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In the course of experimental aerosol infection of mice with influenza A virus, the latter was regularly detected in the blood, liver, salivary glands, spleen, pancreas, kidneys, heart and irregularly in cervical and mediastinal lymph nodes. The findings of extrapulmonary virus were in direct quantitative relationship to the rate of lung involvement.

The clinical utility of ELISA assays for antibodies reactive with a variety of porcine orbital tissue antigen preparations is described using sera from large numbers of patients. Use of such assays does not allow reliable identification of patients with Graves' ophthalmopathy (GO) due to the overlap between patients with and without eye involvement and normal individuals. In 5/6 patients showing high levels of reactivity with porcine eye muscle membrane antigen, a considerable but variable proportion of the binding was found to be species, rather than tissue, specific. No consistent pattern of change in ELISA reactivity with time was seen in studies of serum samples from patients treated medically for hyperthyroidism and patients who received immunosuppressive therapy with cyclosporin A for active, severe ophthalmopathy. Use of previously published ELISA methods using the crude antigen preparations described is therefore not likely to be of use in the routine clinical management of GO patients.

Limiting dilution analysis was performed in the presence of interleukin 2 (IL-2) on lymphocytes isolated from the synovial fluid (SF) and peripheral blood (PB) of patients with rheumatoid arthritis (RA) and PB of normal donors. Clones of these 'spontaneously' IL-2-responsive cells from PB and SF were compared for their reactivity with components of the extracellular matrix (i.e. native or denatured type I or type II collagen and proteoglycan). It was determined that all clones from both PB and SF were activated to produce interferon (IFN) in the presence of any of the connective tissue components (CTC). Clones derived from normal PB behaved in a similar fashion but produced lower IFN-gamma levels. There was a synergy between the CTC and serum or plasma fibronectin, which was more apparent when soluble CTC were used as the stimuli rather than immobilized CTC. The fibronectin alone was unable to induce IFN-gamma production under any of the conditions tested (i.e. soluble or immobilized). These results demonstrate that clones of IL-2-responsive T cells can be activated by interactions with connective tissue components to produce IFN-gamma.

Evidence has been obtained for potential use of total loose connective tissue film preparations during immunofluorescence studies of antinuclear antibodies of serum from patients with systemic lupus erythematosus, antibodies to cytoplasmic components of fibroblasts of serum from patients with rheumatoid arthritis, antibodies to the surface fibroblast antigen fibronectin, and of certain regularities in IgG binding with mast cell components. As compared with other preparations (cryostat, paraffin sections) used in immunomorphology while applying the methods of labeled antibodies, the total preparations have the following basic advantages: simplicity of preparation and possibility to eliminate the denaturative effects on tissue structures.

Electron microscope studies demonstrated that soluble proteins (ferritin and horseradish peroxidase) administered in vitro to strips of guinea pig diaphragmatic muscle under physiological conditions were internalized by vesicles of primitive reticular cells (PRCs) found in the connective tissue. This endocytosis effect was enhanced when the muscle preparations were obtained from animals actively allergized to the protein. Denervation increased the occurrence of peroxidase-positive vesicles in the peroxidase-antiperoxidase system. These results suggest that the antigen-antibody interaction induces endocytosis directed to the specific antigen.

Lymphocyte-dependent antibody cytotoxicity (LDAC) was studied using peripheral blood and in some instances synovial fluid cells from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). No difference from normal controls was observed with peripheral blood lymphocytes from either RA or SLE. Marked decrement in LDAC effector cell activity was present particularly with RA synovial fluid cells. Sera from patients with RA or SLE as well as RA synovial fluids markedly inhibited LDAC.

Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.

The reaction of heterophile antibodies with interstitial connective tissue cells of bovine myocardium was inhibited with alpha-D-galactose. D-glucose, D-mannose, L-fucose, N-acetylgalactosamine and other monosaccharides demonstrated the lack of inhibitory activity. Disaccharide melibiose and trisaccharide raffinose containing terminal alpha-galactose also had an inhibitory effect on the reaction of sera with bovine myocardium. The results show that heterophile antibodies to heterophile bovine antigen revealed in rheumatic patients' sera differ from antibodies to I and i antigens and T-antibodies, whose specificity is associated with terminal beta-galactose. They also differ from other heterophile antibodies described earlier.

The effect of persistent immunostimulation on normal and mechanically injured thoracic aorta was investigated histologically, histochemically and morphometrically. In the uninjured vessel wall no alterations suggestive of acute inflammation were observed following immunization, in accordance with previous biochemical studies. When mechanically elicited vascular injury and repair processes were induced in chronic immunostimulated rabbits, the neo-intimal aortic smooth muscle cell nuclear volume fraction of the vessel wall was significantly repressed, indicating, that the proliferative response to injury was inhibited. Further, the neo-intimal volume fraction of the vessel wall was reduced, suggesting impaired matrix neoformation. A highly significant linear correlation existed between the biochemically estimated DNA concentration and the nuclear volume fraction of smooth muscle cells in the vessel wall (r = 0.6275, P = 5 X 10(-5). Thus, the present study confirms previous biochemical observations, that the early processes of vascular inflammation and repair, i.e. smooth muscle cell proliferation and matrix accumulation, is inhibited following persistent immunostimulation. In addition to describing the histological correlates to the biochemical findings, important regional differences were quantified.

Our major objectives were to determine (1) when mice develop the cellular mechanisms necessary to trap and retain immune complexes in lymphoid follicles, (2) whether the ability to trap and retain immune complexes in lymphoid and collagenous tissues was maintained in old animals, and (3) whether the pattern of antigen localization in lymphoid follicles was altered as a consequence of ageing. Mice were passively immunized with a standardized amount of specific antibody and then challenged in the foot pads with radioactive antigen. The results indicated that newborn, and 1- and 2-week-old BALB/c mice lacked the cells or cellular mechanism necessary for trapping, localizing, and retaining immune complexes in lymphoid follicles. By 3 weeks, however, this ability to trap, retain, and localize antigen became apparent. The ability to trap and retain immune complexes on tendons and in lymphoid tissues was maintained as long as 27-30 months. A comparison of the quantity of antigen retained per mg of tissue in young-adult (6 months of age) and in old (27-30 months of age) mice indicated that old mice retained slightly more antigen in lymphoid tissues and substantially more on tendons. Antigen retained in spleens of both young-adult and old mice was localized in follicles by 24 h. However, the antigen retained in lymph nodes of old mice remained in the subcapsular sinus and adjacent superficial cortex and was not localized in follicles even after a full week. The cellular mechanisms necessary to trap and retain immune complexes in lymphoid tissue appear to develop in BALB/c mice a few weeks after birth and persist throughout life. However, localization of antigen in the lymphoid follicles of lymph nodes diminishes in old mice. The antigen trapping capability of collagenous tissues was not only maintained in old mice but was substantially increased.

It is apparent that significant progress has been made in our understanding of the biosynthesis, modifications, and maturation of collagen and elastin. We now recognize and partially understand special reactions involved in hydroxylations within the cell and complex cross-linking processes occurring outside the cell. Recent experiments (191) have shown that in human diploid fibroblast cultures of limited doubling potential (191) the hydroxylation of collagen prolyl residues appears to be "age" or passage-level dependent. With increasing passage level of these cultures, both the ascorbate requirements and the extent of collagen hydroxylation decrease. "Young" cell cultures have a strong requirement for complete hydroxylation and without ascorbate there is only about 50% of the normal level. "Middle-aged" cultures show higher hydroxylation without and full hydroxylation with ascorbate, whereas "old" (or cultures close to "senescence") are incapable of full hydroxylation with or without ascorbic acid. Although the overall system may show some deterioration with increasing passage levels, it appears that with increasing passage levels other components in the cell replace the ascorbate dependence of the hydroxylase system to a greater exten. In some ways, aging WI-38 cultures begin to resemble some transformed cells in their biochemical reactions, although they continue to remain diploid and eventually lose the ability to replicate. It is not yet known whether old animals can produce collagen, which may now be underhydroxylated, perhaps contributing to certain senescent changes. Careful examination of the hydroxylation index of collagen produced in organoid cultures of tissue biopsies as a function of donor age might be informative, particularly if one looks at the quality of collagen by employing collagenase and other proteolytic digests with collagen (191). One could comare the levels of frequent and characteristic peptide triplet sequences such as Gly-Pro-Hyp to Gly-Pro-Pro, Gly-Ala-Hyp to Gly-Ala-Pro, or Gly-Pro-Hyl to Gly-Pro-Lys and others for evaluation of hydroxylation throughout the entire molecule or at selected sequences.

The distribution of frequencies of HLA-DR alloantigens in HLA-DR4 negative subjects was determined in patients with Rheumatoid arthritis (RA) and normal individuals. An increased incidence of HLA-DR1 alloantigen in DR4 negative RA patients (45.9%) compared with DR4 negative healthy controls (23.6%) was found. The difference became significant when the incidence of DR1 was compared between patients with severe disease stages (III-IV) (75%) in contrast to 32% of incidence in patients of the milder stages (I-II) (p less than 0.05). Using Enzyme Linked Immunosorbent Assay we have determined the incidence of serum antibodies to native bovine type I and type II collagens and proteoglycans in patients with RA. Presence of serum antibodies to native type I collagen was detected in 59% of patients with RA, 60% of sera exhibited reactivity to type II collagen and 12% had antibodies to proteoglycans. There was no correlation between the presence of antibodies to type I and II collagens and disease stages, however, the incidence of serum antibodies to proteoglycans was increased in severe disease stages. On the other hand, the presence of high levels of antibodies to type I collagen was associated to HLA-DR1 antigen, (p less than 0.05).

The fluorescent antibody technique is nowadays mainly a technique, which is routinely carried out in all clinical-immunological laboratories. The main fields of application in clinical immunology are the determination of autoantibodies (e.g. ANA, AMA, smooth muscle antibodies, parietal cell antibodies), detection of B-lymphocytes as well as bacterial antigens (e.g. Treponema pallidum). Furthermore the demonstration of deposits of immune complexes in tissues (e.g. glomerulonephritis, periarteriitis nodosa). The presented paper shows the different fields of application of the fluorescent antibody technique. It is pointed out that the FAT could be only one out of the variety of methods in clinical-immunological investigations.

Immunohistochemical analysis of skin biopsies of 35 psoriatic patients is performed. Adenovirus antigens in the cells of perivascular infiltrates of the syncitial and papillary layers of the antigens were not detected in epitheliocytes and this allows considering the epidermal lesions and epidermal proliferation as caused by connective tissue alteration with subsequent loss of regulation control by basal cells. These processes are followed by immune disorders and inflammatory reaction as local manifestation of general disease. Deposits of immune complexes (C3-component, IgG, viral antigen), fibrin, albumin, C-reactive protein, destruction of cell and fibrillar connective tissue components followed by the inflammatory response result in the alteration of cell interaction in the connective tissue and dermo-epidermal interrelations and this is a basic mechanism of the damage to epidermis and its proliferation.

The immunogenicity of fibrous connective bovine tissue preserved by different methods was tested by a regional lymph node blastic cell reaction. The results were presented after transplantation of 10 experimental groups of grafts.

In a random sample of so-called healthy blood donors under and over 60 years of age (n=196 of 10,593 donations by about 4254 donors) the frequency of antibodies (antibodies against smooth muscles, mitochondria, microsomes, brush border of proximal rat renal tubuli, connective tissue, and parietal cells) detectable by immuno-fluorescence was determined and compared with the incidence thereof in various groups of diseases. In general, two frequency peaks (up to 30 and over 50 years of age) were observed; however, statistical evidence was obtained for the second peak only (with p less than 0.001). In "healthy" blood donors past 60 there was observed a significantly higher frequency of occurrence of antibodies against parietal cells (p less than 0.001) and against connective tissue (p less than 0.01). An attempt has been made to classify antibodies into four groups depending upon age disease, with due consideration being given to the different clinical valency of antibodies. Different frequencies of occurrence of numerous antibodies are believed to be due not only to immune system aging, but especially to the not-yet elucidated disturbance of regulation of autotolerance by alteration of immune cells, which is subject to endogenous (e.g., genetic) and exogenous (e.g. viral) factors that are dependent on age rather than aging.

By means of immunofluorescence using sera against three classes of human immunoglobulins (IgG, IgM, IgA), localization of the components (or one of the components) responsible for reaction of sera from patients with rheumatoid arthritis (RA) with connective tissue antigens was found to be in the cytoplasm of different kinds of cell elements of the connective tissue present in joints. Sera from RA patients collected at different stages of the disease reacted with connective tissue cell elements in titers of 1 : 64 to 1 : 1024, whereas sera from normal subjects (in 33% of specimens) reacted only to 1 : 8--1 : 16.

Sera from patients with chronic inflammatory disease were tested for the presence of connective tissue antibodies. Fetal human skin was used as a substrate in an indirect immunofluorescence assay. Histological staining for connective tissue fibres, antibodies to connective tissue components and immunoelectron microscopy were used to identify the antigenic structures. Some antigenic fibrils were associated with elastin. By immunoelectron microscopy antigenic extracellular microfibrillar structures were identified. Antibodies against these microfibrils were detected in higher titres only in chronic inflammation (14%). Our results suggest that antibodies against connective tissues microfibrils may occur in human sera.

The possibility that Graves' ophthalmopathy and autoimmune thyroid disorders may be associated because of autoimmune reactions against antigens shared between human orbital and thyroid tissues was investigated using anti-thyroglobulin (Tg) monoclonal and autoantibodies. Eleven of 16 mouse monoclonal antibodies (MCAB) tested reacted, in an enzyme-linked immunosorbent assay (ELISA), with an antigen in human orbital connective tissue membranes (OCTmem), but not with the OCT soluble fraction, or with membrane or soluble fractions of human eye muscle, lacrimal gland or skin connective tissue. The anti-OCTmem activity was absorbed by OCTmem and Tg, but not by liver membranes or bovine serum albumin (BSA). In preliminary studies four out of 113 human MCAB against thyroid or orbital tissue antigens showed reactivity restricted to Tg and OCTmem. Sera from approximately 50% of patients with autoimmune thyroid disorders, with or without ophthalmopathy, also reacted with OCTmem. The autoantibody activity correlated closely with serum titres of antithyroglobulin but not with the presence, duration, or severity of the eye disease. The OCTmem reactivity was absorbed by Tg, thyroid membranes, and OCTmem but not liver membranes, membranes prepared from other orbital tissues, or BSA. The OCTmem-Tg shared antigen site appeared not to be native thyroglobulin since, (i) MCAB and serum autoantibodies did not react with the cytosol fraction of OCT, and (ii) because the membrane antigen was not solubilizable. Because not all patients with ophthalmopathy have detectable anti-Tg antibodies and, conversely, because not all patients with detectable anti-Tg antibodies develop ophthalmopathy it is unlikely that autoimmunity against a OCTmem-Tg shared antigen is the primary mechanism of Graves' ophthalmopathy, although this possibility has not been excluded. On the other hand the reaction of anti-Tg autoantibodies with OCT membranes may be a model for other autoimmune reactions against other thyroid-orbital tissue-shared antigens. While the pathogenesis of Graves' ophthalmopathy is likely to be multifactorial, humoral and cellular reactions against primary orbital antigens, thyroid-orbitol tissue shared antigens, or both, are likely to play important roles.

The role of streptococcal infections in initiating the diverse clinical and pathological manifestations of rheumatic fever and rheumatic heart disease is considered in relation to the multiple cross-reactive relations of group A Streptococcus and tissue antigens. Autoantibodies to the following shared antigens have been demonstrated in sera of patients wit rheumatic fever: (1) cardiac, skeletal, and smooth muscle; (2) heart valve fibroblasts; (3) neurons in basal ganglia; and (4) a group A carbohydrate-related determinant in connective tissues. Circulating autoantibodies to these different antigens were present in higher titer or occurred more frequently in patients with rheumatic fever than in those with uncomplicated streptococcal infections. A direct correlation of the presence of these autoantibodies with carditis could not be established. The pathogenetic mechanisms that link streptococcal infection to rheumatic fever and rheumatic heart disease are not yet clear. Among the possibilities to be considered within the above frame of reference are combined cell-mediated and humoral autoimmune mechanisms directed to one or more cross-reactive antigens in the tissues, selective binding of streptococci to tissues, role of circulating immune complexes, and linkage with histocompatibility antigens.

The incidence of connective tissue antibodies was assessed in 3,000 adults and in 388 pediatric patients with gastroenterological disorders. Following a previous classification, connective tissue antibodies were distinguished in 5 different types, R1 and R2 reacting with reticulin components, KC with Kupffer cells, AC and Rs with intra- and extracellular mesenchymal antigens. R1 predominated in children and was observed only in patients with malabsorption; the great majority of them had celiac disease and an abnormal small bowel mucosa. Ac was rarely seen in children and occurred in several unrelated gastrointestinal diseases of the adult; its incidence, however, was significantly higher in disorders with idiopathic and secondary malabsorption. R2, KC and Rs were seen only occasionally in unrelated gastroenterological disorders without malabsorption. R1, therefore, seems to be strongly suggestive of a malabsorption syndrome with abnormal small bowel mucosa and AC is a nonspecific reaction occuring predominantly in primary and secondary malabsorption; the other connective tissue antibodies have no clinical significance in gastroenterological disorders.

Children with rheumatoid arthritis in remission or minimal activity of the disease were examined after sanatorium treatment. It was established that health resort factors had produced a positive capillarotropic effect in rheumatoid arthritis, i.e. capillarotropic insufficiency, tissue hypoxia, inflammation and proteolysis reduced; microcirculation returned to normal.

The article presents a review of literature data and the authors' own investigations dealing with the dynamics of accumulation and distribution of free cells of the connective tissue in various forms of pathological proliferation of the epithelium (inflammatory overgrowth, disharmonal hyperplasia, tumours). Despite the availability of numerous data on morphological characteristics and functional significance of free cells, there are no reliable data on regularities of accumulation and distribution of individual cellular forms depending upon the character of a pathological process, its localization and extent of changes in the parenchymatous elements of the organ. But implementation of a local immunological control and maintenance of structural homeostasis should be linked first of all with reactions of free cells of the stroma. Elucidation of the role of cellular reactions in the stroma of tumour formations is of importance for prognostic evaluation of a possible progression. The authors showed that in experimental reproduction of disharmonal hyperplasia and tumours of the uterus and mammary gland there was a correlation between accumulation and distribution of eosinophilic leucocytes and plasmatic cells and the extent of changes in the epithelium. The presence of the cells of the immunocompetent series may be considered as an evidence of immunological reactions occurring as a responce to changes in antigenic properties of the epithelium in the course of malignization. The authors believe that knowledge of conjugated reactions of the stroma and epithelium in inflammatory, hormonal, and tumour growth may contribute to better understanding of the mechanisms of correlation of tissues in the course of a proliferative process and to ascertaining the causes of impairment of this correlation.

Fourteen patients with rheumatoid arthritis were tested for the presence of cell-mediated immune reactivity to various ocular antigens by means of the leucocyte migration inhibition test. A significant difference in leucocyte migration was found between patients and healthy controls after incubation of their cells with purified rhodopsin, uveal pigment granules or soluble uveal antigens, but not with rod outer segments or soluble retinal antigens. Some indications have been obtained that the sensitization to rhodopsin in fact originates from a sensitization to connective tissue structural glycoproteins.

Adult mice were found to show regional variation in the epithelial expression of some molecules of the blood-group antigen series. To investigate connective tissue influences on such differences, heterotypic recombinants of epithelia and connective tissues from various regions were prepared and examined using monoclonal antibodies directed against bloodgroup antigens H and Ley. The results indicate that epithelia may maintain a preexisting regionally specific pattern following recombination but that, in some recombinant matches, the connective tissue is capable of signalling redirection of the pattern of expression towards that typical of the epithelium with which it is normally associated.

Interleukin-1 (IL-1) is the name given to the polypeptides produced by activated mononuclear phagocytes which were originally defined as lymphocyte activating factors (LAF). Administration of IL-1 in vivo causes fever and synthesis of acute phase proteins. In vitro they have been shown to cause cartilage and bone resorption, and to stimulate fibroblasts and chondrocytes to make prostaglandins and latent collagenase. IL-1 has therefore been proposed to be an important inflammatory mediator and may be involved in the destruction of cartilage and bone that is a feature of rheumatoid arthritis and other inflammatory diseases of joints. We therefore looked for IL-1 receptors on connective tissue cells which might be targets for therapeutic intervention. Here we report the iodination, to high specific activity and with retention of full biological potency, of the two types of natural porcine IL-1. These ligands have been used to demonstrate high affinity dissociation constant (approximately 10(-10) M) specific binding sites on pig chondrocytes and synovial fibroblasts, human dermal fibroblasts and murine osteoblasts (3,000-5,000 sites per cell). Most interestingly, the two different Il-1 proteins show a similar affinity for a common class of receptors.

Unfixed cryostat sections of an organ block comprising the liver, stomach, and kidney of rats as well as the kidney of mice were used as antigenic substrate for detecting antibodies against connective tissues by indirect immunofluorescence. There were found more than 14 different fluorescent patterns. Two types could be distinguished easily: The "type 1" shows a clear fluorescence of fine perivascular fibres and a faint fluorescence of vascular endothelium in the liver and in the kidney. The sinusoids are negative. The "type 2" is characterized by a bright and broad fluorescence of the sinusoids and of the portal tract without perivascular staining of the liver. On the sections of the kidney a fluorescence of vascular endothelium, of the tubular basement membrane or of the peritubular connective tissue fibrils is typical.

Paraffin-embedded surgical pathology specimens from skin (5) and muscle (2) biopsies, from Morton's neuromas (3), traumatic neuromas (8), schwannomas (21), neurofibromas (12), and from one perineurioma and one neurothekeoma were studied by immunoperoxidase histochemistry and antibodies against epithelial membrane antigen (EMA), Leu 7 epitopes (Leu 7), S-100 protein (S-100) and cytokeratins. Normal, reactive and neoplastic perineurial cells stain consistently for EMA, whereas Schwann cells express Leu 7 and/or S-100 positivity. None of the immunoreactive cells stained for cytokeratin. Our findings indicate that perineurial and Schwann cells can easily be distinguished by their different patterns of immunoreactivity with the above markers.