The emulsified perfluorocarbon Oxycyte improves spinal cord injury in a swine model of decompression sickness.

STUDY DESIGN: A prospective, animal model for pharmacological intervention of decompression sickness (DCS), including spinal cord (SC) injury. BACKGROUND: Signs and symptoms of DCS can include joint pain, skin discoloration, cardiopulmonary congestion and SC injury; severity ranges from trivial to fatal. Non-recompressive therapy for DCS may improve time-to-treatment and therefore impact mortality and morbidity. OBJECTIVES: Oxycyte at 5 cc kg(-1) provides both SC protection and statistically significant survival benefit in a swine model of DCS. The purpose of this study was to test whether a reduced dose of Oxycyte (3 cc kg(-1)) would provide similar benefit. SETTING: Silver Spring, MD, USA METHODS: Male Yorkshire swine (N=50) underwent a non-linear compression profile to 200 fsw (feet of sea water), which was identical to previous work using the 5 cc kg(-1) dose of Oxycyte. After 31 min of bottom time, decompression was initiated at 30 fsw per minute until surface pressure was reached. Following decompression and the onset of DCS, intravenous Oxycyte or saline was administered with concurrent 100% O(2) for 1 h. The primary end point was DCS-induced mortality, with Tarlov score and SC histopathology as secondary end points. RESULTS: Oxycyte administration of 3 cc kg(-1) following surfacing produced no significant detectable survival benefit. Animals that received Oxycyte, however, had reduced SC lesion area. CONCLUSION: Further studies to determine the lowest fully efficacious dose of Oxycyte for the adjunct treatment of DCS are warranted.

Dependence of growth, metabolic expression, and pathogenicity of Naegleria fowleri on exogenous porphyrins.

The pathogenicity of the free-living amoeba Naegleria fowleri is modulated by the composition of the medium used for cultivation. The constituents that determine the level of pathogenicity of N. fowleri, however, have not been definitively established. The present study examined the effects of selected porphyrins on N. fowleri amoebae. The iron-containing porphyrins, hemin or hematin, or the iron-free porphyrin, protoporphyrin IX, were effective in supporting growth of N. fowleri in Cline medium lacking serum. Iron-binding proteins, including hemoglobin, could not satisfy the growth requirement of the amoebae for exogenous porphyrin. Expression of biological functions including azocaseinase activity, agglutination, mobility, complement susceptibility, and virulence were altered by the composition of the growth medium. Amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX displayed greater mobility and were more resistant to lysis by complement than those grown in Nelson medium. Similarly, amoebae grown in Cline medium supplemented with either hemin or protoporphyrin IX were more pathogenic for B6C3F1 mice than those grown in Nelson medium. The addition of protoporphyrin IX to Nelson medium resulted in a modest increase in mobility, resistance to complement lysis and virulence when compared to N. fowleri amoebae grown in Nelson medium without added porphyrin.

Subchronic 10 day immunotoxicity of polydimethylsiloxane (silicone) fluid, gel and elastomer and polyurethane disks in female B6C3F1 mice.

Millions of people have been exposed to silicones because of the widespread use in consumer products such as cosmetics and toiletries, food products, household products and paints. Silicones have wide use in medical practice, including lubricants in tubing and syringes, and as implantable devices. The most prevalent silicone in medical use is polydimethylsiloxane. This study was undertaken to determine the subchronic immunotoxicologic potential of the principal constituents of breast implants: silicone fluid, silicone gel and silicone elastomer. An alternative covering for devices containing silicone gels, polyurethane, was also included in the study. Silicone fluid and gel were injected subcutaneously into female B6C3F1 mice (1 ml/mouse) and 6 mm disks of silicone elastomer or polyurethane were implanted subcutaneously. There were no treatment-related deaths or overt signs of toxicity. None of the tested materials had notable effects on body or organ weights, erythrocytes or leukocytes in the blood, blood chemistries such as alanine aminotransferase, urea nitrogen, glucose, albumin or total protein. The cellularity of the bone marrow and responses to CSF-GM and CSF-M were normal. The tested silicones did not alter the distribution of B cells and T cells in the spleen, but polyurethane perturbed the distribution of CD4+CD8+ and CD4-CD8- T cells. The antibody response to sheep erythrocytes was not markedly altered, nor were proliferative responses to concanavalin A, phytohemagglutinin, lipopolysaccharide or allogeneic cells. Reticuloendothelial function was normal, but polyurethane evoked an enhanced phagocytosis of Covaspheres by adherent peritoneal cells. Natural killer cell activity and serum complement were not altered. All silicone materials afforded modest protection to a challenge with Listeria monocytogenes that killed 40 to 58% of control mice. Host resistance to Streptococcus pneumoniae or the B16F10 tumor was not affected by any of the treatments. There is a pattern indicative of some perturbation of T cell differentiation in mice implanted with a polyurethane disk.

Immunotoxicity of 180 day exposure to polydimethylsiloxane (silicone) fluid, gel and elastomer and polyurethane disks in female B6C3F1 mice.

Millions of people have been exposed to silicones which are present in consumer goods such as cosmetics and toiletries, processed foods and household products. In addition, silicones have been used extensively in medical practice as a lubricant in tubing and syringes, and as implantable devices. A silicone widely used in medical practice is polydimethylsiloxane. This study was undertaken to determine the immunotoxicologic potential of long term exposure to the principal constituents of breast implants: silicone fluid, silicone gel and silicone elastomer. An alternative covering for devices containing silicone gels, polyurethane, was also included in the study. Silicone fluid and gel were injected subcutaneously into female B6C3F1 mice (1 ml/mouse) and 6 mm disks of silicone elastomer or polyurethane were implanted subcutaneously. There were no treatment-related deaths or overt signs of toxicity during the 180 day exposure. None of the tested materials had notable effects on body or organ weights, erythrocytes or leukocytes in the blood, blood chemistries such as alanine aminotransferase, urea nitrogen, glucose, albumin or total protein, or serum CH 50 or C3 levels. The cellularity of the bone marrow and responses to CSF-GM and CSF-M were normal. The tested silicones and polyurethane marginally reduced the level of Ig+ cells in the spleen but did not consistently alter the distribution of T cell surface markers. The antibody response to sheep erythrocytes was not markedly altered, nor were proliferative responses to concanavalin A, phytohemagglutinin, lipopolysaccharide or allogeneic cells. Reticuloendothelial function was normal, as was phagocytosis of chicken erythrocytes and Covaspheres by adherent peritoneal cells. Natural killer cell activity was depressed in all silicone treatment groups and in mice implanted with polyurethane. No silicone or polyurethane treatment group displayed altered susceptibility to a challenge with Listeria monocytogenes, Streptococcus pneumoniae or the B16F10 tumor. The only consistent effect of 180 day exposure to silicone materials or polyurethane was a modest depression of natural killer cell activity.

Immunotoxicity of nitrobenzene in female B6C3F1 mice.

Nitrobenzene (NBZ) is primarily employed as an oxidizing agent in the synthesis of analine and benzene compounds. It produces myelotoxic effects and effects on erythrocytes in both animal models and man. Reported hepatosplenomegaly and effects on the bone marrow are indicators that NBZ may be immunotoxic. In these studies, female B6C3F1 mice were exposed to 30, 100 and 300 mg/kg of NBZ in corn oil by gavage for 14 consecutive days. To assess the immunotoxic potential of NBZ, body and organ weights were determined and selected immunologic and host resistance responses were studied. In these studies, the liver and spleen appeared to be the primary target organs. Both liver and spleen weights were dose dependently increased. Gross histopathologic examinations revealed significant changes in the spleen, consisting of severe congestion of the red pulp areas with erythrocytes and reticulocytes. Serum chemistry profiles showed increases in alanine aminotransferase and aspartate aminotransferase activities, indicating liver toxicity. Hematologic studies showed a decrease in erythrocyte number and a concomitant increase in mean corpuscular hemoglobin and mean corpuscular volume. A dose-dependent increase in peripheral reticulocytes was also seen. DNA synthesis was enhanced, as was the number of formed elements and the number of monocyte/granulocyte stem cells in the bone marrow of treated mice. IgM responses were decreased and the phagocytic activity of macrophages in the liver was dose dependently increased with a concomitant decrease in the activities in the spleen and lung. Other immunological parameters examined were unchanged. Host resistance to microbial or viral infection was not markedly altered by NBZ; however, there were trends towards increased susceptibility where T-cell function contributes to host defense. These data indicate that NBZ-induced hemolysis and liver injury are linked to the observed alterations in bone marrow activity.

Immunotoxicity of mono-nitrotoluenes in female B6C3F1 mice: I. Para-nitrotoluene.

para-Nitrotoluene (p-nitrotoluene) is used primarily as an intermediate in the production of various dyes, explosives, pharmaceuticals, and in the production of rubber and agricultural products. Previous investigations indicated that p-nitrotoluene was mutagenic in the Ames Test and that other mono-substituted nitrotoluenes bound covalently to hepatic macromolecules. The objective of these studies was to evaluate the potential immunotoxicity of p-nitrotoluene in mice exposed by the oral route. Mice exposed to p-nitrotoluene (200-600 mg/kg) daily for 14 days showed modest dose-dependent increases in liver and spleen weights. The livers of mice exposed subchronically to 400 and 600 mg/kg showed a mild to moderate swelling of the hepatocytes adjacent to the central veins; this swelling appeared to be reversible and there was no evidence of necrosis. The proportion of monocytes in blood was decreased in mice treated with p-nitrotoluene or toluene. Serum chemistries, bone marrow cellularity and the number of CFU-M and CFU-GM were unaffected. Immunologic investigations showed p-nitrotoluene suppressed the IgM response to sRBC and the DHR response to KLH. There was a 24% decrease in the percentage of CD4+ T lymphocytes in the spleen. There was no dose-dependent alteration of peritoneal macrophage numbers or differential count, unstimulated natural killer cell activity, response to B cell mitogen LPS, C3 activity or interferon levels. Exposure of mice to p-nitrotoluene decreased resistance to Listeria monocytogenes but not to Streptococcus pneumoniae, Plasmodium yoelii or the B16F10 melanoma, and increased resistance to the PYB6 tumor. These studies indicated that the immune system is an important target for toxicity of p-nitrotoluene. The decreased host resistance to L. monocytogenes can be attributed to the decrease in T lymphocytes and to a decreased delayed hypersensitivity response to KLH.

Immunotoxicity of 2,4-diaminotoluene in female B6C3F1 mice.

2,4-Diaminotoluene (DAT) has been demonstrated to be a potent carcinogen. The present studies were carried out to determine the toxic and immunotoxic potential of DAT. Mice exposed to DAT at 25-100 mg/kg per day for 14 days by gavage showed a 42% increase in liver weight and a slight decrease in spleen weight. Histopathologic evaluation of selected organs showed the liver to be the major target with morphological changes which were dose dependent. The high dose (100 mg/kg) was associated with moderate centrilobular necrosis. No abnormal structure was noted in the spleen, lungs, thymus, kidney or mesenteric lymph nodes. The liver toxicity was associated with an elevation in alanine aminotransferase activity. The only change noted in selected hematologic parameters was a 64% increase in peripheral blood leukocytes. Mice exposed to DAT showed a decreased IgM and IgG response to sheep erythrocytes. The decrease was not a function of a decreased number of B cells because the number of B cells increased dose dependently. Proliferative capacity of immunocompetent cells was not impaired by exposure to DAT as measured by the response to several mitogens. The delayed hypersensitivity response to keyhole limpet hemocyanin in mice exposed to DAT was increased. Natural killer cell activity was decreased dose dependently and may represent a spleen cell pool shift because the number of B cells increased in the presence of a decreasing spleen size. Serum C3 was suppressed at the high dose of DAT. Phagocytosis by splenic macrophages, but not peritoneal macrophages, was inhibited by DAT exposure. DAT exposure for 14 days decreased host resistance to the bacteria, Streptococcus pneumoniae and Listeria monocytogenes, while host resistance to the pulmonary tumor model, B16F10, and the PYB6 fibrosarcoma was unaffected by DAT exposure. These data indicate that DAT is hepatotoxic and perturbs the differentiation and maturation of leukocytes.

Immunotoxicity of mono-nitrotoluenes in female B6C3F1 mice: II. Meta-nitrotoluene.

The nitrotoluenes are chemicals used in dyes, agricultural products, pharmaceuticals and explosives. In the present studies, the toxicology and immunotoxicity of meta-nitrotoluene (m-nitrotoluene) were evaluated. Mice, exposed to m-nitrotoluene at dose levels of 200, 400 and 600 mg/kg/body weight for 2 weeks by gastric gavage, gained body weight over the treatment period to a slightly greater extent than the control groups. Of the selected organs weighed, the liver and kidney of mice exposed to m-nitrotoluene were increased in weight while the thymus weight was decreased. The liver of mice exposed to m-nitrotoluene, but not ortho-nitrotoluene, showed slight to moderate swelling of the hepatocytes adjacent to the central veins. The hepatocyte swelling appeared to be reversible and there was no evidence of necrosis. The hematology and serum chemistries examined were unaffected by m-nitrotoluene exposure although there were modest decreases in the percentage of polymorphonuclear leukocytes and eosinophils in differential blood counts. Bone marrow cellularity and the number of CFU/M and CFU/GM were unaffected by m-nitrotoluene exposure. m-Nitrotoluene suppressed the IgM response to sRBC and the DHR response to KLH. There was a slight (8%) decrease in the percentage of B lymphocytes in the spleen. The response to the T cell mitogens was suppressed by as much as 39%. Fc-mediated adherence and phagocytosis of chicken erythrocytes and NK cell activity were increased dose dependently in mice exposed to m-nitrotoluene. Several immune parameters were unaffected by exposure to m-nitrotoluene, including the IgG response to sRBC, responses to the B cell mitogen LPS and to allogeneic cells, and serum interferon levels. Resistance to Streptococcus pneumoniae and Plasmodium yoelii were unaffected also. Resistance to the tumor model PYB6 was increased. Exposure of mice to m-nitrotoluene decreased resistance to Listeria monocytogenes. The decreased resistance to L. monocytogenes may be related to an effect on T cells, evidenced by a decrease in T cell numbers and in the DHR.

Cytopathic action of Naegleria fowleri amoebae on rat neuroblastoma target cells.

The axenically cultured, weakly pathogenic Naegleria fowleri LEE and the highly pathogenic, mouse passaged N. fowleri LEEmp are cytopathic for B103 rat nerve cells in culture. Cytopathogenicity was measured by release of radiolabeled rubidium or radiolabeled chromium from B103 target cells. Cytopathogenicity was time-dependent for up to 18 h and dependent upon amoebae effector to nerve cell target ratios of less than 1:1. Release of 51Cr from B103 cells by either LEE or LEEmp amoebae was enhanced by addition of calcium or magnesium to medium free of these divalent cations but the ion-channel inhibitor, verapamil, or the ionophore A23187 and phorbol myristate acetate did not alter release of 51Cr from B103 cells cocultured with the amoebae. Cycloheximide or actinomycin D impaired release of 51Cr from B103 target cells injured by either LEE or LEEmp amoebae. Both strains of amoebae were fractionated by glass bead disruption and high speed centrifugation into membrane and soluble fractions. Each fraction was incubated with either 86Rb or 51Cr labeled nerve cells. The membrane fraction from LEEmp was more active than the soluble fraction in facilitating rubidium and chromium release. In contrast, the soluble fraction from LEE was more active than the membrane fraction in facilitating rubidium release from radiolabeled target cells. The sequential release of 86Rb and 51Cr from target cells rather than the simultaneous release of the two isotopes indicates that target cell death is due to the release of ions followed later by the release of large macromolecules. The results indicate that N. fowleri amoebae injure nerve cells by two alternate mechanisms, trogocytosis or contact-dependent lysis.

Immunotoxicity of the semiconductor gallium arsenide in female B6C3F1 mice.

The effects of gallium arsenide (GaAs) exposure on immunocompetence of B6C3F1 female mice were investigated. GaAs was administered as a single intratracheal instillation at doses of 50, 100, and 200 mg/kg. Fourteen days after exposure, various cellular and humoral immune parameters were assessed. GaAs exposure increased spleen cellularity in a dose-dependent manner. However, the percentages of Thy 1.2 positive and Ig positive cells were decreased and that of F4/80 positive cells was increased dose dependently. The IgM and IgG antibody-forming cell response of the spleen to the T-dependent antigen sheep erythrocytes was reduced by 66 and 48%, respectively, at 200 mg/kg. Levels of the serum complement protein, C3, were increased by as much as 16% with no significant change in CH50 levels. The mitogenic response of splenic T cells to Con A and PHA was unaffected by GaAs, but that of B cells to LPS was increased by 52%. The delayed hypersensitivity response to keyhole limpet hemocyanin and mixed lymphocyte response were significantly reduced in a dose-dependent manner by GaAs exposure. Natural killer cell activity against the YAC-1 mouse lymphoma was enhanced in treated mice. Analysis of peritoneal exudate cells (PEC) revealed a dose-dependent decrease in number and a shift in the composition of PECs. The percentage of PEC monocytes increased from 53% of the population to 81%, while the lymphocytes decreased from 46 to 20%. The adherent PEC population demonstrated decreased phagocytosis of covaspheres and increased phagocytosis of chicken erythrocytes (CRBC). GaAs exposure had no effect on host resistance to Plasmodium yoelii or Streptococcus pneumoniae, but dose dependently increased resistance of the mouse to Listeria monocytogenes. Treated mice demonstrated a significantly decreased resistance to the B16F10 melanoma with a sevenfold increase in tumor burden at 200 mg/kg. GaAs affects both humoral and cellular immune parameters in mice and impairs the ability of the immune system to protect against B16F10 tumor challenge.

Effects of pyran copolymer on host resistance of mice to Plasmodium yoelii.

Female B6C3F1 mice treated with 25 mg/kg pyran intravenously (i.v.) on days -4 and -3 were more susceptible to nonlethal Plasmodium yoelii 17XNL or lethal Plasmodium berghei ATCC-30090 than untreated mice or mice treated intraperitoneally (i.p.). Female B6C3F1 mice treated with pyran i.p. displayed enhanced resistance to Listeria monocytogenes as compared to untreated mice or mice given pyran i.v. Peritoneal exudate cells (PEC) primed by pyran i.p. possessed enhanced ability to kill Listeria but impaired ability to destroy Plasmodium. Phagocytosis of Covaspheres by PEC was greater for mice given pyran i.p. than those given pyran i.v. Chemiluminescence evoked by zymosan was less for PEC from mice given pyran i.v. than for those from untreated mice or those given pyran i.p. Chemiluminescence was greater for adherent splenocytes from mice treated with pyran i.p. than for those from untreated mice or those from mice treated i.v. Pyran administered i.v. is less effective in modulating the host immune response than pyran administered i.p. Immunomodulatory agents such as pyran have adverse as well as beneficial effects depending upon the route of administration.

Effects of cyclophosphamide and a metabolite, acrolein, on Naegleria fowleri in vitro and in vivo.

Mice challenged intranasally with Naegleria fowleri died of primary amoebic meningoencephalitis. Mice given 30 mg of cyclophosphamide per kg of body weight daily for 10 days starting 2 days before challenge were protected. Neither cyclophosphamide nor serum from cyclophosphamide-treated mice inhibited N. fowleri in vitro. A metabolic product of cyclophosphamide, acrolein, inhibited growth and enflagellation of N. fowleri. Acrolein at 40 microM was amoebicidal. Acrolein injured starved cells and amoebae at 5 degrees C and growing N. fowleri.

An immunotoxicological evaluation of 4,4'-thiobis-(6-t-butyl-m-cresol) in female B6C3F1 mice. 2. Humoral and cell-mediated immunity, macrophage function, and host resistance.

Adult female B6C3F1 mice were gavaged with TBBC in corn oil at doses of 10, 100, or 200 mg/kg daily for 14 consecutive days. All immunological parameters were measured 24 hr after the last chemical exposure. When indicated, animals were immunized during the exposure. TBBC produced a decrease in the peak IgM (44%) and peak IgG (48%) antibody response to sheep erythrocytes (SRBCs), but had no effect on the delayed hypersensitivity response (DHR) to keyhole limpet hemocyanin (KLH). Paradoxically, TBBC caused an overall increase in the number of splenic cells, a decrease in the percentage of splenic T cells and no effect on the percentage of splenic B cells. There were no effects on the lymphoproliferative responses to optimal concentrations of concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS), but there was a significant decrease in the mixed lymphocyte response (MLR). In both the mitogen assays and the MLR there was a dose-related increase in the basal (unstimulated) DNA synthesis of the spleen cells. Innate immunity, as measured by natural killer (NK) cell activity and serum complement, was significantly increased. Effects on macrophage function were complex, as an increase or no effect was observed depending on the parameter measured. In the host resistance models, animals were infected with various pathogens 24 hr after the last chemical exposure. Exposure to TBBC caused an increased resistance to challenge with Streptococcus and B16F10 melanoma, a decreased resistance to challenge with PYB6 tumors, and no effect on the resistance to HSV-2, Listeria or Plasmodium.

Characterization of a neutral aminoacyl-peptide hydrolase from Naegleria fowleri.

An intracellular alpha-aminoacyl-peptide hydrolase (EC 3.4.11.-) from Naegleria fowleri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl-, tyrosyl-, leucyl-, arginyl-, alanyl-, tryptophanyl-, histidyl-, methionyl-, and lysyl-naphthylamide but not benzoylleucyl-, leucylglycyl-, glycylprolylleucyl-, glycyl-, threonyl-, aspartyl-, or glutamyl-naphthylamide. The aminopeptidase activity was inhibited by the cysteine-protease inhibitors--hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate--by the aminopeptidase inhibitors--bestatin and trans-epoxysuccinyl-leucyl-agmatine--by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o-phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine-protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55 degrees C for 30 min, but all activity was lost after 15 min at 80 degrees C. Enzyme activity was inhibited by 100 microM HgCl2 and CdCl2 but not by 1 mM CoCl2, CuCl2, MnCl2, NiCl2, FeCl3, or ZnCl2. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X-100.

Specificity of antibodies from human sera for Naegleria species.

Serum samples from adult humans in North Carolina and Pennsylvania were assayed for antibodies against four Naegleria species: N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis. Agglutinating activities of serum samples from North Carolina subjects were higher for N. fowleri than were those from Pennsylvania subjects. The distributions of agglutination titers of human serum samples for N. australiensis, N. gruberi, and N. lovaniensis were heterogeneous. The agglutination capabilities of selected serum samples absorbed with rounded, killed trophozoites of N. australiensis and N. lovaniensis were distinctly different, as were those of serum samples absorbed with N. fowleri and N. gruberi. N. australiensis and N. gruberi shared some agglutinating antigens, as did N. fowleri and N. lovaniensis. The agglutinating activities of most serum samples correlated with the capability of their immunoglobulin M (IgM) to bind to antigens in extracts of Naegleria species but not with the capabilities of their IgG to bind to antigens of Naegleria species. Absorption of IgM binding capability with rounded, killed trophozoites established that N. gruberi was distinctly different from N. fowleri and N. lovaniensis but that N. fowleri and N. lovaniensis shared surface antigens. The proteins in extracts of the four Naegleria species were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tested for their ability to bind immunoglobulins in a serum sample. The antigens of the four species that bound IgM or IgG in the tested serum sample were separated by SDS-PAGE, and when they were incubated with anti-IgM or anti-IgG, they gave distinct profiles. There was one distinct, shared antigen that had a molecular size of 40,000 daltons. Absorption of the test serum with killed, rounded trophozoites did not markedly change the immunoglobulin binding profile for Naegleria internal antigens separated by SDS-PAGE and did not remove the shared 40,000-dalton protein(s). These results demonstrate that the four Naegleria species have antigenically distinct surfaces and that humans have been individually exposed to antigens of Naegleria species.

Cytopathogenicity of Naegleria fowleri for cultured rat neuroblastoma cells.

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to 51Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44 degrees C for 60 min or at 60 degrees C for 30 min. Cytotoxicity was stable during storage at 4 degrees C or at -20 degrees C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4 degrees C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.