Elastolytic activity of human monocytes from synovial fluid and blood of patients with arthritis. Relations to levels of interleukin 6 and soluble interleukin 2 receptor.

Synovial fluid (SF) and blood from 24 patients with non-traumatic, sterile hydarthron were examined for monocyte elastolysis (MøE) and for levels of interleukin 6 (IL-6) and of soluble interleukin 2 receptor (sIL-2R). Six patients had osteoarthrosis (OA) and 18 patients had inflammatory hydarthron (IH), 10 of whom had rheumatoid arthritis (RA). Blood MøE was lower in OA than in IH, both measured as basal MøE activity and after in vitro stimulation with immune complexes and phorbol myristate acetate (PMA). SF MøE was higher than MøE in blood (p less than 0.01). This increase in SF MøE could be mimicked in vitro by prestimulation of blood Mø with low levels of IC. SF IL-6 and sIL-2R were also elevated (p less than 0.01). All three parameters correlated to the degree of joint inflammation evaluated by SF leucocyte level, complement activation, blood C Reactive Protein, and to the clinical evaluation of the joint. The increase in SF MøE, IL-6 and sIL-2R in patients with IH, points to a stimulation of Mø and lymphocytes in the joint.

Basal and stimulated elastolytic activity of blood monocytes is increased in glucocorticoid-treated giant cell arteritis.

The elastolytic capacity of live human blood monocytes was studied in patients with giant cell arteritis (GA) and in age-matched controls. Despite normalized acute-phase reactants during glucocorticoid (GC) therapy, the basic activity of monocytes from patients with newly diagnosed GA was elevated compared with controls (80 vs. 39 ng/h, p less than or equal to 0.01). The maximum response was enhanced by stimulation with immune complexes (224 vs. 125 ng/h, p less than or equal to 0.01) and with phorbol myristic acetate (324 vs. 214 ng/h, p less than or equal to 0.01). No age difference was found between healthy young and old people. Cell surface related human monocyte elastolytic activity could act as a sensitive marker of cell activation in vivo.

Blood monocyte elastolytic activity enhanced by immune complex stimulation in patients with rheumatoid arthritis and normal controls. Effect of aurothiomalate or D-penicillamine.

Blood monocyte elastolytic activity in patients with rheumatoid arthritis and in normal controls was studied by a new in vitro technique. The enzyme activity of live cells was measured by soluble [3H]elastin hydrolysis first under basic conditions and then after immune complex stimulation. The cells from patients had a higher elastolytic potential than cells from controls (p less than 0.02) and responded to smaller amounts of immune complex (p less than 0.01), even in patients treated with D-penicillamine or aurothiomalate. Treatment of normal monocytes in vitro with aurothiomalate did not influence the elastolytic response. These findings indicate that monocytes from patients with rheumatoid arthritis have an enhanced elastolytic activity compared with cells from normal controls and may invoke greater tissue damage on immune complex stimulation.

Mycoplasma pneumoniae--stimulation of lymphocytes obtained from adenoid vegetations and blood in children with and without serological evidence of Mycoplasma pneumoniae infection.

Thymidine incorporation in lymphocytes obtained from adenoids (AVL) and blood (PBL) and stimulated in vitro with Mycoplasma (M.) pneumoniae antigen, was determined in 16 children with serological indication of a current or recent M.pneumoniae infection, and in 14 controls. Both AVL and PBL gave higher responses in the M.pneumoniae-positive group than in the control group. The best discrimination between the two groups was achieved by AVL stimulated by M.pneumoniae, AVL did not respond, indicating that in this assay recent or actual M.pneumoniae infection is better expressed by AVL than by PBL.

Autoimmune haemolytic anaemia treated with multiple transfusions, immunosuppressive therapy, plasma exchange, and desferrioxamine.

Severe autoimmune haemolytic anaemia in a five year old boy was unaffected by treatment with prednisone and splenectomy, but subsided after combined immunosuppressive therapy and three plasma exchanges. Over five months, a total of 93 transfusions of concentrated erythrocytes was given (equal to 18.6 grams of iron or 1.1 g/kg BW). This resulted in severe iron overload with cardiac, hepatic, and pancreatic complications, together with growth-retardation. These complications disappeared after treatment with desferrioxamine and vitamin C, but despite a normal growth hormone response to glucagon the concentration of somatomedin in serum remained low. Treatment by plasma exchanges and immunosuppressive agents may therefore be of value in severe haemolytic anaemia refractory to corticosteroids and splenectomy. Iron chelating therapy should be considered if multiple transfusions result in iron overload.

Humoral and cellular immunity in typhoid and paratyphoid carrier state, investigated by means of quantitative immunoelectrophoresis and in vitro stimulation of blood lymphocytes.

The humoral and cellular immunity of 8 Salmonella carriers and 9 non-carriers was investigated and compared to findings in normal persons. The antibody response in serum and intestinal secretions was investigated by means of crossed immunoelectrophoresis, and blood lymphocytes were investigated by in vitro stimulation. Both carriers and non-carriers showed increased levels of precipitating antibodies as well as lymphocyte responses, when compared to normal persons. No differences in the antibody responses either qualitatively or quantitatively were found between carriers and non-carriers. Only few precipitins could be detected in intestinal secretions. Lymphocyte responses to S. typhi and S. paratyphi were significantly higher in carriers and non-carriers than in the controls. No significant difference in lymphocyte response to S. typhi, S. paratyphi and mitogens was obtained between carriers and non-carriers. Thus, the carrier state seems not due to detectable major immunodeficiency.

Specificity of lymphocyte stimulation in vitro by microbial preparations: importance of antigen concentration.

Blood mononuclear cells were stimulated in vitro by 7 different microbial preparations, and thymidine incorporation was investigated in 7 groups of persons sensitized to the microorganisms and in 7 groups of controls. The sensitized persons were: 18 BCG-vaccinated, 13 with previous Mycoplasma pneumoniae pneumonia, 22 who had serum antibodies to Herpes simplex virus, 14 with previous typhoid fever, 13 with previous acute pyelonephritis caused by Eschrerichia coli, 6 with previous Haemophilus influenzae meningitis, and 15 tetanus-vaccinated persons. Employing microbial preparations corresponding to the type of sensitization, higher responses were obtained with PPD, Mycoplasma pneumoniae, Herpes simplex virus, Salmonella typhi and tetanus toxoid in sensitized than in controls, but the responses to E. coli and H. influenzae were similar. In each experiment a dose titration of the microbial preparation was carried out. The best separation between sensitized and non-sensitized persons was obtained at low concentrations, giving submaximal stimulation. It is concluded that lymphocyte responses to many microbial preparations are combined of antigen-specific components and of responses to cross-reacting or polyclonally activating moieties, and that the antigen-specific response is best investigated at low concentrations of these preparations.

Quantitative immunoelectrophoretic analysis of Salmonella typhi antigens and of corresponding antibodies in human sera.

By quantitative immunoelectrophoretic methods, 86 different antigens were found in sonicated preparations of a Salmonella typhi, using corresponding rabbit antiserum. Lipopolysaccharide was identified, and 5 other antigens characterized. Antibodies against a total of 19 S. typhi antigens were found in human sera. The antibody response in patients with typhoid fever was significantly more pronounced compared to that in normal persons. When the antibody response was expressed as a precipitin score, the predictive value of both a positive (precipitin score greater than or equal to 8) test and a negative test was 100%, when sera from patients suffering from typhoid fever were compared with sera from normal persons.

Mycoplasma pneumoniae antigens demonstrated by crossed immunoelectrophoresis.

Soluble Mycoplasma pneumoniae antigens were analysed by crossed immunoelectrophoresis. At least 15 precipitation arcs were developed between the soluble antigens and rabbit anti-Mycoplasma pneumoniae hyperimmune sera. Sera from Mycoplasma pneumoniae infected patients formed precipitation arcs which were identified with those formed by a chloroform-methanol extract of Mycoplasma pneumoniae. Low concentrations of human antibodies, not detectable in a complement fixation test, were found to react with these antigens. The antigens were shown to cross-react with antigens of Mycoplasma neurolyticum. Emphasis was placed on the technical development of the crossed immunoelectrophoresis for the analysis of immune-precipitable antigens of Mycoplasma pneumoniae.

In vitro stimulation of blood lymphocytes from Mycoplasma pneumoniae infected patients with pneumonia and with disorders of the central nervous system.

In 23 patients with Mycoplasma pneumoniae (MP) infection (13 with pneumonia and 10 with an acute febrile, non-bacterial disorder of the central nervous system (CNS)) and in 26 healthy control persons, thymidine incorporation of blood lymphocytes stimulates stimulated in vitro by killed MP was studied. The lymphocyte response to MP was significantly higher in the pneumonia patients than in the controls. In the patients with an acute disorder of the CNS, lymphocyte responses to MP tended to be low or normal in lack of pleocytosis in the spinal fluid, but were predominantly high when either pleocytosis or a pulmonary infiltrate was present. Lymphocyte responses to the mitogens PHA, PWM and Con-A were normal in all groups. The lack of increased responses to MP antigen in some of the neurological patients, despite a current MP infection, may reflect an antigen-specific depression or a lack of specific sensitization of their lymphocytes.

The invitro response of lymphocytes from adenoid vegetations and tonsils to PPD. Influence of autologous blood monocytes, T lymphocytes and unseparated lymphocytes.

Thymidine incorporation in lymphocytes obtained from adenoids (AVL), tonsils (TL) and blood (PBL) and stimulated by PPD was compared in 19 BCG-vaccinated patients. Responses were approximately tenfold higher in vaccinated than in unvaccinated patients. The responses obtained for AVL/TL were lower than for PBL in BCG-vaccinated as well as in unvaccinated patients. In autologous mixtures of AVL/TL and PBL a linear increase in thymidine incorporation was obtained with increasing numbers of PBL. The monocyte concentration in cell suspensions obtained from adenoid and tonsil tissue was lower than in cells prepared from blood; addition of autologous monocytes purified from blood to cultures of AVL and TL was followed by a small increase in response to PPD, but not to the level of responsiveness of PBL. Addition of autologous T lymphocytes purified from blood to cultues of AVL and TL was followed by a small increase in thymidine incorporation, of similar proportions in cultures with and without PPD. It is concluded that the proportion of PPD-sensitized lymphocytes is smaller in AVL/TL than in PBL.

Studies on lymphoid cells of adenoid tissue in relation to clinical findings.

Adenoid tissue was obtained at operation in 27 children admitted for adenoidectomy and in 6 controls. The occurrence of cells with cytoplasmic immunoglobulin was studied by immunofluorescence of tissue sections, and localization, number of cells and class of immunoglobulin were determined. No differences were found between patients and controls. Mononuclear leucocytes isolated from adenoid tissue and from blood were compared. In the patients, the proportions of B lymphocytes were higher in the tissue than in the blood, in particular IgG-, IgM- and IgD-carrying cells. Stimulation in vitro with polyclonal activators induced less thymidine incorporation in adenoid cells than in blood cells. Stimulation with heat-killed Haemophilus influenzae induced a high response inadenoid cells from 7/20 patients with negative nasopharyngeal culture for H. influenzae, whereas all 7 patients who harboured H. influenzae in the nasopharynx were low responders.

Lymphocytes from adenoid vegetations: proliferative responses in vitro as compared to blood lymphocytes.

Thymidine incorporation by lymphocytes obtained from adenoids (AVL) and blood (PBL) were compared in 27 children undergoing adenoidectomy. Optimal conditions as regards cell number and duration of culture were worked out. The spontaneous thymidine incorporation was higher in PBL than in AVL. In cultures stimulated by polyclonal activators or by PPD, the responses of PBL were higher. The dose-response curves for PBL and AVL after stimulation with killed H. influenzae were different: PBL showed a higher response, the optimal antigen concentration was lower for PBL, and the responsiveness to suboptimal antigen concentrations wash higher in PBL than in AVL.

Salmonella typhi--induced stimulation of blood lymphocytes from persons with previous typhoid fever.

In 21 persons with previous typhoid fever and in 15 controls thymidine incorporation of blood lymphocytes stimulated in vitro by killed Salmonella typhi was studied. The optimal culture conditions were established to be a cell density of 10(5) cells per vial, stimulated with 10(8) S. typhi and incubated for 5 days. The lymphocytes response to S. typhi was significantly higher in the typhoid group than in the controls. The lymphocyte responsiveness was not correlated to the time elapsed since the attack, and was not found different in patients who had had typhoid relapse. No difference was found in lymphocyte response to the mitogens PHA, PWM and Con-A between the typhoid group and the controls.

Lymphocytes of adenoid tissue.

Adenoid tissue was obtained at operation from 27 children admitted for adenoidectomy and from 6 controls. The occurrence of cells with cytoplasmic immunoglobulin was studied semiquantitatively by immunofluorescence microscopy of tissue sections, by which localization, number of cells and class of immunoglobulin were determined. No differences were found between patients and controls. Cells isolated from adenoid tissue were compared with mononuclear leukocytes obtained from the blood. More B lymphocytes were found in the tissue, in particular IgM-carrying cells. This was more pronounced in the patient group. Cells were stimulated in culture with polyclonal activators and microbial antigens. The response of adenoid lymphocytes to Haemophilus influenzae (HI) was high in 7/27 patients; all patients in whom throat culture was positive for HI were low responders.

Lymphocyte transformation studies in Mycoplasma pneumoniae infections.

The in vitro transformation of lymphocytes stimulated by a Mycoplasma pneumoniae preparation was measured by the uptake of 14C-thymidine. The lymphocytes from five patients with M.pneumoniae pneumonia showed a high degree of responsiveness when they were compared to the lymphocytes taken from eleven healthy control subjects who lacked M.pneumoniae antibodies. Another four patients with an acute affection of the central nervous system and serological evidence of an actual or recent M.pneumoniae infection had a lymphocyte response within the same range as that of the controls. The transformation of lymphocytes was studied at intervals for seven months after the onset of the illness in one of the patients with pneumonia. These studies showed an increasing response to a small dose of mycoplasma antigen. Lymphocyte transformation induced by other microbial antigens was studied in three pneumonia patients during and after convalescence. The first responses were low and increased more steeply than the response to M.pneumoniae. The later responses to the mycoplasmal and to the other microbial antigens increased in parallel. The usefulness of incorporating other microbial antigens in the evaluation of the patient's immune response to a relevant antigen in this type of experiment is discussed.