Pancreatic involvement in patients with Sjögren's syndrome and primary biliary cirrhosis.

Serum pancreatic enzyme activities, exocrine pancreatic function, and pancreatic ductal morphology were evaluated in patients with one or both of Sjögren's syndrome and primary biliary cirrhosis. Ten of 20 patients with Sjögren's syndrome (50%), 6 of 17 patients with primary biliary cirrhosis (35%), and 4 of 11 patients with both diseases (36%) had an elevated level of at least one pancreatic enzyme, including elastase-1, lipase, and trypsin. Diminished excretion of N-benzoyl-L-tyrosyl-para-aminobenzoic acid was observed in 3 of 17 patients with Sjögren's syndrome (18%), 4 of 16 with primary biliary cirrhosis (25%), and none of 7 with both diseases. Endoscopic retrograde pancreatograms demonstrated an abnormal pancreatic ductal configuration in 3 of 11 patients with Sjögren's syndrome (27%), 2 of 9 with primary biliary cirrhosis (22%), and 3 of 4 with both diseases (75%). Only minimal changes in branches of the pancreatic duct were observed in the pancreatogram. Finally, 9-30% of patients with Sjögren's syndrome and/or primary biliary cirrhosis had a mild and intermittent abdominal pain. These findings support the concept of a disease complex, "autoimmune exocrinopathy," in patients with Sjögren's syndrome, primary biliary cirrhosis, and chronic pancreatitis.

Effects of carbamylcholine chloride on human antral gastrin mRNA levels.

The effects of the muscarinic receptor agonist, carbamylcholine chloride (carbachol), on gastrin release and gastrin mRNA levels in human antral mucosa (n = 15) were determined. During a-2-h incubation period, carbachol (10(-6)-10(-4) M) decreased gastrin mRNA levels to 71 +/- 8% (10(-6) M), 40 +/- 8% (10(-5) M), and 33 +/- 5% (10(-4) M) of control levels. Carbachol (10(-5) M) decreased intracellular gastrin (from 1634 +/- 103 to 1272 +/- 126 pg/mg tissue protein), while it increased gastrin release into the medium (from 609 +/- 48 to 918 +/- 68 pg/ml per mg tissue protein). After 6- and 9-h culture, carbachol gradually increased gastrin mRNA levels, by 96 +/- 12% and 126 +/- 23%, respectively. Atropine sulfate (10(-5) M) completely inhibited the carbachol-induced changes. Cycloheximide markedly decreased tissue gastrin concentration, but increased gastrin mRNA levels, whereas it had no effects on gastrin release. These findings suggested that carbachol may have a time-related biphasic action on human antral gastrin biosynthesis.

Identification of autoantibodies to a pancreatic antigen in patients with idiopathic chronic pancreatitis and Sjögren's syndrome.

A serum autoantibody to a pancreatic antigen was identified in patients with idiopathic chronic pancreatitis and Sjögren's syndrome by radioimmunoassay and Western immunoblotting. Antigen from porcine and human pancreas extracts was partially purified using a monoclonal antibody, SP3-1, which recognizes the antigen in duct cells of various exocrine organs. Solid phase radioimmunoassay of the pancreatic antigen showed a positive result in 6 of 20 patients with idiopathic chronic pancreatitis (30%), 3 of 11 patients with Sjögren's syndrome (27%), and 1 of 15 patients with alcoholic chronic pancreatitis (7%). Among seven patients with stone-related chronic pancreatitis, six patients with autoimmune thyroiditis, and 14 normal controls, none showed evidence of autoantibodies to the pancreatic antigen. Western immunoblotting showed that serum antibody commonly reacted with a 60-kD molecule of either porcine or human pancreatic antigen, with which SP3-1 also reacted. These results show the existence of the autoantibodies to pancreas, especially to an antigen expressed in ductal cells of exocrine glands, in idiopathic chronic pancreatitis and Sjögren's syndrome, and suggest the possibility of an autoimmune mechanism in the pathogenesis of idiopathic chronic pancreatitis.

Distribution of collagen IV in human ocular tissues.

PURPOSE: To examine the distribution of the collagen alpha 1(IV) chain and a novel collagen alpha(IV)-related chain in human ocular tissue. METHODS: Two monoclonal antibodies, JK199 and M3F7, against the alpha 1(IV) chain, and one monoclonal antibody, JK132, against a novel alpha(IV)-related chain were used in the avidin biotin peroxidase complex procedure of immunohistochemical studies. In situ hybridization and reverse transcription polymerase chain reaction were used to examine the presence of alpha 1(IV) messenger RNA in corneal epithelium. RESULTS: Our data indicate that monoclonal antibodies JK199 and M3F7 react with most ocular basement membranes, but not with those of corneal epithelium. Similarly, monoclonal antibody JK132 reacts with most basement membranes of ocular tissues, with the exception of the inner limiting membrane of neural retina, Bruch's membrane, and corneal epithelial basement membrane. To examine if the epitopes recognized by the monoclonal antibodies were masked in corneal epithelium, the tissue sections were subjected to limited enzyme digestion, that is, pepsin, hyaluronidase, trypsin, and pronase E, or chemical treatments such as 0.1 N NaOH or 6 M urea. Proteinase treatment removed the JK132 epitope from all ocular basement membranes examined. Despite the pretreatment, corneal epithelial basement membrane was not stained by any of the monoclonal antibodies. However, the alpha 1(IV) messenger RNA was detected in corneal epithelium by in situ hybridization and reverse transcription polymerase chain reaction. Western immunoblotting indicates the presence of the alpha 1(IV) and the novel alpha(IV)-related chain in the basal lamella of corneal epithelium. CONCLUSIONS: The epitopes recognized by JK199, M3F7, and JK132 are masked in basement membrane of corneal epithelium. Based on the tissue distribution and partial amino acid sequences of peptides recognized by JK132, the novel alpha(IV)-related chain differs from other known alpha(IV) chains.

Semiquantitative analysis of the effects of fixation and paraffin embedding on immunoreactivity of renal basement membranes to a monoclonal antibody against type IV collagen.

The effects of formalin fixation and paraffin embedding on the immunoreactivity of human kidney to a monoclonal anti-type IV collagen antibody (JK-199) were examined semiquantitatively by a modified enzyme-linked immunosorbent assay (ELISA). The intensity of immunoreactivity in paraffin sections of the tissue fixed overnight with 10% formalin was approximately 70% of that in frozen sections. Immunoreactivity reduced to this extent did not impair the specific staining of basement membranes. Paraffin sections of tissues fixed 2 days showed 50-60% of the reactivity in the frozen sections of the tissue fixed overnight; the basement membranes in Bowman's capsules were stained positively, but those in other sites were not. The paraffin sections of tissues fixed 7 or 14 days showed no specific immunostaining. The immunoreactivity for type IV collagen in the basement membranes was restored after treatment with pronase E. The immunoreactivity after the enzymatic treatment was about 150% of that in the frozen sections of the overnight fixed specimens.

A novel chain of basement membrane-associated collagen as revealed by biochemical and immunohistochemical characterizations of the epitope recognized by a monoclonal antibody against human placenta basement membrane collagen.

Biochemical and immunohistochemical characterizations of the epitope recognized by a monoclonal antibody, JK-132, originally produced against human type IV collagen showed that it was distinct from the previously reported monoclonal antibody, JK-199 (Kino et al, J Biochem 1988, 103:829-835). The bound fraction of a crude pepsin extract of human placenta on JK-132 antibody-coupled resin showed close similarity to type IV collagen in a triple-helical conformation in terms of the amino acid composition and circular dichroism spectrum. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the fraction showed six peptide bands with molecular weights of 50,000 or below, both before and after reduction. Four of the peptides reacted with JK-132 on immunoelectroblotting, but none reacted with JK-199. JK-132 reacted with two additional bands with molecular weights of 100,000 and 120,000, which were not visible on direct staining with Coomassie Brilliant Blue R-250. Two peptides (molecular weights 40,000 and 15,000) bound on a JK-199 antibody affinity column were sequenced, and both contained the same amino-terminal sequences as alpha 1(IV) chain. Conversely the sequences of three of the peptides (molecular weights 50,000, 32,000, and 23,000) eluted from a JK-132 antibody affinity column did not match either the alpha 1(IV) or the alpha 2(IV) sequence reported. Immunohistochemically, JK-132 reacted strongly with basement membranes of blood capillaries in skeletal muscle tissues but not with the basement membranes of muscle fibers in frozen sections of periodate-lysine-paraformaldehyde-fixed tissue, suggesting heterogeneity or tissue specificity of basement membrane collagen. By immunoelectron microscopy, the reaction products were found on the basal laminae of endothelium and of smooth muscle cells around blood vessels. These findings suggest the presence of a new collagen chain associated with basal laminae.

[Serum level of vascular basement membrane associated collagen by the sandwich ELISA with monoclonal antibodies and its clinical significance in various diseases].

A sandwich ELISA system for detecting vascular basement membrane associated collagen (BAC) was developed. Serum levels of BAC were determined in patients with liver diseases (N = 53), various cancers (N = 65) and other diseases (399). Serum levels of procollagen type III (PIIIP) amino propeptide, type IV collagen.7s domain (7s domain) and other parameters (TP, ALB, GOT, GPT, CHE, gamma-GTP, ALP, LDH, CHE, TG, GLU) were also determined in those patients. In the whole patients, serum concentrations of BAC showed a weak correlation with GOT, GPT, ALB and CHE but not with gamma-GTP and ALP. There was no correlation between BAC and PIIIP or 7s domain. Although serum levels of BAC were elevated in both liver diseases and cancers, the increase in liver diseases was more marked. Markedly increased serum levels of BAC with low levels of CHE were found only in liver cirrhosis and liver cirrhosis plus hepatocellular carcinoma. Increased BAC may reflect capillarization of the liver sinusoid or remodeling of the vascular basement membrane which is observed in the progression of liver fibrosis. Serum BAC is thought to be a promising new marker, different from PIIIP or 7s domain for diagnosing fibrosis state in the organs, particularly in the liver.

The effect of fixatives and paraffin embedding on the histochemical reactivity of a monoclonal antibody against human type IV collagen (JK-199).

A new monoclonal antibody (JK-199) was found to react with basement membranes on paraffin-embedded tissue sections without prior enzyme digestion. JK-199 was shown to react with isolated type IV collagen treated by any of four different fixatives--PLP, 4% formalin, modified Zamboni's (0.2% picric acid, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) or Bouin's--applied for 6 h at room temperature and incubated at 60 degrees C for 30 min to simulate routine tissue processing. None of the fixatives was able to alter the reactivity of JK-199 with isolated type IV collagen. In the human placenta, specific and intense staining of basement membranes was demonstrated on paraffin sections fixed with any of the four fixatives. In human skin, basement membranes were fully demonstrated on paraffin sections fixed by PLP fixative or by 4% formalin, but only partially on sections fixed by picric acid-containing fixatives. Optimal results, i.e., with the least non-specific or incomplete staining, were obtained on PLP-fixed paraffin-embedded tissues. In PLP-fixed paraffin sections of the kidney, skeletal muscle, and small intestine, all basement membranes were stained intensely by this antibody (JK-199) at the expected locations. The results indicate that JK-199 may be widely applicable for the analysis of basement membrane kinetics, including developmental processes or pathological conditions.

Partial characterization of an unusual 185 kDa protein synthesized by dermal fibroblasts from patients with Marfan syndrome: identification of the protein as type IV collagen.

Production of an unusual collagenous protein was observed in culture of dermal fibroblasts from four patients with Marfan syndrome. The apparent molecular weight of the protein was about 185 kDa after reduction with 2-mercaptoethanol and 175 kDa after limited pepsin treatment. The 185 kDa protein was susceptible to the bacterial collagenase but resistant to the animal collagenase. Immunoprecipitation revealed the specific interaction of the pepsin-treated 175 kDa collagenous protein with monoclonal and polyclonal antibodies to human type IV collagen. From the patterns of CNBr peptide mapping the 185 kDa band was identified as alpha 1 (IV) chain. Type IV collagen in the skin is generally considered to be of non-fibroblastic origin. However, in "diseased" condition, dermal fibroblasts might produce type IV collagen. The clinical manifestation in relation to production of type IV collagen by cultured skin fibroblasts from Marfan patients is discussed.

Characterization of a monoclonal antibody against human placenta type IV collagen by immunoelectroblotting, antibody-coupled affinity chromatography, and immunohistochemical localization.

We have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260,000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.

Immunohistochemical study of beta-adrenergic receptors in the psoriatic epidermis using an anti-alprenolol anti-idiotypic antibody.

In order to study the beta-adrenergic receptor-cyclic AMP system in the human skin, we have developed a monoclonal antibody against the receptor binding site in two fusion steps. We produced an anti-alprenolol monoclonal antibody using a hybridoma technique after the immunization of a mouse with alprenolol-BSA conjugate. This antibody was then used to immunize mice, whose splenocytes were used for the second fusion. Colonies were screened by enzyme linked immunosorbent assay (ELISA) using turkey erythrocyte membrane as an antigen. One of the positive colonies was grown in mice to obtain ascites fluids. This antibody showed the ability to compete with [125I]-iodocyanopindolol for the binding to beta-adrenergic receptors of turkey erythrocytes. The binding of the antibody to the turkey erythrocytes was prevented by the existence of the anti-alprenolol antibody and also L-alprenolol. This antibody showed a prominent inhibition of the adrenaline-stimulated adenylate cyclase activity of the turkey erythrocyte membrane. The data indicate that the anti-idiotypic monoclonal antibody against alprenolol binds to the beta-adrenergic receptors and acts like an antagonist. We further studied the localization of the beta-adrenergic receptors with this antibody in the normal human skin as well as in the psoriatic skin using immunohistochemical technique. A prominent decrease of the beta-receptors was observed in the psoriatic epidermis.

Monoclonal antibodies against epidermal cell surface and dermal-epidermal junction: antigens which may be epidermal cell spreading factors.

Two monoclonal antibodies which reacted with the epidermal cell surface (SF-1) and the dermal-epidermal-junction (SF-2), respectively, were obtained by immunizing mice with partially-purified human epibolin. The corresponding antigens were partially purified from fetal bovine serum by affinity chromatography using these antibodies. SDS-polyacrylamide gel electrophoresis showed that these antigens contained polypeptide components with molecular weights different from that of epibolin (mol. wt. 65,000 daltons); SF-1 antigen had a 68,000 dalton main component, and SF-2 antigen a broad 58,000-61,000 dalton main component. Both of these partially-purified antigens promoted the spreading of dissociated pig epidermal cells. SF-2 antigen also promoted the spreading of Pam cells (a murine keratinocyte line). The results suggest that proteins capable of promoting epidermal cell spreading may be present on the epidermal cell surface and at the dermal-epidermal junction. However, their physiological role in keratinization remains to be elucidated.

A cell-spreading inhibitor exists in serum and in epidermal basal cells.

A unique monoclonal antibody was obtained by immunizing mice with complement-inactivated fetal bovine serum (FBS). This antibody, named SI-1, stained epidermal basal cells of humans, pig, guinea pig, and rat by an indirect immunofluorescence technique after pretreatment of cryostat sections with alkali buffer (pH 9.6). After dissociating pig epidermal cells by trypsin, the SI-1 antibody stained exclusively and strongly one type of uniquely shaped cells. They were small and hanging-bell or columnar in shape with one convoluted side on the base, consisting of less than 2.8% of the dissociated epidermal cell population. The antigen contained in FBS was partially purified by affinity chromatography using the SI-1 antibody. The affinity-purified antigen inhibited the spreading of PAM cells, a spontaneously transformed murine keratinocyte line, in serum-free medium in a dose-dependent manner at concentrations of 10(-5) to 10 ng/ml. The antigen also inhibited the spreading of trypsinized pig epidermal cells in the range of 10(-2) to 10(3) ng/ml in the presence of 0.05% FBS. Although there have been a few reports indicating that serum inhibited both spreading and attachment, a specific factor in serum has not been purified before. This is, to our knowledge, the first presentation of a cell-spreading inhibitor contained in serum.

Monoclonal antibodies to the beta-adrenergic receptor: modulation of catecholamine-sensitive adenylate cyclase by the antibody.

We have developed two types of hybridomas producing monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor in order to study the beta-adrenergic-cAMP system of epidermis. Splenic cells from BALB/c mice immunized with partially purified turkey erythrocyte beta 1-receptors were fused with mouse myeloma cell line SP2/0-Ag14. Five hybridomas of 17 positive cells producing antibodies which could precipitate soluble turkey erythrocyte beta 1-receptors were cloned by the limiting dilution method. The antibodies cross-reacted with beta 1- and beta 2-adrenergic receptors and stained epidermal basal cells with immunocytochemical techniques. Neither type of antibody interfered with the antagonist binding, i.e., all antibodies bound to sites other than the ligand binding site on the surface. One type of antibody inhibited epinephrine-stimulated adenylate cyclase activity in our "leaky" epidermal cell system. The data suggest that the antibody interferes with the coupling of the receptor to the regulatory protein.

Successful preparation of cobalt(II)-substituted hemocyanin.

The cobalt(II)-substituted hemocyanin was first prepared by dialysis of apohemocyanin against Tris-HCl buffer (pH 8.0) containing cobalt(II) ion. The amount of cobalt(II) introduced into apohemocyanin reached 47% of the total sites for copper ion in native hemocyanin, being estimated as nearly complete formation of the half-filled cobalt(II)-hemocyanin (Co(II)-Hc). The Co(II)-Hc did not bind oxygen molecule even under O2-atmosphere. The spectral data indicated that the Co(II) is in tetrahedral geometry (high-spin state).

Studies on the examination of imported laboratory monkey, Macaca fascicularis for E. histolytica and other intestinal parasites.

Infection rate of imported monkeys, under this study, with intestinal protozoa is higher than the infection rate with intestinal nematodes. Mostly encountered protozoa were E. coli, E. nana, E. histolytica and I. butschlii. Formalin-Ether concentration method yielded higher % positive for E. histolytica than the culture method using Tanabe-Chiba medium, but the results obtained from these 2 methods correlate perfectly well with each other. Efficacy of thiabendazole against intestinal nematodes such as Strongyloides, Trichuris, Physaloptera, Oesophagostomum and Capillaria was very satisfactory.