Dependent Lysis Research Articles

C Dependent Immune Lysis of Virus Altered Plasma Membranes: Dependence on the Alternative C Pathway and Factor B

Abstract Antibody-mediated C dependent lysis of cell lines infected with herpes type 1 (HeLa cells), herpes type 2 (HeLa), influenza Å (Chang), measles (HeLa) and mumps (Vero) were analyzed for C participation. Lysis occurred with immune human sera, Mg++ EDTA immune sera, and immune sera depleted of C4 or treated with Fab anti-C4. Lysis did not occur with non-immune sera, Mg++EDTA sera, sera heated 50°C for 25 min, sera depleted of Factor B, or sera treated with Fab anti-Factor B. Lysis was restored to heated sera and Factor B immunodepleted sera by addition of Factor B but not by C2. Properdin and Factor D depleted immune sera lost significant lytic activity, but to a lesser extent than serum depleted of Factor B. Addition of depleted factors in physiologic concentrations partially restored the lytic activity. Further studies showed that lysis of measles infected HeLa cells was induced by both immune IgG and F(ab')2 but not Fab' in the presence of a non-antibody containing human C source.

An immunocytotoxic assay for Trypanosoma cruzi

Trypanosoma cruzi culture forms grown for 8 days in LIT medium fortified with 14C-labeled amino acids will incorporate the isotope into membranous and soluble components of the parasite. On lysis, labeled cytoplasmic elements released into the surrounding medium can be measured and reflect the degree of destruction of the parasites. Materials released as a result of trypanosome destruction are not re-utilized by mouse spleen cells which supports the use of this assay for studies on lymphocyte mediated cytotoxicity in experimental Chagas' disease. Experiments on complement dependent antibody-mediated lysis of T. cruzi demonstrate utility of the assay in examining both agglutinating and cytotoxic capacities of immune and normal serums. It is suggested that this assay is applicable to other protozoan parasite systems and may have some utility in diagnosis.

BPO-Specific, Complement-Dependant Cell-Lysis of Differently Sensitized Sheep Red Cells: Evaluation of Haptenic Groups and their Influence on IgM- and IgG-induced Lysis

Sheep erythrocytes were coated with bencylpenicilloyl-(BPO)groups. Different incubation periods resulted in erythrocyte preparations with different hapten density. Complement dependent lysis induced by IgM or IgG antibodies was studied with the cell preparations. The calculation of hapten density on the erythrocyte surface was not possible by direct measurement of coupled radioactive BPO, since more than 90% of radioactive material was found in the soluble supernatant after osmotic cell lysis and less than 10% was fixed to the cellular membrane. Measurement of membrane bound immunologically relevant BPO-groups was achieved, therefore, by comparison of the inhibitory capacity of the test cells with that of a standard cell preparation. The latter consisted of tannic acid treated erythrocytes coated with protein complexed radioactive BPO. Surface hapten density of the different target cell preparations varied between 1.9 × 105 and 4.8 × 105 BPO-groups per cell depending on the time of incubation. Complement dependent antibody mediated cell lysis was significantly reduced by reduction of haptenic sites per target cell, IgG induced lysis being much more affected than hemolysis induced by IgM antibodies. Statistical calculations led to the conclusion that 18,000 protein islets per cell bearing 4 or more BPO-groups are not sufficient for hemolysis induced by IgG antibodies. 48,000 protein islets with this hapten density are necessary for «optimal« sensitization. IgG antibodies must be apparently bound to the cell surface in bivalent form.

Measurement of virus antigens on the surface of HeLa cells persistently infected with wild type and vaccine strains of measles virus by radioimmune assay.

Persistent states of measles virus infection have been established in HeLa cells by using Edmonston strain virus and two types of measles virus vaccine (M-VAC and Schwarz). The absolute amount of surface viral antigens expressed on these cells infected separately with the three viruses has been assessed by a newly developed method which employs [125I]-labelled Fab fragments of immunoglobulin G (IgG) from immune human sera. This method was used to determine the level of viral antigenic expression on acutely infected HeLa cells harvested at a time when 95 to 100% of cells could be lysed by antiviral antibody and complement. From our data, more than 1 X 10(6) antibody molecules must bind to each cell infected with measles virus before complement dependent lysis can occur in a homologous test system. Persistently infected cells bind 2 to 3 times less antibody than acutely infected cells and correspondingly exhibit less susceptibility to humorally-mediated immune lysis.

Estimation in sera by radioimmunoassay of a specific membrane antigen associated with a murine lymphoma.

Material with a molecular weight of less than 10(5) daltons has been isolated and partially purified from the ascitic fluid of DBA2 mice bearing a syngeneic lymphoma (SL2). This substance inhibits the cytotoxic action of an allogeneic antiserum directed specifically against SL2 cells. Material has been rendered radioactive with 125I and between 20 and 25% of the radioactivity is bound in a specific manner to the antiserum. The material which is referred to as 125I-TSTA has been used in a radioimmunoassay to measure the level of TSTA in the sera of mice bearing both ascitic and subcutaneous SL2 tumours. The level of circulating TSTA was found to be high immediatley following inoculation of live SL2 cells, probably because a large proportion of the injected cells autolyse. The serum concentration of TSTA then falls but 6-10 days later begins to rise again in parallel with the growth of the SL2 tumour either in the peritoneal cavity or subcutaneously. Following surgical removal of an intradermal SL2 tumour the level of TSTA in the serum falls rapidly. No evidence could be found that a significant proportion of the TSTA in the serum of tumour-bearing mice is completed with antibody. However, in the serum of DBA2 mice which have been hyperimmunized with irradiated SL2 cells there are antibodies which bind 125I-TSTA although syngeneic anti-SL2 sera, unlike alloantisera, do not show complement dependent lysis of SL2 cells.

Enhanced in vitro cytotoxic anti H-2 responses in the presence of Ia antigens

Because surface Ig and Ia antigens cap independently, A.TH anti-A.TL serum combined with the indirect immunfluorescence technique could be used to test defined murine cell populations ofH-2k haplotype for the presence of Ia antigens. Mitogen induced T- and B-cell derived blast cells, purified by velocity sedimentation at 1g, were tested for the expression of Iak antigens and then used both as stimulator cells and as target cells, in primary and secondary in vitro cytotoxic allograft responses. Fibroblasts, cortisone-resistant thymocytes, and nylon column purified splenic T cells were also included in these tests. Ia antigens were detected on 100% of LPS-induced blast cells, on 20%–30% of ConA-induced blast cells (100%Θ Thy-1 or antigen positive), but only to 5%–10% on PHA-blasts (100% Thy-1 antigen positive). Fibroblasts and nylon column purified splenic T cells were essentially Ia negative. Ia-positive allogeneic stimulator cells induced a far stronger in vitro cytotoxic T-cell response compared to Ia-negative stimulator cells; that is, there was a positive correlation between the expression of Ia antigens on the stimulator cells and the magnitude of cytotoxicity induced. This correlation was restricted to primary allograft responses. Ia antigens could not be detected as a target for killing in the cytotoxic effector phase, using both different target cells as well as the approach of “PHA dependent lysis” for detecting cytotoxic T lymphocytes.

Differentiation between B cells, T cells, and histiocytes in melanocytic lesions: Primary and metastatic melanoma and halo and giant pigmented nevi

Membrane properties of infiltrating mononuclear cells in melanocytic lesions were studied. In frozen sections of six primary melanomas and six halo nevi, the infiltrating cells lacked C3 receptors characteristic of B cells and IgG receptors characteristic of histiocytes. Viable infiltrating cells extracted from two primary melanomas formed rosettes with sheep erythrocytes, providing direct evidence of their T cell identity. In contrast, the majority of cells infiltrating melanoma metastases in four patients were identified as histiocytes by their IgG receptors and ultrastructural appearance. Focal aggregates of B cells and plasma cells were found in a giant pigmented nevus in areas of disrupted nevus cells. This suggests a B cell mediated response against nevus-associated antigens, possibly involving antibody dependent target cell lysis. It appears that a spectrum of cellular reactions to melanocytic lesions occurs in vivo and that the type of infiltrating mononuclear cell is related to the type of lesion.

Effector cell activity in antibody mediated cell dependent immune lysis: II. Evidence for different populations of effector cells for different targets

Using the adhering property of certain types of cells to glass or plastic in human peripheral blood lymphocyte preparations, two populations of effector cells can be identified in the ABCIL system, each with a selective killing directed toward a different antibody coated target. The adherent cells which are radioresistant functionally, with the morphology of macrophages, exhibit strong cytotoxicity against the antibody-coated erythroid target while lacking any killing effect toward the antibodycoated target lymphocytes. The nonadherent cells which are radiosensitive and with the morphology of small lymphocytes, selectively kill the antibody-coated lymphocyte target without possessing any cytotoxicity against the antibody-coated target erythrocytes.

Antibodies in Parous Women Detected by Antibody Dependent Cell Mediated Cytotoxicity

Feto‐maternal symbiosis during pregnancy induces the production of antibodies against fetal alloantigens. In this study, the presence of antibodies in the sera from parous women was checked by Complement dependent Lysis (CdL) at 20°C and at 15°C, and by Lymphocyte Antibody Lytic Interaction (LALI). The percentage of positive sera in CdL at 20°C is 15.3%, at 15°C 25.8% and in LALI 41.0%. This difference is due to the higher sensitivity of the LALI technique in detecting antibodies directed against histocompatibility antigens expressed on target cell surface. The high percentage of positive sera found suggests that alloimmunization during pregnancy is a generalized and physiological phenomenon and that this fact should be taken into account in the investigation of the feto‐maternal relationship.

PSEUDOMONAS AERUGINOSA INFECTION IN CYSTIC FIBROSIS. Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response

The occurrence of Pseudomonas aeruginosa in the respiratory tract of 70 cystic fibrosis patients and the occurrence of precipitins against Ps. aeruginosa in sera from the same patients have been investigated during one year by means of monthly bacteriological examinations of tracheal secretions and by means of crossed Immunoelectrophoresis of a polyvalent Ps. aeruginosa antigen against sera from the patients. The one‐year period prevalence rate of patients harbouring Ps. aeruginosa was 64 per cent. In newly colonized patients and in intermittently colonized patients non‐mucoid strains were predominating, whereas mucoid strains were predominating in chronically colonized patients. The occurrence of mucoid strains, especially in chronically colonized patients, was associated with a significantly higher number of precipitins against Ps. aeruginosa than the occurrence of non‐mucoid strains. Males chronically colonized with mucoid strains presented a significantly higher number of precipitins than females chronically colonized with mucoid strains and the number of precipitins was correlated with the duration of the chronic colonization with Ps. aeruginosa in males in contrast to females. The results are in accordance with the hypothesis that mucoid substance could be a virulence factor because it might inhibit the opsonizing effect of the precipitins on the mucoid cells and the complement dependent lysis of the cells hereby favouring mucoid strains at the expense of non‐mucoid strains in the respiratory tract of cystic fibrosis patients.

Phagocytic Cells as Effectors in a Cell-Mediated Immunity System

Abstract Cell-mediated immunity, using a xenogeneic system, i.e., mouse spleen cells attacking chicken erythrocytes (CRBC) is studied. In this system target cell lysis by spleen cells from immunized animals is complement independent, hence cell-mediated. It is specific because trinitrophenyl-(TNP) sensitized spleens lyse TNP-CRBC but not CRBC. CRBC-sensitized thymus-derived (T) lymphocytes cooperate with bone marrow-derived lymphocytes in induction of CRBC plaque-forming cells but cannot lyse CRBC as target. However, anti-CRBC antibody at concentrations too low to be detected by complement dependent lysis do induce cytotoxicity to CRBC in spleen cell suspensions containing T cells as well as in spleen cell suspensions depleted of T cells. Anti-ϑ serum absorbed to remove non-anti-ϑ antibody does not abrogate the cytotoxicity of immune spleen cells. These results indicate that the effector cell in this system is not a ϑ-antigen-bearing T lymphocyte. The effector cell is abundant in spleen and in the peritoneal cavity but not in lymph nodes of normal animals: it is bone marrow-derived, radiation resistant, and sticks to siliconized glass bead columns. Since, in addition, the effector cell can be separated from other spleen cells by treatment with carbonyl iron powder and a magnet, it is, we suggest, a phagocytic cell.

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca 2+, induced the lysis of red blood cells after an induction period. In the absence of Ca 2+, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca 2+ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.

Lysis of an Aspergillus nidulans mutant blocked in chitin synthesis and its relation to wall assembly and wall metabolism.

1. Mutant Aspergillus nidulans hyphae containing 7–15% of the normal chitin complement did not lyse in dilute buffer but lysed on continued incubation in growth medium without osmotic stabiliser. Distortion and lysis could occur anywhere along the hypha and were not confined to the apex. 2. Hyphae with a normal chitin content started to distort and lyse 90 min after transfer to medium lacking osmotic stabiliser and at the temperature inhibiting chitin synthesis. Lysis was not confined to the growing tips. 3. Hyphae lacking wall chitin and transferred to conditions where chitin synthesis could proceed, formed stable mycelium at the growing tips and distorted in the older, chitin-poor, regions. Pulse-labelling with N-acetylglucosamine and radioautography showed that, in such hyphae, chitin was incorporated into the wall in the older regions undergoing distortion. 4. The relation between growth—dependent distortion and lysis and the activity and location of autolytic enzymes is discussed.

Haemolytic efficiency of rabbit IgG anti-Forssman antibody and its augmentation by anti-rabbit IgG.

Complement dependent lysis of an erythrocyte can be achieved in optimal conditions by reaction of a suitable anti-IgG with a single molecule of IgG antibody on the surface of an erythrocyte. Optimal conditions require special precautions to avoid antibodies which prevent C′ activation. For lysis of a sheep erythrocyte without augmentation, about 800 molecules of an IgG antibody were needed.