Immunohistochemical demonstration of age-related deposition of vitronectin (S-protein of complement) and terminal complement complex on dermal elastic fibers.

Immunoreactivity of vitronectin was investigated in 100 skin specimens from different body regions in 87 individuals of different ages using monoclonal and polyclonal anti-vitronectin antibodies in an avidin-biotin-peroxidase complex technique. Vitronectin immunoreactivity was found in conjunction with dermal elastic fibers in all subjects older than 13 years. No vitronectin immunostaining was detected in subjects younger than six years, suggesting deposition of vitronectin during late childhood or early adolescence. Using an immunogold staining procedure, vitronectin immunoreactivity was ultrastructurally localized to the periphery of elastic fibers. The blood level of vitronectin in 20 healthy newborns was 67% of the adult level, suggesting active biosynthesis already in the fetus. To investigate whether vitronectin is deposited as part of the SC5b-9 complex or as uncomplexed protein, the immunoreactivity of vitronectin was compared with that of C9, using monoclonal and polyclonal antibodies against the C9 neoantigen. Distinct C9 neoantigen immunoreactivity was demonstrated in association with dermal elastic fibers in human skin in adults but only in subjects older than 30 years. The intensity of C9 neoantigen immunoreactivity appeared to increase with age and was found to be stronger in sun-exposed skin than in sun-protected skin. These findings indicate that uncomplexed vitronectin is deposited during childhood or early adolescence and that terminal complement complexes (C5b-9 and/or SC5b-9) are deposited on elastic fibers later on in life. Hypothetically, the tissue form of vitronectin may be involved in the prevention of tissue damage in proximity to local complement activation. In addition, it may be physiologically important as substratum for cells, stimulating cell migration and anchorage.

Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis.

Fibrillin, a 350-kD glycoprotein, was recently localized to elastin-associated 10 nm microfibrils. Here, the distribution of fibrillin immunoreactivity was determined in normal skin in individuals of different ages and in lesions of solar elastosis or anetoderma. It was compared with the distribution of orcein-stainable fibers and with the immunoreactivities of vitronectin and amyloid P component. These glycoproteins are known to occur in conjunction with the orcein-stainable elastic fibers in adults, but not in the young. Fibrillin immunoreactivity was associated with orcein-stainable fibers in normal skin of both adults and the young. In addition, the fibrillin immunoreactive fiber network comprised fine fibers that were unstainable by orcein, anti-vitronectin, or anti-amyloid P component. Such fine fibers were especially abundant close to the dermal-epidermal junction zone. Immunoreactivities of anti-vitronectin and anti-amyloid P component were not always associated with fibrillin immunoreactivity but were consistently found to co-localize with orcein-stainable fibers in adults. This suggests vitronectin and amyloid P component to be associated with the amorphous elastin rather than with the microfibrils, although alternative interpretations are possible. In elastotic lesions, fibrillin immunoreactivity was generally fainter than that obtained using anti-vitronectin or anti-amyloid P component. In contrast, an extensive network of dermal fibers stained by anti-fibrillin, but not by anti-amyloid P component, anti-vitronectin, or orcein, was seen in an anetoderma lesion. In conclusion, fibrillin immunoreactivity is associated with a unique dermal network, which ultrastructurally is composed of microfibrils. These fibers are proposed to have an important structural and functional role in anchoring the dermal elastic fibers in the extracellular matrix and to the lamina densa.

gamma-trace in human pituitary adenomas.

gamma-Trace, a small protein occurring in body fluids and in secretory and neuroendocrine cells, was demonstrated by immunohistochemical techniques in the cytoplasm of the tumor cells of 13 pituitary adenomas obtained at surgery and autopsy. Seven of the adenomas also contained LH immunoreactivity. FSH, TSH, and ACTH were each found in one gamma-trace-containing adenoma. gamma-Trace was also demonstrated in extracts of 1 pituitary adenoma and of 5 nontumorous adenohypophyses. The immunoreactive protein found in the extracts had a molecular weight and electrophoretic mobility characteristic of gamma-trace. Computerized amino acid sequence comparisons between the primary structure of gamma-trace and those of known hormonal peptides showed no significant similarities.

The cerebrospinal fluid and plasma concentrations of gamma-trace and beta2-microglobulin at various ages and in neurological disorders.

The concentrations of gamma-trace and beta2-microglobulin in cerebrospinal fluid (CSF) and plasma were determined in 64 individuals of various ages without signs of organic disorder in the central nervous system (CNS). A strong connection was found between the CSF level of gamma-trace and the age of the individual, with the CSF level of newborns being 3--4 times that of adults. A similar, but less marked, connection was found for the CSF level of beta2-microglobulin and the age of the individual. The plasma levels of the two proteins also varied with the age of the individual, but the variations were not as great as those of the CSF levels. The results strongly emphasize the necessity of using age-matched reference values when CSF and plasma levels of the proteins are to be evaluated in different groups of patients. Thirteen children and 98 adults with various neurological disorders were also examined. Significantly increased CSF levels of gamma-trace and beta2-microglobulin as well as increased plasma concentration of gamma-trace and CSF/plasma gradient of beta2-microglobulin were found in infectious disorders. Increased gamma-trace concentration in plasma and beta2-microglobulin concentration in CSF were seen in cerebrovascular disorders. The mechanisms which regulate the turnover of proteins in CSF are discussed.

Human gamma-trace. Structure, function and clinical use of concentration measurements.

The discovery, tissue distribution, concentration in extracellular fluids and structure of human gamma-trace are reported. The use of determinations of the cerebrospinal fluid concentration of gamma-trace in the diagnosis of hereditary cerebral hemorrhage with gamma-trace-amyloidosis is described. The physiological function of gamma-trace as a cysteine proteinase inhibitor is accounted for an it is suggested that the six trivial names used for gamma-trace so far are replaced by the functional designation cystatin C.

Human gamma-trace, a basic microprotein: amino acid sequence and presence in the adenohypophysis.

The amino acid sequence of human gamma-trace, a basic microprotein without known function, was determined by automated Edman degradations of the carboxymethylated polypeptide chain and of fragments obtained by cyanogen bromide treatment and tryptic digestion after blocking of lysine residues. The single polypeptide chain contained 120 residues, and the calculated Mr was 13,260. A proline residue at position 3 was partly hydroxylated. The presence of gamma-trace in a significant proportion of the cells in the anterior lobe of simian and human pituitary glands was demonstrated by immunohistochemical procedures with a rabbit antiserum against human gamma-trace. The tissue localization and amino acid sequence of gamma-trace indicated that this protein is connected with the peptidergic gastroenteropancreatic neuroendocrine system.

Quantitation of gamma-trace in human biological fluids: indications for production in the central nervous system.

Gamma-Trace was purified in large amounts from urine and used for the production of a specific rabbit antiserum. An enzyme immunoassay for quantitation of gamma-trace was developed using the pure protein as a primary standard. Its sensitivity was approximately 30 microgram/l. An enzyme amplified single radial immunodiffusion was developed as well. Its sensitivity was approximately 0.3 mg/l. These assays allowed quantitation of gamma-trace in normal human biological fluids. The following results were obtained (mean +/- SD): cerebrospinal fluid: 5.8 +/- 2.2 mg/l, plasma: 1.1 +/- 0.42 mg/l, saliva: 1.8 +/- 0.88 mg/l and urine: 0.095 +/- 0.057 mg/l. Plasma samples from patients with advanced renal failure revealed gamma-trace values up to 13 times the normal mean plasma value. The results indicate a production of gamma-trace in the central nervous system and that the protein is primarily catabolized by the kidney.

Localization of vitronectin (S-protein of complement) in normal human skin.

Vitronectin, now known to be identical to serum spreading factor and to S-protein of complement, is a multifunctional glycoprotein involved in the adhesion and spreading of cells and in the complement and coagulation pathways. The distribution of vitronectin in normal human skin was investigated with immunofluorescence using polyclonal antibodies, and with an avidin-biotin peroxidase complex technique, using polyclonal as well as monoclonal antibodies. Vitronectin immunreactivity was found to be localized on the elastic fibres in the dermis. Both the thin fibres in the papillary dermis and the thicker elastic fibres in the reticular dermis were stained. No crossreactivity was found between vitronectin and serum amyloid P component, known to bind to elastic fibres. The two proteins were immunohistochemically localized to the same structures in the skin. The distribution of vitronectin in the dermal tissue established in this study provides a basis for further studies of the function and behaviour of vitronectin in health and disease.

Immunohistochemical studies on vitronectin in elastic tissue disorders, cutaneous amyloidosis, lichen ruber planus and porphyria.

Vitronectin, identical with serum-spreading factor and S-protein of complement, is a glycoprotein present in both plasma and tissue. It stimulates cell adhesion and spreading and affects the complement and coagulation pathways. Vitronectin immunoreactivity was recently found in conjunction with dermal and renal elastic fibres, in renal amyloid deposits in cases of AL- and AA-amyloidosis, and in sclerotic glomerular lesions. Skin specimens from lesions of patients with selected skin diseases were investigated with an avidin-biotin peroxidase technique using both monoclonal and polyclonal anti-vitronectin antibodies and an alkaline phosphatase anti-alkaline phosphatase technique using monoclonal anti-vitronectin antibodies. Vitronectin immunoreactivity was found in association with the abnormal elastic tissue in solar elastosis and pseudoxanthoma elasticum. It was also found in conjunction with dermal amyloid deposits in primary localized cutaneous amyloidosis and in Civatte bodies in cases of lichen ruber planus. In cases of erythropoietic protoporphyria and porphyria cutanea tarda, hyaline perivascular deposits also demonstrated positive vitronectin immunoreactivity. The presence of vitronectin immunoreactivity not only in normal and degenerated elastic fibres but also in various pathological tissue deposits suggests that vitronectin occurs both in elastic fibres and in different types of abnormal protein deposits.

Immunohistochemical demonstration of vitronectin in association with elastin and amyloid deposits in human kidney.

The multifunctional glycoprotein vitronectin, also called serum spreading factor and S-protein of complement, is a potent inducer of cell adhesion and spreading in vitro, and also has a regulatory function in the complement and coagulation pathways. It is present both in plasma and tissue. Recently, vitronectin immunoreactivity was demonstrated in the elastic fibres of normal human skin. Normal and amyloid kidney tissue was investigated for vitronectin immunoreactivity using polyclonal and monoclonal antibodies in an avidin-biotin-peroxidase complex technique and in an alkaline phosphatase anti-alkaline phosphatase complex technique. Vitronectin was found in the elastic layers of normal vessel walls, and in glomerular sclerotic lesions in cases of benign nephrosclerosis, but not in normal glomeruli. Strong specific vitronectin immunoreactivity was found in the amyloid deposits in kidneys from cases with amyloid A type amyloidosis, and in cases with amyloid light chain type amyloidosis. Structures immunostainable with anti-amyloid A antiserum were invariably immunostainable with anti-vitronectin. An antiserum against serum amyloid P component stained the same structures as did the anti-vitronectin antibodies, and in addition stained normal glomerular basement membranes. In conclusion, vitronectin immunoreactivity was demonstrated in elastic tissue, in amyloid deposits and in sclerotic lesions in human kidney.

Vitronectin colocalizes with Ig deposits and C9 neoantigen in discoid lupus erythematosus and dermatitis herpetiformis, but not in bullous pemphigoid.

C9 neoantigen immunoreactivity has been found to colocalize with C3 immunoreactivity at the dermal-epidermal junction zone (DEZ) in skin specimens from patients with bullous pemphigoid, lupus erythematosus and dermatitis herpetiformis. The present study was designed to elucidate whether the C9 neoantigen immunoreactivity represents deposition of membrane attack complexes or non-lytic SC5b-9 complexes. Skin specimens from 11 patients with pemphigoid, five patients with discoid lupus erythematosus and from nine patients with dermatitis herpetiformis were studied with immunofluorescence using both monoclonal and polyclonal antibodies against C9 neoantigen and against vitronectin (S-protein), an inhibitor to the membrane attack complex of complement. Specimens from the pemphigoid patients demonstrated C9 neoantigen reactivity along the DEZ without detectable colocalized vitronectin. This suggests deposition of membrane attack complexes in the pemphigoid lesions. Immunoreactivity of both C9 neoantigen and vitronectin was detected in the DEZ in specimens of discoid lupus erythematosus and in the tips of dermal papillae in specimens of dermatitis herpetiformis. The combined presence of C9 neoantigen- and vitronectin immunoreactivity may indicate deposition of C9 as part of the non-lytic SC5b-9 complex. The finding reported here of differential deposition of vitronectin and C9 in different diseases indicates that the presence of C9 neoantigen immunoreactivity in tissue per se does not represent the deposition of membrane attack complexes, but that it may also be C9 deposited as part of the nonlytic SC5b-9 complex.

The gamma-trace concentration of normal human seminal plasma is thirty-six times that of normal human blood plasma.

Fresh human seminal plasma was demonstrated to contain a basic microprotein with the same size, electrophoretic mobility, isoelectric point and immunochemical properties as isolated human gamma-trace. The concentration of gamma-trace in 24 normal seminal plasma samples was found to be (mean +/- SD): 51 +/- 8.1 mg/l which is 36 times higher than the normal human blood plasma concentration of gamma-trace.

IgM deposition in skin biopsies from patients with primary biliary cirrhosis.

Immunofluorescence studies on skin biopsies from 14 patients with primary biliary cirrhosis (PBC) showed granular papillary deposition of IgM in all. In addition, 6 patients had C3 deposition. Control patients with various other liver diseases, idiopathic high plasma levels of igM and extrahepatic cholestasis were only sporadically positive for IgM and not at all for C3. IgM deposition in dermal papillae in PBC does not merely reflect high plasma IgM levels or cholestasis but probably represents an immunochemically abnormal IgM population.

Fixed drug eruption: an immunohistochemical investigation of the acute and healing phase.

Five patients were each challenged orally with a drug which had previously induced a fixed drug eruption. A positive reaction occurred in all the patients. Punch biopsies were taken 6-12 h, 24 h and 3 weeks after challenge. The specimens were tested with different mouse anti-human monoclonal antibodies to identify T lymphocytes and phenotypic subsets, natural killer cells, B lymphocytes, OKT-6 and HLA-DR-positive cells. T suppressor/cytotoxic cells seemed to play a major role in initiating the flare-up reaction and preserving the cutaneous memory function of the fixed drug eruption.

Production, characterization and use of monoclonal antibodies against the major extracellular human cysteine proteinase inhibitors cystatin C and kininogen.

Murine monoclonal antibodies against the major cysteine proteinase inhibitors of human biological fluids, cystatin C and kininogen, were produced. The cystatin C antibody, HCC3, with a Ka of 2 x 10(7) l/mol, increased the inhibition of papain by cystatin C and was suitable for use in immunoblotting, immunohistochemistry and in the construction of a sensitive sandwich enzyme immunoassay for quantification of cystatin C. It recognized not only free cystatin C but also cystatin C in complexes with cysteine proteinases. The kininogen antibody, HK4, was directed against the third, cysteine proteinase inhibitory domain of the heavy chain of kininogen (Ka = 1 X 10(7) l/mol), but did not influence the papain inhibitory activity of kininogen. It reacted with free kininogen as well as kininogen in complex with cysteine proteinases. Both antibodies could be used for the production of specific immunosorbents.

Renal arteriovenous shunting in rejecting allograft, hydronephrosis, or haemorrhagic hypotension in the rat.

We studied the occurrence of arteriovenous (A-V) shunting in three experimental rat models, namely in rejecting allograft kidney, in uni- or bilateral ureteral obstruction, and in haemorrhagic hypotension. Isografted or sham-operated rats served as controls. Radiolabelled microspheres were injected into the renal artery and the increase in the amount of radioactivity in the lungs was considered to reflect A-V shunting in the kidney. In animals exposed to haemorrhage, with a blood pressure not less than 70% of the initial blood pressure, practically no shunting was seen. When animals were bled to a hypotension beyond the autoregulation, A-V shunting occurred inversely correlated to the degree of hypotension. In ureteral obstruction, a less marked but significant increase in shunting of microspheres to the lungs was found after 24 h of unilateral obstruction, irrespective of whether the spheres were injected into the obstructed or the contralateral kidney. Significant A-V shunting during the allograft rejection process was also demonstrated. Histologically, microspheres were found in afferent arterioles less frequently in kidneys with A-V shunting than in controls. These results indicate that A-V shunting is involved in haemorrhagic hypotension, renal graft rejection, and hydronephrosis. In the latter situation A-V shunting is probably regulated by a humoral factor.

Demonstration of gamma-trace in normal endocrine cells of the adrenal medulla and in phaeochromocytoma. An immunohistochemical study in monkey, dog and man.

gamma-Trace was demonstrated by the immunohistochemical peroxidase/anti-peroxidase technique to occur in normal chromaffin cells of the adrenal medulla from African green monkey, capuchin monkey and beagle dog and in the cells of a norepinephrine-producing human phaeochromocytoma. The presence of gamma-trace in the endocrine epithelial cells of the adrenal medulla suggests that gamma-trace might be related to the neuroendocrine system.

Demonstration and classification of amyloidosis in needle biopsies of the kidneys, with special reference to amyloidosis of the AA-type.

To examine whether sequence-specific antibodies directed against serum amyloid A were useful in the demonstration and classification of amyloidosis, needle biopsy specimens from the kidneys of 152 cases with renal disorders were investigated using the avidin-biotin-peroxidase complex technique of immunohistochemistry. A distinct immunoreactivity of protein AA was seen in biopsies from all 42 individuals who were clinically classified as having the AA-type of amyloidosis. The stained areas coincided with deposits stained by Congo red. Four of these cases demonstrated immunoreactivity of both protein AA and light immunoglobulin chains and all biopsies except one showed immunoreactivity for the amyloid P-component. After treatment with potassium permanganate, the amyloid deposits in the biopsies of all 42 cases lost their affinity for Congo red. Ten patients with clinical and laboratory findings compatible with the AL-type of amyloidosis were also investigated. All their biopsies demonstrated Congophilic amyloid deposits but none of them showed any immunoreactivity of protein AA. Amyloid deposits of lambda light immunoglobulin chains-but not kappa-were demonstrated in biopsies from four patients. The amyloid P-component was found in biopsies from six individuals and positive Congo red staining after treatment with potassium permanganate was seen in biopsies from four of the cases. Biopsies of 100 patients suffering from non-amyloid renal disorders were also examined. None of them displayed any immunoreactive deposits of protein AA. The investigation shows that amyloid deposits of the AA-type can be identified in needle biopsies when sequence-specific antibodies against serum amyloid A are used in the avidin-biotin-peroxidase complex technique. Both the diagnostic sensitivity (42 of 42) and specificity (110 of 110) of the assay were optimal (1.0). The method was found to be superior to other investigated techniques and useful for classifying amyloidosis in formalin-fixed renal biopsies.

Linear IgA dermatosis: a study of ten adult patients.

Ten adult patients with homogeneous-linear deposition of IgA along the basement membrane zone have been studied. The direct immunofluorescence IgA pattern was stable, and there was no deposition of IgG or IgM. The clinical presentations were heterogeneous and resembled dermatitis herpetiformis (DH) (3 cases) or bullous pemphigoid (5 cases). Two patients had widespread gyrate blistering lesions of acute onset. Pruritus was constantly present. The course of the disease was chronic except for one patient who had a spontaneous remission after 5 years. The histology was indistinguishable from "classical" DH with granular IgA in dermal papillae. The patients studied in the present investigation did not show the high incidence of atrophic intestinal villi found in "classical" DH. Five of 9 cases carried the haplotype HLA-A1, B8, DR3. In spite of a close similarity between linear IgA dermatosis and DH, differences exist which indicate discrepancies in etiopathogenesis.