High-affinity binding of interferon-gamma to a basement membrane complex (matrigel).

Recently it was demonstrated that growth factors are bound to the extracellular matrix, and can regulate cell behavior. Using three different types of binding assays, we have examined the interaction of interferon-gamma with a basement membrane produced by the Engelbreth-Holm-Swarm tumor. Basement membrane was found to bind interferon-gamma in both a time- and concentration-dependent manner. Equilibrium binding analysis revealed a high-affinity site with a dissociation constant of 1.5 10(-9) M and a maximum binding capacity of 1.6 10(9) sites/mm2 of basement membrane. Competition studies show that the binding is inhibited by heparan sulfate, suggesting that basement membrane-heparan sulfate proteoglycan could be the binding site. This interaction was clearly confirmed by native polyacrylamide gel electrophoresis and dot-blot analysis with purified basement membrane molecules. Furthermore, the carboxy-terminal part of the interferon-gamma molecule contains an amino acid cluster, very closely related to a consensus sequence, present in more than 20 proteins known to bind sulfated glycosaminoglycans such as heparin. These data demonstrate a possible role of extracellular matrix components in storing cytokines and in modulating the cellular response to such factors.

Tumor cell surface-associated binding site for the M(r) 72,000 type IV collagenase.

We have studied the capacity of two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, to bind exogenous M(r) 72,000 type IV collagenase by both morphological and radioreceptor binding assays. By indirect immunofluorescence, staining with a specific anti-M(r) 72,000 type IV collagenase antibody was strongly induced when cells were preincubated with the purified enzyme. Scatchard plot analysis indicated the existence of a binding site for the M(r) 72,000 type IV collagenase with high affinity for both cell lines (Kd = 2 x 10(-9) M). These results are the first demonstration of the existence of a tumor cell membrane-associated putative receptor for a member of the matrix metalloproteinase family, as previously evidenced for the urokinase-type plasminogen activator.

Binding of interferon-gamma to heparan sulfate is restricted to the heparin-like domains and involves carboxylic--but not N-sulfated--groups.

Interferon-gamma binds to the glycosaminoglycan part of basement membrane proteoglycan. To obtain a greater insight into this interaction, different glycosaminoglycans and their subfractions were used in various binding assays. High affinity binding occurs with heparin and heparan sulfate only, the latter being the predominant basement membrane glycosaminoglycan. Furthermore, using heparan sulfate and heparin treated with heparinases I and III, we have shown that the interferon-gamma binding sites are localized on the N-sulfated glucosamine rich domains of the molecule. Interestingly, interferon-gamma and fibroblast growth factor compete for the same binding domain on heparan sulfate, although they are unrelated proteins. This last point is discussed in the light of the conformational flexibility of the glycosaminoglycan molecules.

Mechanical properties and structure of carotid arteries in mice lacking desmin.

OBJECTIVE: Our aim was to determine in desmin homozygous mutant mice the viscoelastic properties, the mechanical strength and the structure of the carotid artery. METHODS: To assess the viscoelastic properties of large arteries, we have performed an in vivo analysis of the diameter-, and distensibility-pressure curves of the common carotid artery (CCA) in homozygous (Des -/-), heterozygous (Des +/-) and wild-type (Des +/+) mice. To evaluate the mechanical strength, we have measured the in vitro intraluminal pressure producing the rupture of the carotid artery wall. The structure analysis of the arterial wall was based on histology and electronic microscopy. RESULTS: A lower distensibility and an increase of arterial wall viscosity were observed in Des -/- compared with Des +/+. Arterial thickness of Des -/- was similar to those of Des +/+, without changes in elastin and collagen contents. Electron microscopy revealed that the perimeter of cellular fingerlike-projections was smaller in Des -/-, indicating that the cells have lost part of their connections to the extracellular matrix. The rupture pressure was significantly lower in Des -/- (1500+/-200 mmHg) compared with Des +/+ (2100+/-80 mmHg) indicating a lower mechanical strength of the vascular wall. No significant difference was found between Des +/- and Des +/+. CONCLUSION: The desmin is essential to maintain proper viscoelastic properties, structure and mechanical strength of the vascular wall.

Interactions between fibroblasts and a reconstituted basement membrane matrix.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the interactions between normal calf skin fibroblasts and basement membranes. Within 6 h after seeding, fibroblasts initiated a migration that resulted in the formation of a cellular network after 1 day of culture on top of the gel. Electron microscopy revealed that fibroblasts were able to remodel the basement membrane matrix by penetrating into the gel (from day 3), depositing fibronectin and collagen fibers, and retracting this extracellular matrix. Fibroblasts cultured on the Engelbreth-Holm-Swarm reconstituted basement membrane matrix displayed ultrastructural features characterized by a poor synthetic apparatus (rough endoplasmic reticulum and Golgi vesicles), a large cytoskeleton, and intracytoplasmic vesicles containing laminin. Thus the reconstituted basement membrane matrix is remodeled by skin fibroblasts, and reciprocally their ultrastructural morphologic features are affected by this matrix.

Interferon-gamma binds to heparan sulfate by a cluster of amino acids located in the C-terminal part of the molecule.

Using three different approaches (domain mapping with monoclonal antibodies, limited enzymatic digestion and competition with synthetic peptides), we demonstrated that a cluster of basic amino acids on interferon-gamma is involved in its binding to heparan sulfate. This cluster (Lys125-Arg131) is localized in the C-terminal part of IFN-gamma. Once bound to heparin sulfate, IFN-gamma is protected against protease attack.

Interferon-gamma inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts.

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with D-[1-(3H)]glucosamine and Na2(35)SO4. Considering 3H incorporation, we found that IFNgamma increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNgamma inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.

Thymic extracellular matrix in myasthenia gravis. II. Immunohistochemical evidence for increased type I collagen degradation.

By immunohistochemistry, we studied the breakdown of type I collagen in frozen sections of normal and hyperplastic (myasthenia gravis-associated) human thymuses. This analysis was carried out using a specific polyclonal antibody directed against the alpha 2-CB(3,5) peptide, a degradation product of type I collagen. In the normal thymus, virtually no labeling was observed within thymic septae or intralobular regions. In contrast, bright fluorescent staining was consistently detected in myasthenia gravis-associated hyperplastic thymuses. Such immunoreactivity was found not only in septal regions but also in the intralobular connective tissue meshwork that exists in these thymuses. Our results represent further evidence for a defect in extracellular matrix metabolism in hyperplastic thymuses, which may be related to the abnormal intrathymic penetration of B cells, known to occur in this disease.

Collagen immunotyping in human liver: light and electron microscope study.

Types I, III, IV, and AB collagens have been extracted from human cirrhotic livers and specific antibodies have been raised in rabbits and purified. Histological immunofluorescent staining of collagen types in normal and fibrotic human livers reveals the respective distribution of the various collagens among the hepatic connective matrix and the modification of the normal pattern in fibrosis: types I and III appear to be the main components of the fibrotic connective matrix in enlarged portal spaces and of the Dissian reticulin framework; type IV collagen deposits are thickened around portal vessels and ducts and outline lobular capillarized sinusoids; type AB collagen appears as thin punctual deposits in portal and Dissian fibrotic connective matrix. Ultrastructural immunoperoxidase labeling of type I and III collagen makes it possible to identify the typical collagen fibers, using 65 nm periodicity, as type I collagen and the fibrillar associated network as type III collagen. Fibers of type I collagen are preferentially organized in large dense bundles in Dense Connective Matrix Organization (DCMO), since fibrillar type III collagen network is predominant in Loose Connective Matrix Organization (LCMO) surrounding vascular and biliary tracts.

Ultrastructural immunoenzymatic study of alpha-fetoprotein-producing cells in the human fetal liver.

Cells producing alpha-fetoprotein in human fetal liver have been studied with specific horseradish peroxidase labeled immunoglobulins. Under light microscopy, the alpha-fetoprotein is strictly localized in the cytoplasm of certain hepatocytes, distributed randomly in the hepatic lobule. Ultrastructural examination of thesame cells shows that the alpha-fetoprotein is present within the cytoplasm. Ultrastructural differences are described in hepatocytes according to whether or not the cell is producing alpha-fetoprotein at the time of sampling. These observations lead to the hypothesis that alpha-fetoprotein may correspond to a particular functional state of the hepatocyte in human fetal liver.

A procedure for light and electron microscopic intracellular immunolocalization of collagen and fibronectin in rat liver.

Experimental conditions have been designed that permit both extracellular and intracellular immunolocalization of various collagen types and fibronectin in rat liver. The procedure involves paraformaldehyde fixation by perfusion of the organ, use of saponin as a membrane permeabilizing agent, and visualization of the matrix components by indirect immunoperoxidase. Intracellular demonstration of collagens was particularly sensitive to the composition of the fixative and the duration of fixation. Hepatocytes contained fibronectin and types I and IV collagen, whereas fat-storing and endothelial cells evidenced type III collagen in addition. All the components were specifically located in the endoplasmic reticulum and/or the Golgi apparatus.

Hepatic connective tissue changes in hepatosplenic schistosomiasis.

Destruction of intrahepatic portal vein branches with dispersion of smooth muscle cells into the periportal fibrosis and preservation of arterial and ductal structures were the main characteristic findings seen in 66 surgical liver biopsies from patients with the hepatosplenic form of schistosomiasis. Besides these diagnostic features, the present histologic, immunocytochemical, and ultrastructural study revealed the presence of a complex matrix forming the portal and septal fibrosis in advanced schistosomiasis. There was marked hyperplasia of elastic tissue, presence of several collagen isotypes (I, III, procollagen III, IV, and V), actin, desmin, fibronectin, and laminin in a richly vascularized connective tissue. Signs of multifocal matrix (collagen) degradation were observed both at light and electron microscopic levels, suggesting a predominance of a fibrolytic process, at the time parasite-related lesions had almost disappeared. The latter findings are related to the involution of periportal fibrosis now being observed in patients who have undergone antischistosomal chemotherapy. They exemplify morphologic changes connected with chronic collagen degradation in human schistosomiasis that are similar to those first seen in experimental material. Evidence of either persistent or active chronic hepatitis was seen in several cases but its etiology could not be determined.

Perisinusoidal fibrosis and basement membrane-like material in the livers of diabetic patients.

A direct correlation exists between collagenization of Disse's space and the presence of diabetic microangiopathy in type I diabetes. To confirm and extend this finding, we studied four liver biopsy samples from two patients with type I diabetes (one with retinopathy) and two patients with type II diabetes (no retinopathy). All had normal or subnormal results on liver function tests and normal liver architecture. Levels of collagen types I, III, and IV, laminin, and fibronectin, as determined by immunocytochemical techniques, appeared increased in all patients. Liver biopsy samples were perfusion fixed for electron microscopy of sinusoids and sinusoidal cells. Numerous and thick collagen bundles could be seen in Disse's space, as could the increase of basement membrane-like material underlying the endothelial cells, perisinusoidal cells, and sinusoidal membrane of hepatocytes. Perisinusoidal cells were active and had abundant rough endoplasmic reticula and thick processes. This preliminary study indicates that collagenization of Disse's space is not specific to a certain type of diabetes. The increase of basement membrane-like material raises the question of whether liver sinusoids are truly different from other capillaries as far as diabetic microangiopathy is concerned.

Fibrosing vasculitis in Wegener's granulomatosis: ultrastructural and immunohistochemical analysis of the vascular lesions.

This study of two cases of pulmonary Wegener's granulomatosis (WG) focuses on the ultrastructural aspects of the vascular wall injury and on the immunohistochemical characterization of the perivascular connective matrix. The iterative waves of endothelial cell necrosis and regeneration are demonstrated by the multilamellar appearance of the basal lamina. Neutrophils infiltrate the vessel wall and myofibroblasts are recruited to injured vessels. The perivascular connective matrix associates basement-membrane like and fibrillar material with fibrin deposits. The initiation of the fibrosing process was assessed by the visualization of matrix molecules involved in targeting (p-fibronectin), organizing (cellular fibronectin and tenascin) and stabilizing (lysyl-oxidase) the fibrogenic activity. These elementary lesions affect different levels of the vascular tree, and capillaritis is involved in the extension of the pathological process. Lysyl-oxidase labelling reveals the fibrosing front which is located on the border of dense fibrosis. The markers of fibrosing activity disappear in the areas of fibrosis following vasculitis and/or ischaemic necrosis and/or granulomatosis. Vasculitis plays a major role in both the genesis and progression of the fibrosis observed in the late stage of WG.

Organization of the connective matrix of the sarcoid granuloma. Evolution and cell-matrix interactions.

The connective matrix of sarcoid granulomas is polymorphic. Immunolabeling techniques show that it is partly composed of fibronectin and collagen types I and III and is found at all stages of the granulomas' evolution. This suggests that the process known as hyalinization is not due to changes in the composition of the matrix but results from a gradual accumulation of connective tissue components. Fibronectin seems to play an essential role as mediator between different cell populations during the evolution of the granulomas.

Pyridinoline, a mature collagen cross-link, in fibrotic livers from Schistosoma mansoni-infected mice.

The amount of pyridinoline, a cross-linking amino acid found in mature collagen, was measured in murine liver for a period of up to 40 weeks following Schistosoma mansoni infection. The pyridinoline level (expressed as pmol per 100 micrograms of liver) markedly increased in the course of murine schistosomiasis, and the number of pyridinoline residues per collagen molecule was also higher in the livers of infected mice than that of control livers. This increase in the extent of intermolecular collagen cross-linking might play a role in the modulation of collagen catabolism as an auxiliary mechanism of extracellular matrix accumulation during fibrogenesis.

Sequential changes of the connective matrix components of the myocardium (fibronectin and laminin) and evolution of cardiac fibrosis in mice infected with Trypanosoma cruzi.

Interstitial matrix alterations due to chronic Trypanosoma cruzi myocarditis were studied in mice by immunofluorescent microscopy with specific purified antibodies against the main different collagen isotypes, laminin and fibronectin. During the early subacute stage (26-30 days postinfection), sarcolemmal and perivascular deposits of laminin and fibronectin were prominent. The presence of fibronectin appeared to correlate with the presence of inflammatory cells. By the late subacute phase and early chronic phase (50-90 and 80-90 days postinfection, respectively), laminin and Type IV collagen were present. These were the principal features, although fibronectin continued to be found among inflammatory cells, and pro-III and III collagens formed irregular bands and periarteriolar deposits. During the late chronic phase (150-200 days postinoculation) the interstitium was enlarged and irregular, with positive staining for laminin, Types III, pro-III, and IV collagens; fibronectin appeared as focal, subendocardial, interstitial, and perivascular deposits. The relative absence of Type I collagen and the apparent positive correlation between interstitial matrix amplification and the presence of mononuclear inflammatory cells suggest that fibrotic changes in chronic T. cruzi myocarditis can be reversed if the inflammatory changes subside.

Collagen isotypes, laminin, and fibronectin in granulomas of the liver and intestines of Schistosoma mansoni-infected mice.

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.

In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods.

The development of new in situ assessment of HCMV disease on endoscopical gastrointestinal biopsies from AIDS patients is described and compared with the viral load measured by semiquantitative solution-phase PCR (SQ-PCR). Ten biopsies were examined by viral isolation, standard histology, in situ hybridization (ISH), in situ PCR-hybridization (PCR-ISH) and SQ-PCR, using the same target sequence. The methods developed for in situ HCMV detection were HCMV primers, the plasmid pCMV 406-S, a vector-free-digoxigenin-labelled HCMV-362 probe and the pSK + MCS nonsense probe. Paraffin-embedded MRC5 cells, either HCMV-infected or uninfected served as controls of specificity for ISH. beta-Actin primers were designed as markers of DNA integrity. Computerized models of the PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine biopsies were negative for HCMV by histology and virus isolation. SQ-PCR revealed 80,000; 80 and < 80 HCMV genomic equivalents in 6, 2 and 2 biopsies, respectively. In 8 biopsies, both ISH and PCR-ISH identified positive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can help the timely diagnosis of HCMV infection. Direct in situ PCR with beta-actin primers showed a positive signal in all the nuclei in the tissue sections, whereas omission of Taq polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-stage reactivation of HCMV, possibly harboured in the intestinal epithelium.

Identification and detection of long incubation non-A, non-B hepatitis virus and associated antigens or antibodies.

Three distinct antigen/antibody systems supposedly associated with an HBV-like virus of non-A, non-B hepatitis have been identified. Because of previously demonstrated cross-reactivity with HBe/3 and HBc antigens and other analogies the following terminology is tentatively used. 1. The previously reported serum antigen has been redesignated non-A, non-B e antigen, since it is equivalent to HBe/3 Ag and cross-reacts with it. Non-A, non-BeAg or Ab were detected in 51/62 post-transfusion and 11/56 sporadic acute non-A, non-B hepatitis cases, and in 12/14 cases affecting staff members. In non-A, non-B chronic persistent or active hepatitis and cryptogenic cirrhosis, the prevalence was similarly high: 14/18, 22/48 and 12/18 respectively. Ten out of 26 implicated blood donors were found positive for non-A, non-BeAg accounting for 7 out of 8 post-transfusion cases. A high prevalence of non-A, non-BeAg was also found in haemophiliacs (11/48) and haemodialysed patients (6/42), whereas anti-non-A, non-Be was respectively detected in 4/48 and 6/42 of these cases. 2. Using immunofluorescence, a second antigen termed non-A, non-BcAg has been identified in liver biopsies from 55/84 non-A, non-B chronic hepatitis or cryptogenic cirrhosis cases. All 8 positive biopsies examined by electron microscopy revealed clusters of 22--25 nm intranuclear particles identical to those described in chimpanzees. Anti-non-A, non-Bc detectable by counter-electrophoresis and indirect immunofluorescence was found in the serum of all patients of which biopsy was positive for non-A, non-BcAg. Anti-non-A, non-Bc was also detected in 5/5 non-A, non-BeAg positive cases of post-transfusion hepatitis, 2--6 weeks after onset end remained positive for the 6 month follow-up period. 3. A third antigen, tentatively designated non-A, non-BsAg, has been found less frequently than non-A, non-BeAg in serum. However, it was detectable in 3/18 and 2/12 washed ultracentrifugation pellets of sera positive for non-A, non-BeAg or anti-non-A, non-Be, respectively.