin binding sites in skeletal muscle from normal and dystrophic (dy/dy) C57 BL/6J mice were demonstrated by use of histochemistry and electrophoresis combined with electron microscopy. The following lectins were used: Canavalia ensiformis Con A, Triticum vulgaris (WGA), Glycine max (SBA), Griffonia simplicifolia (GS II), Arachis hypogaea (PNA), Pisum sativum (PSA) and Lens culinaris (LCA). After incubation of frozen sections with Con A, WGA, GS II, PSA and LCA a sarcoplasmic staining was observed in both normal and dystrophic muscle. The most consistent light microscopic observations in the dystrophic muscles were a decreased staining intensity of the sarcoplasm after incubation with Con A, WGA, PSA and LCA, but not with GS II, and a strong staining of the interfiber connective tissue. Supernatants, deprived of organelles and membranes, were prepared from normal and dystrophic muscle by high speed centrifugation. Lectin stained Western blots of the supernatant from dystrophic muscle showed two bands (120 and 67 K) with high affinities to avidin. Further this supernatant contained two glycoprotein bands (180 and 140 K) with affinities to Con A and a number of glycoprotein bands with apparent molecular weights below 67 K showing affinities to LCA and PSA. None of these glycoprotein bands could be detected in the supernatant from normal muscle. These changes of the muscle carbohydrate components might be involved in the expression of the dystrophic syndrome This seems to be the first report on changes of soluble glycoproteins in muscular dystrophy.

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures.

Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) alpha-naphthylphosphate and Fast Blue BB; (4) beta-glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and beta-glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, an capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.

Collagen distribution in developing experimentally induced granulation tissue. A morphometric study.

Viscose cellulose sponges were implanted subcutaneously on the back of full-grown Sprague-Dawley rats. Seven, 14, 21, 28, 42, 60 and 90 days after implantation, groups of 12 animals decapitated and the sponges were removed and processed for light microscopy. Five microns sections were stained with Picro-Sirius Red. Morphometry was performed on the zone of ingrowth and the collagen. The intersectional variation in the morphometrically determined collagen density within the sponges was below 20%. The hydroxyproline content was determined biochemically in 5 microns sections of sponges implanted for 14, 42, 60 and 90 days. A positive correlation (rho = 0.79, p less than 0.0001) was observed between the biochemically and morphometrically determined collagen contents. The morphometric determinations showed a steady increase in the granulation tissue ingrowth. At day 60 the ingrowth was complete. There was an increasing collagen density from days 7 and 14 through days 21 and 28, followed by a nearly steady state up to day 90 and a significantly higher collagen density peripherally than centrally in the day 42 sponges. The study has shown that morphometric collagen determination at light microscopical level using Sirius Red-stained sections may add quantitative data describing the dynamic changes in collagen content and distribution within developing granulation tissue.