Polymorphonuclear cell interaction with immunoglobulin in situ in human rheumatoid synovium.
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Biomedical subjects
Publications and source records attributed to S Reitamo.
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Middle ear mucosal biopsies were taken from 11 patients with middle ear effusion. In 8 cases the specimens were sufficiently large to allow detailed studies of the submucosal cellular components. It appears that in noncomplicated serous middle ear effusion, due to mechanical obstruction of the Eustachian tube, the submucosa is not infiltrated by inflammatory cells. In all types of mucoid effusion of variable duration, various lymphocyte classes, i.e. T- and B-lymphocytes and plasma cells, were present, suggesting a normal cellular immune response. The lack of granulocytes seems to indicate that there is no submucosal infection.
T Lymphocytes and mononuclear phagocytes were measured quantitatively with the histochemical acid alpha-naphthyl acetate esterase (ANAE) method from paraffin sections of skin affected by systemic and discoid lupus erythematosus and by Jessner's lymphocytic infiltrate. The composition of the patchy cutaneous mononuclear cell infiltrates was similar in these three disorders.
Histochemical and immunohistochemical techniques were used to identify T lymphocytes, mononuclear phagocytes and plasma cells in situ from allergic and toxic epicutaneous test reactions. Intracellular alpha-naphthyl acetate esterase (ANAE), endogenous peroxidase and immunoglobulin were used as markers for inflammatory cells. In allergic contact dermatitis 76 +/- 7% of all cells were ANAE-positive T lymphocytes, 13 +/- 6% mononuclear phagocytes and 12 +/- 6% ANAE-negative cells. In toxic skin lesions the corresponding values were 64 +/- 20%, 18 +/- 15% and 18 +/- 6%, respectively. There were no statistically significant differences between the allergic and toxic skin reactions. The basic reaction type in allergic and toxic contact dermatitis seems to be similar, with possibly some qualitative and quantitative differences.
Skin lesions were produced by application of 50% potassium iodide to twelve patients with dermatitis herpetiformis (DH). Perivascular cellular infiltrates were found to be characteristic of developing lesions. The cells were mainly round cells; alpha-naphthyl acetate esterase staining revealed that in 24-h lesions the mean percentage of T-lymphocytes was 43%, that of mononuclear phagocytes 6% and that of non-T/non-M cells (mainly B-lymphocytes) 44%. The percentage of the latter was highest (mean 81%) in 6-h specimens, suggesting that these cells are participating in the early stages of lesion formation. The infiltrating cells in dermal papillae and within subepidermal vesicles were predominantly polymorphonuclear leukocytes (mean 86%) with some mononuclear phagocytes and non-T/non-M cells. Immunofluorescence examination confirmed that fibrin deposition is characteristic of the initial lesions of DH and showed that the same is true of fibronectin. Seven out of eight patients had fibronectin deposits in dermal papillae. IgA was found in all and C3 in most of the specimens and, with the exception of papillary vesicles and blister cavities, the intensity of IgA and C3 fluorescence showed no marked alterations during the development of lesions.
We used an immunoperoxidase method to study the distribution of lactoferrin in seven human fetuses from 11 to 21 weeks of age. Lactoferrin was found in mononuclear cells, presumably granulocytes, in various organs from thirteen weeks of gestation onwards. In addition to the liver, spleen, lung and thymus, lactoferrin-positive mononuclear cells were also found close to vessel walls in human pancreas in fetuses more than 20 weeks old. This finding might suggest that there is significant granulopoiesis in the pancreas at this stage of maturation. In addition to the mononuclear cells, lactoferrin could be found in Hassal's corpuscles of the thymus from 13 weeks and in glandular cells of the tongue from 20 weeks onwards. The present results are in good agreement with the maturation of humoral and cellular acquired immunity and the onset of synthesis of lysozyme and interferon.
This is the first reported case in which neoplastic cells not of granulocytic or monocytic origin have been shown to contain lysozyme. A highly differentiated metastasizing adenocarcinoma of the jejunum is presented. Areas of the tumour contained cells resembling Paneth cells, that is, they contained cytoplasmic granules with 1) stained red with Masson's trichrome and 2) were shown to contain lysozyme by an immunoperoxidase technique. No argentaffin cells could be identified within the tumour. Staining of mucosubstance revealed large intestine-type sulfated glycoproteins in the tumour tissue. The presence of lysozyme-containing neoplastic Paneth cells suggests that the tumor: 1) originated from the mucosal crypts, and 2) had a high degree of cellular differentiation.
A nephelometric lysozyme (LZM) assay method was compared to two commonly used enzymatic LZM assay methods. LZM concentrations obtained with the nephelometric method correlated well with those obtained with the spectrophotometric (r = + 0.98) and the lysoplate (r =+ 0.93) methods. The precision of the nephelometric method was 5.1% (CV). The nephelometric LZM assay offers a simple, reproducible and rapid assay method for serum LZM determinations.
A patient with Waldenström's macroglobulinaemia presenting with numerous red or brownish-red, round, papulous lesions of the skin is reported. Skin biopsy revealed a massive infiltrate of immature lymphatic cells of the dermis down to the subcutaneous fat. Approximately 80% of these cells were positive for intracellular IgM and kappa light chains as shown by the immunoperoxidase method. This finding established the skin lesions as part of Waldenström's macroglobulinaemia.
An immunoperoxidase technique was used to study the distribution of lactoferrin (LF) in human salivary glands from autopsy tissues fixed in Carnoy's fluid for the optimal preservation of LF antigenicity. Specific LF staining was seen in the intralobular ducts of all salivary glands but never in the interlobular ducts; acinar LF was detected in most serous demilunes of the mixed glands and in some but not all acinar cells of the pure serous glands. No LF was detected in the acinar cells of the pure mucous glands. In the oral tissues studied the only additional cells containing LF were polymorphonuclear leucocytes. Whether LF in the salivary glands is synthesized locally and/or ultrafiltrated from the blood is at present unclear. Present evidence indicates that the biological role of salivary LF is antibacterial.
Optimally fixed paraffin enbedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.
Immunoperoxidase identification of intracellular immunoglobulins from cell smears. Am J Clin Pathol 73: 248--249, 1980. Optimal fixation procedures for intracellular immunoglobulins (Ig) were defined by using cytocentrifuged or conventional cell smears of pokeweed-mitogen-stimulated human lymphocytes. The Ig was detected by the unlabeled-antibody-enzyme technic. The results indicate that superior preservation of Ig antigenicity combined with good morphologic integrity is obtained with 2--4 min fixation in Baker's formol-calcium at 20 C with subsequent wash in distilled water. This technic is currently used for the detection of Ig isotypes from bone marrow aspirates and from B cell mitogen-stimulated cell cultures.
Histochemical and immunoperoxidase techniques were used to characterize the spatial relationships of various inflammatory cell types to the different transplant structures in human renal allograft rejection. T lymphocytes were identified by acid alpha-naphthyl acetate esterase (ANAE) staining, plasma cells by intracytoplasmic immunoglobulin, mononuclear phagocytes by intracytoplasmic 'dispersed' ANAE reaction and/or lysozyme staining and granulocytes by intracellular lactoferrin. In the two cases of acute rejection the infiltrate around the blood vessels consisted mainly of lymphocytes, whereas the infiltrate around the tubules and within the glomerular tufts consisted mainly of mononuclear phagocytes. In acute rejection only a few plasma cells and granulocytes were seen. In the single case of chronic rejection studied, the lymphocytes were no longer concentrated exclusively around the blood vessels, but diffusely distributed throughout the kidney parenchyma. The different distribution of various inflammatory cells may reflect differences in the functions of these cell types in graft destruction.
The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.
Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.
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An immunoperoxidase staining method was used to identify lysozyme in biopsy or transplantectomy specimens of human renal allografts during reversible and irreversible rejection of the grafts. Proximal tubules in apparently functioning nephrons showed lysozyme staining. In irreversibly rejected grafts, infiltrating mononuclear phagocytes in and near peritubular and glomerular capillaries also stained intensely for lysozyme. In acute necrotizing arteritis, lysozyme-positive cells (mononuclear phagocytes) infiltrated the blood vessel wall. The presence of infiltrating lysozyme-positive cells in the transplant was consistent with poor graft survival. The variation in lysozyme staining of proximal tubular cells apparently was a reflection of the differences in the reabsorption capacity of the tubular cells, attributable to the tubular dysfunction of renal allografts. The infiltrating lysozyme-positive cells probably contribute to the increased urinary excretion of lysozyme during acute rejection.
The preservation of lysozyme (LZM) antigenicity was studied in paraffin embedded tissue blocks. The reactivity for LZM varied with the type of tissue studied, the fixative used, the osmolarity and pH of the fixative, fixation time and temperature, and the method of dehydration. In both rat and human tissues aqueous fixatives were superior to nonaqueous fixatives in retaining LZM antigenicity. Brief fixation in fixatives of low osmolarity enhanced LZM staining in the parenchymatous tissues but diminished staining in human cartilage; prolonged fixation in fixatives of high osmolarity gave opposite results. Least affected by fixation was the LZM antigenicity in the serous cells of the glands of the respiratory tract. These cells also stained most intensely for LZM of all autopsy material studied.