Enhanced lymphocyte stimulation by bacterial lysates after treatment of probands.

The proliferative response of lymphocyte cultures upon the addition of a mixture of antigens from the lysates of different bacteria (Paspat) to the culture medium was investigated using lymphocytes of groups of probands that investigated using lymphocytes of groups of probands that had been treated with the i.c. test preparation (T.P.1, group I), the lyophilized test preparation given orally (T.P.2, group II), a commercially available lyophilized bacterial lysate of similar composition given orally (T.P.3, group III), or with placebo (group IV). Lymphocyte cultures were set up on day 0 (before treatment), day 40 and day 70 (after treatment). The results show, that T.P.1 and T.P.2 produce a dose dependent proliferative response in lymphocyte cultures with a peak reactivity between 70 and 210 micrograms/ml. The degree of stimulation obtained with the bacterial lysate in different concentrations is increased in groups I and II over the stimulation obtained before treatment. Groups I and II which were treated with T.P.1 (i.c.) and T.P.2 (orally) are significantly different in their response to the bacterial lysate from the groups treated with T.P.3 or placebo on days 40 and 70. Evidence is presented that the stimulation obtained is predominantly a proliferation of T-cells.

Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.

[Plasma levels and tissue concentrations in rabbits following cutaneous administration of an indomethacin solution]. / Plasmaspiegel und Gewebskonzentrationen beim Kaninchen nach kutaner Applikation einer Indometacin-Lösung.

After cutaneous application to rabbits of a solution containing 1% of indomethacin (Elmetacin), concentration-time curves were determined in the skin-layers, in the subcutaneous tissue, in the superficial musculature, in the knee-joint capsule and in plasma. The quantitative assay used on-line fluorimetric detection after separation by HPLC and alkaline hydrolysis. Levels of indomethacin decreased with the depth of the tissue layers at the site of application. Indomethacin concentrations in the examined tissues exceeded plasma concentration at least twice for up to 8 h. Indomethacin was also detectable in the contralateral untreated tissue specimens and in the knee joint capsule.

[Detection of cutaneously administered flufenamic acid in the inner space of the knee joint]. / Nachweis von kutan applizierter Flufenaminsäure im Innenraum des Kniegelenkes.

Ten patients who were scheduled for surgery due to an internal damage of the knee joint, were treated 2 days before operation 3 times daily with an ointment containing flufenamic acid (Mobilisin spezial). At the time of operation, plasma and synovial fluid, tissue samples from the synovial membrane, Hoffa's fat pad and-- as far as possible--meniscus and articular cartilage were taken. Until 72 h after the operation, urine samples were collected. The flufenamic acid level was determined by means of high pressure liquid chromatography associated with fluorometric detection. Flufenamic acid was detected in all tissues of the knee joint and in the synovial fluid. The highest concentration of flufenamic acid was found in the synovial membrane.

Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane.

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.

Identification of binding site for heparin and other polysulfated glycosaminoglycans on human thrombocytes.

Heparin and other polysulfated glycosaminoglycans (GAGPS) can evoke an immune response when complexed to the surface of platelets in vivo. We have previously reported on the detection and characteristics of such antibodies leading to thrombocytopenia. Here we report data concerning the biochemical characterization of the heparin-/GAGPS-binding site on human thrombocytes. By means of heparin affinity chromatography, GAGPS chromatography and subsequent 'Western blotting' of specifically eluted proteins we were able to detect two proteins of apparent molecular mass of 207 and 170 kilodaltons, which could be reduced to 57 and 142 kilodaltons, respectively. The 170-kilodalton glycoprotein was stained by the carbohydrate specific PAS-reagent and by surface labeling with 3H. The second molecule binding to heparin (GAGPS)-Sepharose was detected by platelet surface labeling with 125I, protein-specific staining Coomassie brilliant blue and PAS staining. Binding studies using enzyme-labeling techniques with five monoclonal antibodies (MCA) against platelet surface proteins GPI, GPIIb/IIIa complex, GPIIIa and CRI (C3b receptor) revealed specific interaction of the 170-kilodalton heparin-binding glycoprotein with an MCA to GPI, suggesting identity or immunological cross-reactivity of these two moieties. No specific reactions were observed between the 207-kilodalton glycoprotein and any of the MCA tested. For further evaluation of the antigenicity of separated platelet proteins, an ELISA in microtiter plates was developed, and a cellular sandwich ELISA for serological detection of antibodies against whole native thrombocytes or GAGPS-derivatized proteins was set up.(ABSTRACT TRUNCATED AT 250 WORDS)

Cutaneous absorption of indomethacin from two topical preparations in volunteers.

The percutaneous absorption of indomethacin in 0.5 % or 1 % solution or 1 % gel at a dose of 50 mg or 100 mg indomethacin was compared in a randomized complete block design in seven healthy volunteers. The formulations were applied over an area of 12 dm(2) under an 8 h occlusion dressing. In addition, in the same volunteers the plasma concentration curves were determined after a single oral dose of 50 mg indomethacin. Indomethacin and some of its metabolites were determined with modified, existing assays using HPLC-fluorescence or gas chromatography-mass spectrometry. On the basis of a newly developed method, it was possible to separate and quantify O-desmethylindomethacin and N-deschlorobenzoyl-O-desmethylin-domethacin. After cutaneous administration of the two drug formulations, peak indomethacin plasma concentration of 95 ng/ml and 130 ng/ml were found between 4 and 8 h; the cutaneous bioavailability was approximately 20 % of the oral dose, as judged by comparing the areas under the plasma concentration time curves (AUC) and the amount of metabolites excreted into the urine. Percutaneous absorption did not change the metabolic pattern in the urine that is obtained after oral administration.

Nidogen: a new, self-aggregating basement membrane protein.

Nidogen was purified from a mouse tumor basement membrane where it accounted for 2-3% of the total proteins. It was isolated as two forms (A and B) of a monomer (Mr = 80000) each consisting of a single polypeptide chain folded into a globular head connected to a small tail. The B form of the monomer was shown to be capable of aggregating into a nest-like structure (Mr greater than 250000). A smaller form (Mr = 45000) was observed in some of the extracts. The amino acid composition of nidogen was different to that of other basement membrane proteins. It contained about 10% carbohydrate, with N-linked and O-linked oligosaccharide chains in similar proportions. Isoelectrofocussing demonstrated a limited heterogeneity of nidogen with pI in the range 6.5 - 7. Monomeric nidogen failed to interact with other basement membrane components and heparin. Aggregation could be induced by limited proteolysis and was reversed by detergents or high salt concentrations. Together with the observation that most of the nidogen could be solubilized only after destroying the collagenous matrix, the data indicate that aggregation of nidogen reflects an activity involved in matrix assembly. Specific antibodies raised against nidogen did not distinguish between the monomeric and aggregated form of the protein but showed that the fragment was antigenically deficient. These antibodies did not cross-react with collagen type IV, laminin, entactin and heparansulfate proteoglycan. Immunofluorescence staining and absorption studies demonstrated that nidogen is a common component of authentic basement membranes. Larger forms of nidogen (Mr about 100000 and 150000) were found in organ cultures of Reichert's membrane suggesting that it is synthesized in precursor forms.

Detection of antibodies interacting with glycosaminoglycan polysulfate in patients treated with heparin or other polysulfated glycosaminoglycans.

A new method has been developed for the demonstration of the antibody nature of a factor occurring in some patients during treatment with heparin or other polysulfated glycosaminoglycans (GAGPS). So far, antibodies reacting with GAGPS, except heparin, have not been described. We used heparin- or GAGPS-coated normal blood group O thrombocytes for the detection of suspected antibodies. After addition of patients' plasma or serum to a suspension of purified heparin- or GAGPS-coated platelets, agglutination was determined microscopically. In indirect immunofluorescence, positive staining of agglutinated platelets was observed with fluorescein isothiocyanate anti-Ig and anti-IgG conjugates, but not with anti-IgA or anti-IgM, with one exception in which IgM antibodies were also involved. Binding of complement was also shown using a tetramethylrhodamin isothiocyanate-labelled anti-C3 conjugate in double-staining experiments. Fibronectin could be excluded as a possible factor responsible for thrombocyte agglutination.

[Transdermal salicylate absorption and behavior of corticosteroid plasma levels during epidermal administration of an antiphlogistic ointment]. / Transdermale Salicylat-Resorption und Verhalten des Corticosteroid-Plasmaspiegels nach epidermaler Applikatiion einer antiphlogistisch wirksamen Salbe.

After repeated epidermal application of an ointment containing salicylic acid, mucopolysaccharidepolysulfate and suprarenal extract (Mobilat) the transdermal absorption of salicylate was determined in seven healthy volunteers by measuring the renal excretion of salicylate as well as the plasma level. The determination of the corticosteroid plasma level was to elucidate a possible effect caused by the suprarenal extract contained in Mobilat ointment. By means of high pressure liquid chromatography the concentrations of salicylic acid and salicyluric acid in the collected urine and in the plasma were established. The quantitative determination of hydrocortisone, 11-desoxycortisol and 17 alpha-hydroxyprogesterone, selected as indicator steroids, was performed by radioimmuno assay. After repeated application of Mobilat ointment a constant level of salicylic acid in plasma of approximately 0.2 microgram/ml was observed. The total excretion of salicylate reaches a constant level of approximately 12 mg/day. About 6.9% of the amount of salicylate was renally excreted after 7 days. The corticosteroid plasma level showed no significant change.

Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen.

Localization of antigenic activity and immunogenic capacity in different conformational domains of procollagen peptide.

The antibody response to the amino-terminal CNBr peptide of sheep procollagen which consists of a globular and a collagenous segment, was studied in inbred strains of mice. The determinants reacting with antibody could be localized in the globular domain and were lost by reduction of disulfide bridges. The ability to induce an antibody response required the collagen-like sequences and was independent of the triple-helical conformation of this segment. The data were interpreted as indicating a different conformation dependence of hapten and carrier determinants.

Isolation and structure of the amino-terminal cross-linking region in insoluble type III collagen.

A collagenous peptide T1X was isolated from a tryptic digest of the insoluble matrix of calf skin. The peptide consists of two identical polypeptide chains each with a length of 72 amino acid residues joined by a cross-link. Absorption spectra obtained from hydrazone and azine derivatives of T1X indicated that the peptide contains an aldol-type of cross-link (X). The sequence of 23 amino acid residues in the amino-terminal region was determined as Glx-Tyr-Glu-Ala-Tyr-Asp-Val-X-Ser-Gly-Val-Ala-Gly-Gly-Gly-Ile-Ala-Gly-Tyr-Hyp-Gly-Pro-Ala. This sequence overlaps the previously described amino-terminal sequence of alpha1 (III) chain obtained from pepsin treated, insoluble type III collagen. Thus, the present data demonstrate a nonhelical segment of 14 amino acid residues in type III collagen important for cross-linking.

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.

Production and specificity of antibodies against the aminoterminal region in type III collagen.

A cross-linked fragment (peptide T1X) with a molecular weight of 13,000 could be isolated from a tryptic digest of insoluble type III collagen of calf skin. Peptide T1X was conjugated on to bovine serum albumin by glutaraldehyde and used for immunization of rabbits. The antisera reacted in passive haemagglutination and radioimmune assay with peptide T1X, type III collagen and its constituent alpha1(III) chain. Little or no reaction was observed with type I collagen and alpha1(I) chain. While rabbit antisera to neutral salt-soluble type III Collagen also showed a strong binding for 125I-labelled peptide T1X much less reaction was observed with antisera to type I collagen. The antigenicity of type III collagen was largely destroyed by pepsin treatment suggesting that it resided in non-helical segments. A fragment of peptide T1X produced by digestion with collagenase retained antigenic activity. The data indicated that the aminoterminal region of type III collagen contains strong antigenic determinants located in a non-helical sequence of about sixteen amino acids. Antibodies to these antigenic determinants were purified and rendered specific for type III collagen by immunoadsorption. The antibodies stained in indirect immunofluorescence tests particularly those regions in various connective tissues which are rich in reticulin fibres. Different staining patterns were observed with antibodies to type I collagen.

Visualization of type I and II collagens in tissue sections by immunohistologic techniques.

Rabbit and rat antibodies were prepared against Type I and II collagens derived from bovine skin and articular cartilage, respectively. As judged by passive hemagglutination and radioimmune assays, these antibodies could be rendered generally specific for the type of collagen used for immunization by immunoadsorption. Thus, antibodies to Type II collagen did not cross-react with Type I and III collagens from skin. However, antibodies to Type I collagen still showed some cross-reaction with Type III collagen. Antibodies to Type I procollagen showed a negligible degree of cross-reaction with Type III procollagen. These purified antibodies reacted strongly with bovine and human tissue collagen as demonstrated by indirect immunoflourescence. Antibodies to Type I collagen stained dermal tissue, perichondral tissue, kidney stroma, aortic tissue, and annulus fibrosus. Antibodies to Type II collagen stained mainly the hyaline matrix of rib cartilage and nucleus pulposus. The staining patterns with anti-Type I procollagen were similar but not identical to that found with antibodies to Type I collagen. Neither of these antibodies reacted with kidney glomerular basement membrane. These antibody reagents are recommended as a sensitive and rapid screening tool for studying tissue distribution of collagen under normal and pathologic conditions.