The detection of IgG antibodies to silicone.

Following a recent report of an ELISA test for the detection of antibodies to silicone, we attempted to use the same assay in four patients with known exposure to silicone. These patients all gave similar positive results as did a number of control sera with no known silicone exposure. We conclude that this assay does not measure serum levels of antibodies to silicone.

Production of tumor necrosis factors alpha and beta by human mononuclear leukocytes stimulated with mitogens, bacteria, and malarial parasites.

Tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta) are multifaceted polypeptide cytokines which may mediate some of the significant changes in cellular homeostasis which accompany the invasion of the mammalian host by viruses, bacteria, and parasites. Although it is well established that bacterial lipopolysaccharide is a potent inducer of TNF-alpha, there is still very little known of the types of agents which can trigger the production of TNFs in mononuclear leukocytes. Using an enzyme-linked immunosorbent assay for measuring TNF-alpha and TNF-beta, we examined the capacity of various T-lymphocyte and beta-lymphocyte mitogens as well as microbial components to stimulate production of these cytokines in culture. The mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen induced production of both TNF-alpha and TNF-beta, while whole-killed Staphylococcus aureus and Bordetella pertussis, like lipopolysaccharide, were potent inducers of TNF-alpha but failed to stimulate TNF-beta production. TNF-alpha production was detectable within 1 h after stimulation, while TNF-beta production was not detected until after 8 h of culture. The bacterial products tetanus toxoid, purified protein derivative, pertussis filamentous hemagglutinin, and pertussis toxin were all able to induce TNF-alpha and TNF-beta production. Disrupted (frozen-thawed) Plasmodium falciparum-infected erythrocytes were also potent inducers of TNF-alpha and TNF-beta. The results demonstrated that a wide variety of microbial components are inducers of TNF-alpha. Some may not only be more effective than lipopolysaccharide but can also induce TNF-beta production. Furthermore, evidence is presented showing that TNF-beta but not TNF-alpha production correlates with lymphoproliferation.

Tetrandrine, a plant alkaloid, inhibits the production of tumour necrosis factor-alpha (cachectin) hy human monocytes.

Human mononuclear leucocytes (MNL) or the adherent fraction (monocytes) produced tumour necrosis factor-alpha (TNF-alpha) (by ELISA) in culture when stimulated with killed Staphylococcus aureus. The bisbenzylisoquinoline alkaloid, tetrandrine inhibited the capacity of MNL and monocytes to produce TNF-alpha at a concentration range of 0.1 to 5 micrograms/ml. Tetrandrine may be potentially useful in the treatment of inflammatory diseases in which TNF-alpha plays a major role.

Depression of immunity to Naegleria fowleri in mice by selective depletion of neutrophils with a monoclonal antibody.

In an attempt to define the role of neutrophils in immunity to Naegleria fowleri in vivo, we examined the effects of treating immunized (with amoeba culture supernatant antigen) mice with the monoclonal antibody NIMP-R10, which binds to neutrophil complement receptor type 3bi (CR3) and causes selective neutrophil depletion in mice. Mice in the nonimmunized group challenged with amoebae all died by day 12, while 97% in the immunized group survived. By contrast, the immunized group treated with NIMP-R10 showed only 25% survival. The immunized group treated with "control" mouse ascites, WEM-G11, was highly resistant (90% survival). There was a significant neutrophil response in the nasal mucosa and olfactory lobes of immunized, NIMP-R10-treated mice, despite a marked degree of neutropenia similar to that seen in immunized, untreated mice. Nonimmunized mice showed virtually no neutrophil response. Despite this response in the NIMP-R10-treated mice, amoebic proliferation was not depressed, and there was no evidence of neutrophil degranulation or amoebic killing, despite the close apposition of large numbers of neutrophils to amoebae. The results indicate that neutrophils are necessary for the expression of immunity to N. fowleri.

The role of antibody in immunity against experimental naegleria meningoencephalitis ('amoebic meningitis').

Mice immunized with amoeba culture fluid (ACF) from axenically cultured Naegleria fowleri showed marked protection against a lethal amoeba challenge, a result consistent with previous observations from this laboratory. The nature of this acquired resistance is not known. The data presented show that the degree of protection conferred to mice by immunization is related to the levels of antinaegleria antibodies. These antibodies react with the surface of the amoeba. The data also show that serum (and the IgG serum fraction) from immunized mice confer protection to normal mice against a lethal N. fowleri challenge. Spleen cells from immunized animals were only capable of conferring protection to recipients, when the challenge time was delayed (10 days), at which time anti-naegleria antibodies appeared in the serum of the mice. The studies suggest that antibodies play an important role in the ACF-induced resistance to experimental naegleria meningoencephalitis.

An enzyme-linked immunosorbent assay for the quantitation of human IgG subclasses using monoclonal antibodies.

An enzyme-linked immunosorbent assay was established for the quantitation of human IgG1, IgG2, IgG3 and IgG4 using IgG subclass-specific monoclonal antibodies. The method could detect 1-10 ng/ml of the Ig subclasses. The technique is suitable for measuring IgG subclass concentration in sera of healthy adults and in supernatants from human lymphocytes cultured in the presence of pokeweed mitogen.

Depression of human polymorphonuclear leucocyte function by anti-malarial drugs.

The effect of the anti-malarial drugs quinine, chloroquine, pyrimethamine, mefloquine and quinacrine on human polymorphonuclear leucocyte (PMN) function was examined in vitro. In general, all drugs had their greatest effect on PMN iodination reaction and locomotion, intermediate effects on PMN hexose-monophosphate shunt activity, and least effect on PMN adherence. The most potent of these were pyrimethamine and mefloquine. The PMN iodination reaction and locomotion were inhibited between 0.5-1 microgram/ml (congruent to 2-4 X 10(-6) M) pyrimethamine and 1-4 micrograms/ml (congruent to 0.25-1 X 10(-5) M) mefloquine. The study demonstrates that anti-malarial drugs depress PMN functions associated with antimicrobial activity of the cell.

Inhibition of in vitro human lymphocyte response by the pneumococcal toxin pneumolysin.

The effects of pneumolysin, a sulfhydryl-activated cytolytic toxin produced by Streptococcus pneumoniae, on the in vitro human lymphocyte response was examined. The toxin, at concentrations of one to five hemolytic units per ml, caused marked inhibition of the response of lymphocytes to concanavalin A, phytohemagglutinin, pokeweed mitogen, and protein A. The response was assessed by measuring both [3H]thymidine incorporation and the ability of lymphocytes to produce immunoglobulins and lymphokine activity. The effects of pneumolysin were irreversible, could be prevented by pretreatment of the toxin with cholesterol, and were not related to a direct cytotoxic effect on the lymphocytes. Pneumolysin appeared to act at the initiation phase of the immune response and had no effect on lymphocytes committed to DNA synthesis or to the synthesis and secretion of immunoglobulins. Furthermore, pneumolysin-mediated inhibition of the lymphocyte response was not due to the inhibition of binding of mitogens to leukocytes and is likely to be related to effects on membrane-mediated signals essential for lymphocyte triggering. This may be one means by which pneumolysin plays a role in the pathogenesis of pneumococcal infections.

Activation of human complement by the pneumococcal toxin pneumolysin.

Highly purified pneumolysin (at a concentration of 10 micrograms/ml) caused significant activation of human complement, as measured by conversion of C3. Complement activation in the presence of pneumolysin was not observed in sera chelated with a combination of Mg2+ and ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, and activation was only slight in C2-deficient sera. This suggests that the toxin is capable of activating the classical complement pathway. Treatment of normal human serum with pneumolysin also significantly reduced its opsonic activity for Streptococcus pneumoniae.

Modification of polymorphonuclear leucocyte function by imidazoles.

The effect of five imidazole derivatives (metronidazole, tinidazole, clotrimazole, miconazole and ketoconazole) on human polymorphonuclear leucocytes (PMNL) was examined in vitro. Metronidazole and tinidazole had no apparent effect on either PMNL chemotactic response or PMNL fungal/bacterial killing. In contrast, clotrimazole, miconazole and ketoconazole inhibited PMNL chemotaxis. In addition, miconazole and ketoconazole were shown to depress the ability of PMNL to kill bacteria and fungi.

Immunization with killed Acanthamoeba culbertsoni antigen and amoeba culture supernatant antigen in experimental Acanthamoeba meningoencephalitis.

Mice immunized with killed (sonicated) Acanthamoeba culbertsoni A-1 antigen displayed marked resistance to intranasal challenge with the amoeba. A primary immunization produced a survival rate of approximately 40%, and survival values of greater than 80% were obtained by multiple immunizations. Similarly mice immunized with fluids from A-1 cultures were highly resistant to infection.

Activation of the alternative pathway of complement by Acanthamoeba culbertsoni.

Normal human serum (NHS) contained an amoebicidal property for Acanthamoeba culbertsoni. Killing was quantitated by measuring the ability of the amoebae to undergo cell division subsequent to exposure to NHS, and also by microscopical examination. Plasma membrane disruption and extrusion of intracellular components occurred within 5-10 min following exposure to NHS. Adsorption of specific antibody did not remove the amoebicidal activity while heating serum at 56 degrees C/30 min or treatment with zymosan prevented the killing of A. culbertsoni. Haemolytic complement was consumed and C3 conversion occurred during the incubation of NHS with amoebae. Killing required the presence of the late complement components. The findings that (a) amoebae were killed in C2 deficient human serum and ethylene glycol tetra-acetic acid (EGTA), but not ethylenediamine tetra-acetic acid (EDTA) treated NHS; (b) haemolytic complement consumption, which occurred by incubating NHS with the amoebae, could be prevented by addition of EDTA, but not EGTA and (c) conversion of C3 occurred in the presence of EGTA, but not EDTA, indicated that activation of the alternative pathway of complement was involved. This may be of importance as a natural defence mechanism in humans against A. culbertsoni infections.

Site of expression of immunity to Naegleria fowleri in immunized mice.

An experiment was performed which confirmed a previous finding that mice are protected against Naegleria fowleri infection by immunization with amoeba-free supernatant from amoeba cultures. Histological observations suggested that this protection is expressed mainly at the nasal mucosa and possibly results from the combined effects of polymorphonuclear leucocyte-mediated killing of the amoeba and mechanical elimination of the organisms by extensive shedding of necrotic epithelium.

The chemotherapeutic value of sulphadiazine in treatment of Acanthamoeba meningoencephalitis in mice.

The chemotherapeutic value of three sulphonamides, sulphamethizole, sulphamethoxazole and sulphadiazine, was examined in experimental Acanthamoeba meningoencephalitis in mice. Only sulphadiazine was capable of protecting mice challenged with A. culbertsoni A-1 strain. However, protection to mice was only afforded if the drug was given early after infection. Once the amoebae reached, and became established in, the central nervous system, sulphadiazine displayed no chemotherapeutic value.

Inhibition of mitogen-induced human lymphocyte responsiveness by polymixin antibiotics.

Polymixin antibiotics, polymixin B and polymixin E (colistin) inhibited the mitogen-induced lymphoproliferative response of human lymphocytes. Inhibition of the lymphocyte response to PHA, PWM and Con A was evident at a low concentration of 1 U/ml of antibiotics. Lymphocytes in which the signals for proliferation had occurred were similarly prevented from proliferating. The effects were not due to cell death (toxicity). Since polymixin concentrations at which inhibition of lymphocyte proliferation was observed are employed in tissue culture medium and are also attained in plasma of patients, the results suggest that the use of the antibiotics in lymphocyte cultures limits lymphocyte responsiveness and that patients receiving polymixin antibiotics may experience a state of immunosuppression.

Immunization with live amoebae, amoebic lysate and culture supernatant in experimental Naegleria meningoencephalitis.

Immunization with two doses of live Naegleria fowleri produced a survival of 34% of mice compared to 0% in unimmunized controls, whereas multiple doses of live N. fowleri resulted in loss of protective immunity. In contrast, multiple doses of N. fowleri lysate produced a survival of 30%, and multiple doses of N. fowleri culture supernatant produced a survival of 67 to 78%. Fractionation of the culture supernatant by column chromatography showed that all six fractions contained protective antigens, but the best protection occurred from immunization with the high molecular weight fraction (greater than 200,000 daltons).

Activation of complement by Naegleria.

Neither Naegleria nor its culture supernatant was found to be directly chemotactic for human neutrophils. Interaction of Naegleria with human serum, however, resulted in the generation of a strong chemotactic stimulus. The reduction of serum activity by heat-inactivation indicated a dependence on serum complement for the interaction. The ability of Naegleria to activate the alternative complement pathway was demonstrated with the use of C2-deficient serum.

Suppression of immunological responses in mice by treatment with amphotericin B.

The polyene antibiotic amphotericin B (AmB) caused a marked suppression of the cell-mediated immune response in mice. Similar treatment did not effect the humoral antibody response. The immunosuppressive property of the drug was related to its ability to inhibit the manifestation rather than the induction phase of the delayed-type hypersensitivity response. In vitro AmB suppressed mitogen-induced lymphocyte proliferation. The drug seemed to act at the proliferative phase of the response. Results presented show that the T cell response was much more sensitive to the action of AmB than was the B cell response. During AmB chemotherapy consideration must be given to the immunosuppressive properties of this drug.

Effect of mefloquine on the immune response in mice.

The effect of mefloquine, a new antimalarial compound, on the immune response in mice was studied in vitro and in vivo. Slight inhibition of mitogen-induced lymphocyte proliferative responses was observed at a concentration of 1 microgram/ml, and marked cytotoxicity at 4 microgram/ml. In contrast, a higher proportion of human lymphocytes remained viable at the same concentration of mefloquine. Antibody responses to sheep erythrocytes were impaired in mice receiving a total dose of 60 mg/kg. At this dosage schedule, delayed-type hypersensitivity responses to the same antigen were not affected.