Immunohistochemical detection of factor XIIIa and factor XIIIs in synovial membranes of patients with rheumatoid arthritis or osteoarthritis.

In spite of differences in etiology, RA and OA lead to astonishingly similar synovitic alterations. Fibroblastic transformation of the synovial membrane and an increase in monocytes constitute a rare but highly characteristic feature of RA. Monocytes synthesize factor (F) XIII, implying that FXIII (a and s) in synovial tissue might help to differentiate between RA and OA. Biopsies were obtained at open surgery from 98 unselected patients with the clinical diagnosis of RA (n = 54) or OA (n = 44). In a three-stage (ABC) immunoperoxidase technique, polyclonal antisera against factor XIIIa and factor XIIIs were investigated. Compared to OA sections. RA synovium showed more FXIIIa-positive cells-monocytes, fibrocytes, fibroblasts and synovial lining cells. In the subsynovial layer, band-like structure of FXIIIa-stained cells was observed in 27.8% of the RA patients, but in only one OA specimen. Higher proportions of FXIIIa-positive monocytes, macrophages, histiocytes and fibroblasts, as well as positive Langhans giant cells and vascular wall regions (except endothelial cells), were observed in RA. OA specimens revealed more intense FXIIIa labeling of these cells with a lower percentage of stained cells. Overall, labeling with FXIIIs antibody resulted in less intense staining. In conclusion, distinction between synovitis caused by RA and synovitis due to OA is possible, as the former show higher numbers of FXIIIa-positive cells, including monocytes, fibroblasts, fibrocytes and synovial lining cells. Further more, RA tissue is stained less intensely than OA tissue. There is evidence for continuous excretion of FXIII in the synovial membrane by the above-mentioned cell systems.

Quantitative photometrical assessment of iron deposits in synovial membranes in different joint diseases.

We investigated 86 synovial membranes from patients suffering either from rheumatoid arthritis (RA) or osteoarthritis (OA). Iron deposits in the synovial membrane were stained by the Prussian blue reaction, and the amount of stained iron was quantitatively assessed by microscope photometry. We found a statistically significant increase in iron deposits in the synovial membrane of RA patients when compared to OA patients. The amount of iron deposits correlated with the histological subtype of synovitis, those presenting with more exudative and proliferative features showing greater amounts of iron deposits. We also observed an inverse correlation between the haemoglobin concentration and erythrocytes in the serum and the amount of iron in the synovial membrane. From our data we concluded that iron deposits in the synovial membrane can contribute by several mechanisms, including activation of oxygen radicals, to the chronic inflammatory reaction in RA synovitis.

Morphometric analysis of the angioarchitecture of the synovial membrane in rheumatoid arthritis and osteoarthritis.

Using three different immunohistochemical methods we measured the number of vessels, vessel area and diameter and their form factor in the synovial membrane of 102 patients suffering from different joint disease. The variables were evaluated by means of immunomorphometric analysis. We found UEA (Ulex europeus) immunostaining to be the optimal method for quantification of data characterizing the vasculature of the synovial membrane. Irrespective of causes of the given joint disease, we found increases in the number of vessels, vessel perimeter, vessel area and the product of the number of vessels and vessel area over and against the control group (patients without arthritis). Consequences are discussed regarding local bioavailability of medicaments in the synovial membrane.

Two-dimensional electrophoresis of synovial tissue, synovial fluid and serum in patients with rheumatoid arthritis and related diseases.

Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.

The neo-synovial membrane of patients with active rheumatoid arthritis at resynovectomy. Histomorphological and immunohistological evaluations.

Neo-synovial membranes, which formed after "primary" synovectomy in 21 patients with rheumatoid arthritis (RA), were studied at resynovectomy. The clinical, histomorphological, and immunohistological data were compared with data derived from "primary" synovial membranes from RA and osteoarthritis (OA) patients. The clinical data suggest a less active rheumatoid inflammatory response after synovectomy. Histomorphologicaly, the synovitis in resynovectomized neosynovial membranes of RA revealed no qualitative differences when compared with synovitis in the "primary" synovium. However, the degree of the inflammatory rection evaluated by the different parameters was found to be distinctly lower. The immunohistological data correlated with these findings.

Histological classification of synovitis.

A simple system of synovitis classification is proposed based on histomorphological evaluation of 400 random specimens of synovial tissue. It discriminates between two types of diagnosis: 1) one with a high degree of information, being called C-synovitis (synovitis with a characteristic or diagnostic histomorphological pattern), where the clinical diagnosis can be predicted on the basis of the histomorphological pattern, 2) a second type of diagnosis with a lower degree of information, being called NC-synovitis (synovitis with non-characteristic or non-diagnostic morphological findings in the synovial membrane). We subdivided NC-synovitis into 4 subgroups and determined the RA-probability of each subgroup in order to permit clinical use of the proposed histomorphological classification. In addition, by using immunoperoxidase staining methods for immunoglobulins the subgroups were shown to have different quantities of extravasally deposited immunoglobulins in the synovial membrane. The subgroup with the highest RA-probability also turned out to have the highest amount of extravasally deposited immunoglobulins.

[Clinical value of histomorphologic studies of joint capsules]. / Klinische Wertigkeit der histomorphologischen Untersuchungen von Gelenkkapseln.

The clinical significance of the histomorphological evaluation of synovial tissue from 389 patients with joint diseases was studied. From these data we conclude: (1) the histomorphological diagnosis in joint diseases is rarely of decisive value (6.2%); (2) in 23.1% of cases the histomorphological diagnosis is judged to be of clinical assistance; (3) the histomorphological grading of the inflammatory reaction in the joint under study corresponds in approximately two out of three cases to the clinical judgement.

Immunophotometric quantification of extravascular immunoglobulin deposits in the synovial membrane of patients with osteoarthritis and rheumatoid arthritis.

The amount of extravascular immunoglobulin deposits in the synovial membrane of patients with osteoarthritis and rheumatoid arthritis was studied in comparison with that of patients suffering from non-joint diseases. Immunoglobulin deposits were immunostained using the three-layer immunoperoxidase method. The staining results were quantified with the help of a microscope photometer connected with a scanning stage. Several experiments involving artificial test substrates, diseases with allegedly increased extravascular deposits or diseases not exhibiting extravascular deposits of immunoglobulins are used to validate the described microspectrophotometrical approach for measuring extravascular immunoglobulin deposits. The scanning photometry demonstrates significantly higher amount of extravascular immunoglobulin deposits in rheumatoid arthritis as compared with osteoarthritis and non-joint diseases.

[Results of long-term D-penicillamine therapy in chronic polyarthritis]. / Ergebnisse der D-Penicillamin-Langzeittherapie bei chronischer Polyarthritis.

In 34 patients with severe rheumatoid arthritis the therapeutic efficacy of 600 mg/die of D-penicillamine was evaluated for five years. Patients were included in the study only if the duration of their disease exceeded 6 months (upper limit 12 months), and there was involvement of 6 or more joints and an elevated erythrocyte sedimentation rate (1 Hour values greater than or equal to 25 mm). In 9 patients (26%) a decline in the activity index could be observed, but 7 of these subjects had a deterioration of findings as assessed by x-ray. The increase of the activity index indicated no therapeutic benefit in 25 patients (74%), which could be confirmed in all subjects by x-ray.

In situ evaluation of immunoglobulin synthesis of synovial plasma cells in rheumatoid arthritis and osteoarthritis by plug photometry.

Synovial membranes of patients with osteoarthritis--a disease with unaltered immunoregulation--and rheumatoid arthritis were immunostained for the different immunoglobulin isotypes using the PAP-method and a two-layer alkaline phosphatase technique. Randomly selected plasma cells with positive immunostaining were measured by plug photometry. The average extinction of the single plasma cell is considered to be indicative of the immunoglobulin synthesis of the measured plasma cell and the isotype under study. When we compared both disease entities, patients with rheumatoid arthritis were characterized (i) by higher average extinction values for all Ig-isotypes except IgG (ii) by a higher rate of high producer plasma cells. From this data, we conclude that immunophotometry is a new in vivo method for discriminating two types of synovitis (i) one without increased Ig-synthesis at the single cell level and (ii) a second one with increased Ig-synthesis.

[Effectiveness of D-penicillamine therapy in chronic polyarthritis]. / Untersuchungen über die Wirksamkeit der D-Penicillamintherapie bei chronischer Polyarthritis.

In 52 patients with severe rheumatoid arthritis the therapeutic efficacy of 600 mg/die of D-penicillamine was evaluated for two years in a prospective trial. Patients were included in the study only if the duration of their disease exceeded 6 months (upper limit 12 months), and there was involvement of 6 or more joints and an elevated erythrocyte sedimentation rate (1 hour values greater than or equal to 25 mm). In 26 patients (50%) a decline in the activity index could be observed, but 18 of these subjects had a deterioration of findings as assessed by X-ray. The increase of the activity index indicated no therapeutic benefit in 26 patients (50%), which could be confirmed in 23 subjects by X-rays.

Distribution of lysozyme in synovial tissue of patients with osteoarthritis and rheumatoid arthritis demonstrated by different enzyme histochemical methods.

Lysozyme-producing cells were analysed by enzyme histochemistry in paraffin sections of synovial tissue of 60 patients with rheumatoid arthritis (RA) and 20 patients with osteoarthritis (OA). For lysozyme detection three enzyme histochemical systems - peroxidase-anti-peroxidase, alkaline phosphatase and biotin-avidin - were used in parallel experiments. Lysozyme was found to be produced by polymorphonuclear cells, mononuclear phagocytes and part of synovial lining cells. All types of lysozyme-producing cells were increased in RA compared with OA. Subgrouping of RA synovitis according to histomorphological criteria allowed the demonstration of an inverse relationship between the number of lysozyme-producing cells and the grade of proliferation of fibroblasts, called mesenchymoid transformation by Fassbender [19]. The different methods of lysozyme detection differed in specificity and sensitivity. The immunoenzymatic staining of lysozyme allows specific and quantitative evaluation of phagocytizing cells in RA and OA.

[The effect of basic therapeutic procedures on the immunoregulatory T-cell apparatus in patients with chronic polyarthritis]. / Der Einfluss von Basistherapieverfahren auf den immunregulatorischen T-Zellapparat bei Patienten mit chronischer Polyarthitis.

Five by five patients with rheumatoid arthritis were treated with gold salts, D-penicillamine and cyclophosphamide. The T-lymphocyte subpopulations were measured. In patients, treated with gold salts or D-penicillamine, we did not find alterations of the number of TG- and TM-lymphocytes. The daily treatment with 100 mg cyclophosphamide will result in a significant reduction of TG-lymphocytes.

[Distribution of immunoglobulin classes in joint capsules in osteoarthritis (arthrosis) and chronic polyarthritis]. / Beitrag zur verteilung der Immunglobulin-Klassen in Gelenkkapseln bei Osteoarthritis (Arthrose) und chronischer Polyarthritis.

The distribution of the various classes of immunoglobulins (IG) in synovial membranes in rheumatoid arthritis (RA) and in osteoarthritis (OA) is analyzed. Both plasma cells and extravascular deposits of immunoglobulins are stained using class-specific antisera and the peroxidase-antiperoxidase technique. It can be shown that the local humoral immune response in RA-Synovialitis occurs at a higher level than that of OA; inflammatory infiltrates are denser and plasma cells are activated to a higher degree which is evident in the greater intensity of cytoplasmic staining. While there is no difference in the relation between singular classes of IG in these two groups of diseases, the fraction of plasma cells synthesizing an immunoglobulin is greatly elevated in RA-Synovialitis and there is a correlation of this to the morphological degree of RA-Synovialitis. This applies even more to extravascular deposits of IG with rheumatoid-factor-activity. The determination and staining of extravascular immunoglobulins with a polyvalent anti-human-serum or with antisera specific for the gamma, alpha, and mu-antigens of heavy chains as well as the demonstration of rheumatoid-factor activity of these IG constitute a possibility to histochemically discriminate between RA-Synovialitis and OA-induced synovialitis. In OA, intensely stained areas of IG with rheumatoid-factor-activity located extravascularly are never found.

[Problems of therapy control in arthroses and suggestion of a method for the non-invasive identification of changes in joint morphology (knee joint) (author's transl)]. / Probleme der Therapiekontrolle bei Arthrosen und Vorschlag eines Verfahrens zur nicht-invasiven Erfassung von Vernderungen der Gelenkmorphologie (Kniegelenk).

The article explains the fundamental problems in therapeutic studies of the primary and secondary processes in arthrosis. Many drugs are on the market for treating the secondary phenomena; however, such phenomena can often be assessed only on the basis of "soft" action criteria (verbal information given by the patients). Biochemical examinations of the synovial fluid and methods to assess joint morphology, are particularly suitable for evaluating the primary changes taking place in the joint cartilage. An assessment of the "joint noises" is known in literature as a non-invasive method. An improved method of recording and evaluating these joint noises is suggested. It is characterized by the following features: - registration of signals with defined joint load - classification of the noise phenomena with reference to the joint position, i.e. to joint areas - by separation of the signal into movement cycles elimination of artificial noise producers - evaluation of those signal periods only which are disease-specific. The article shows examples by quoting signals with the respective frequencies for healthy and diseased persons, and explains the suitability of the method in therapy control by describing two examples of surgical treatment.

[Enzyme histochemical demonstration of immunoglobulins in joint capsules in chronic polyarthritis and active inflammatory arthritis]. / Beitrag zum enzymhistochemischen Nachweis von Immunglobulinen in Gelenkkapseln bei chronischer Polyarthritis und entzündlich aktivierter Arthrose.

In rheumatoid arthritis, immunoglobulins are detectable in joint capsules within the subsynovial space and in superficial cell layers in significantly greater frequency as compared with osteoarthritis. These extravascular immunoglobulins were demonstrated in good agreement by a three-step immuno-peroxydase technique and a two-step alkaline phosphatase technique, the former being preferable because of reduced unspecific background staining. Both techniques are at least as sensitive as conventional immunofluorescence while in addition rendering the histological evaluation of the entire joint capsule possible. Thus, the detection of extravascular immunoglobulins can be related to their activity as mediators of inflammation. Accordingly, hypertrophy and hyperplasia of superficial cells as well as stromal proliferation could be consequences of immunoglobulins or immune-complexes persisting in the joint capsule. The histochemical techniques described here are suitable to morphologically distinguish osteoarthritis from chronic polyarthritis in histological sections. The presence of immunoglobulins in superficial cell layers and/or extravascular spaces argues in favour of rheumatoid arthritis.