J. Štefanovič

Quantification of proliferative and suppressive responses of human T lymphocytes following ConA stimulation

The mitogenic response of human T lymphocytes to graded doses of concanavalin A (ConA) has been measured by means of an MTT tetrazolium dye metabolic assay. Three groups of healthy subjects, representing children, younger adults and elderly persons, were investigated. It was shown that a typical bell-shaped course of the ConA dose-response curve is the result of a proliferative response to suboptimal concentrations of ConA and a toxic action of ConA at supraoptimal concentrations. The ascending part of the response curve reflects in its shape the regulatory interaction of responding cells. A decrease in suppressive functions is accompanied by a shift of this part of the curve to lower concentrations of ConA. By means of a mathematical model derived from enzyme kinetics, an attempt was made to quantify the suppressive functions from the course of the individual dose-response curve. It was found that after suitable data processing, suppression-related shape changes can be assigned to a single parameter. The value of this parameter as a diagnostic tool was tested in a study of the age dependence of human T lymphocyte responses to ConA. While the proliferative response decreased with age, the suppressive functions exhibited their maximum effect in the group of adults. Thus it could be demonstrated that ConA induced proliferative and suppressive responses are due to two different pathways which can be independently extracted from the dose-response curve.

Family Study of Natural Killer Cell Activity in C1qDeficient Patients with Systemic Lupus Erythematosus-Like Syndrome: Association between Impaired Natural Killer Cell Function and C1q Deficiency

Impaired natural killer (NK) cell activity has been found in patients with systemic lupus erythematosus (SLE)-like syndrome. The mechanism by which NK cell function is impaired in SLE patients is not quite clear. We report here a family study of NK cell activity in C1q-deficient patients with SLE-like syndrome. In both SLE-active and SLE-inactive stages of the disease, NK cell function was significantly impaired when compared with the healthy controls (10.6 +/- 2.3% and 16.9 +/- 4.8% to 34.7 +/- 9.6%, p less than 0.025). On the other hand, differences in NK cell cytotoxicity between SLE-active and SLE-inactive members of the family were not statistically relevant (p less than 0.1). Further, we found no correlation between NK cell activity and clinical or laboratory values, except for a positive correlation between function of NK cells and C1q and CH50 values, respectively (rs = 0.93, 0.01 less than p less than 0.02). To our knowledge, this is the first report on a notable association between impaired NK cell activity and C1q deficiency. The type of inheritance of C1q deficiency in this family is also discussed.

Lysosomal enzyme activities in polymorphonuclear leukocytes, macrophages, serum, and spleen of conventional, germ-free, and antigen-free minnesota miniature swine

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of guotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.

Lysosomal enzymes and metabolic activity of polymorphonuclear leukocytes from patients with systemic lupus erythematosus and from experimental animals after levamisole treatment.

The activity of β-d-glucuronidase (BDG) was lowered and the activity of myeloperoxidase (MPO) was elevated in polymorphonuclear leukocytes (PMNs) of SLE patients compared with normal subjects. After levamisole treatment, the activities of BDG, MPO, and lysozyme rose in PMNs of SLE patients. A higher activity of lysozyme was also observed in peritoneal PMNs of rabbits after levamisole administration. During incubation of human phagocytizing and non-phagocytizing PMNs, no effects of levamisole at concentrations of 10−3 to 10−5M were observed on the release of lysosomal enzymes. These concentrations of levamisole also did not influence INT reductase activity and production of superoxide by normal human PMNs in the presence of zymosan particles. These findings suggest that levamisole might have an effect on the actual level of lysosomal enzymes in PMNs rather than on their release.

Difference in the proteolytic activity of spleen cells of young and immunologically mature rabbits

During the early stages of postnatal ontogenesis, young rabbits have low antibody-forming capacity. This is explained either as inability of the young to react to antigen or as the outcome of lowered ability to synthesize gamma globulin (Sterzl, 1960; Sterzl & Riha, 1957). The authors examined the proteolytie activity of spleen cell extracts from 28 young rabbits aged 3--4 days and from 10 adult rabbits, in an at tempt to find an explanation for this difference in the immunological reactivity of young and adult rabbits. After killing the animals, cells obtained from the spleens of 3--4 young in the cold were washed by spinning in cooled saline for 10 minutes at 1,000 rpm. Cells from the spleens of immunologically mature rabbits were obtained in a similar manner. The cells were adjusted to a concentration of 15--25 X 10e/ml and were destroyed with saponin in a concentration of up to 0.5%. The proteolytic activity of these extracts was tested on a human serum albumin substrate at pH 3.5 and pH 2. Proteolytic activity was determined by the method of Lapresle and Webb (1962), by which cathepsin D activity and 75--85% of cathepsin E activity is detected at pH 3.5, while 90% of cathepsin E activity is detected at pH 2 (Stefanovi6, 1964). Proteolytic activity was expressed by the optical density (0. D.) and by units calculated from the tangent led from the start of the curve prepared from different concentrations of purified cathepsin D. Since lymphocytes contain very little cathepsin E (~tefanovi6 et al., 1962), activity at pH 2 was very low and no differences were recorded; these findings are therefore not given in the results. The results of the proteolytie activity of spleen cell extracts at pH 3.5 are given in Table 1.

Antibacterial and Proteolytic Activity of Rabbit Phagocytes and the Effect of Temperature and Endotoxin on this Activity.

Proteolytic activity at pH 2 (caused by cathepsin E) and at pH 3.5 (caused by cathepsin D and E) was found in extracts of rabbit polymorphonuclear leucocytes, macrophages and lymphocytes, while antibacterial activity was detected only in polymorphonuclear leucocyte extracts, against microorganisms of theEnterobacteriaceae family. Comparison of the character of antibacterial and proteolytic activity showed that the two systems were quite different. If polymorphonuclear leucocyte extract was heated at 56° C for 30 minutes, the effect on antibacterial activity was non-significant, while proteolytic activity at pH 2 and 3.5 fell by about 60%. The addition of a heat-inactivatedEscherichia coli suspension or of purifiedSalmonella paratyphi B bacterial lipopolysaccahride to leucocyte extract completely destroyed antibacterial activity, while proteolytic activity was unaffected.

The interaction of protein substrates (antigens) during anin vitro degradation by rabbit cathepsin D

The rate of degradation of human serum albumin (HuSA) by rabbit cathepsin D, measured on the basis of the amount of degradation products soluble in trichloroacetic acid, was shown to depend on the original concentration of the substrate and on the presence of other proteins in the system. Both human and rabbit IgG decreased the rate of cathepsin degradation of HuSA, whereas bovine haemoglobin, on the contrary, increased the rate of degradation. Immunochemical analysis (i.e. immunoelectrophoresis and radial immunodiffusion) have shown that the mixture of two proteins was degraded at a slower rate than either of these proteins alone. This phenomenon was studied using even a substrate possessing an enzymatic activity (catalase) and it was shown that the presence of a further substrate in the mixture decreased the degree of inactivation of catalase by cathepsin D. It suggests that competition between individual proteins and their fragments takes place in the course of the proteolytic reaction and, in addition, their association also occurs. Therefore the kinetics of catheptic degradation of a mixture of protein antigens is rather complicated. These results are discussed in view of antigen degradation within the cells and mechanism of killing of phagocytosed microorganisms.

The inhibitory effect of homologous immunoglobulin G on the degradation of antigen (human serum albumin) by rabbit cathepsin D

The dynamic of degradation of antigen (human serum albumin) by rabbit cathepsin D was studied in the presence of homologous immunoglobulin G having antibody activity to substrate (antigen) as well as without antibody activity. Both immunoglobulins possessed an inhibitory effect on the intensity of antigen degradation, however, immunoglobulin G with antibody activity had a more pronounced effect. The studies on the kinetics of inhibition showed that we were probably dealing with a special case of inhibition of the mixed type which is known as coupling or anticompetitive inhibition.

Changes in antibacterial and proteolytic activity of polymorphonuclear leucocytes following the whole body x-irradiation of rabbits

In whole body x-irradiated rabbits with 150 r, 500 r and 1000 r an antibacterial and proteolytic activity in extracts of polymorphonuclear leucocytes obtained from peritoneal exudate was tested immediately and 1, 3, 6, 10 and 21 days following irradiation. The changes in antibacterial activity tested inEscherichia coli, Bethesda andSalmonella adelaide strains depended on the intensity of radiation and time interval between radiation and collection of leucocytes. With increasing radiation the antibacterial activity inBethesda andSalmonella adelaide strains was decreased. In rabbits irradiated 150 r and 500 r a decrease or even a disappearance of bactericidal activity on the sixth day in all strains tested was found, whereas after an irradiation with 1000 r the antibacterial activity was found to be low immediately after irradiation and this decrease could be detected up to the tenth day. UsingEscherichia coli strains the antibacterial activity of leucocyte extracts did not show such a regular dependence on the intensity of radiation and time as with other strains used. The proteolytic activity of leucocyte extracts tested at pH 3.5 (cathepsin D and E) and pH 2.0 (cathepsin E) within 3 days after irradiation was higher with increasing radiation. At other time intervals the activity did not show regular changes. The dynamics of changes in proteolytic activity at both pH was different and also a relation between proteolytic and antibacterial activity was not found.

A standard microcytotoxicity technique for quantitative analysis of lymphocyte subsets: A comparison with indirect immunofluorescence, evaluated by microscopy or flow cytometry

A standard complement-dependent microcytotoxicity (CDC) technique was used for quantitative analysis of T-lymphocyte subsets in human peripheral blood and the results compared to those obtained by indirect immunofluorescence microscopy and flow cytometry. The monoclonal antibodies OKT3, OKT4 and OKT8 were used in the CDC method for detection of total-T cells, T-helper and T-suppressor cells respectively. The CDC technique provided reproducible results (CV, 3-7%) correlating well with both immunofluorescence techniques. This observation was valid both for healthy persons (n = 21) and for patients (n = 10) with immunological disorders. The correct antibody dilution, correction for background and the use of eosin staining are considered critical for the usefulness of this technique. The method has several advantages: it is widely used for histocompatibility testing, only simple equipment is necessary, and the amount of monoclonal antibody required per test is small.