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.

Immunotoxic effects of diethylstilbestrol on host resistance: comparison with cyclophosphamide.

To evaluate the usefulness of host resistance assays for measurement of immunotoxicologic effects of chemicals, the immunosuppressive effects of exposure to diethylstilbestrol (DES) were compared with the effects of treatment with the known immunosuppressive drug cyclophosphamide (CPS). A panel of six host resistance models was evaluated, including infection with the bacterium Listeria monocytogenes, herpes simplex virus type 2 (HSV-2), and encephalomyocarditis virus (EMC), the yeast Cryptococcus neoformans, the parasite Naegleria fowleri, and transplantation of the B16F10 melanoma tumor. The results demonstrate a general correlation between the effects of CPS and DES on host resistance. Acute treatment with CPS (200 mg/kg) markedly depressed resistance to the microbial infections with L. monocytogenes and HSV, and exposure to DES usually also decreased resistance in a dose dependent manner. Moreover, CPS had no marked effect on resistance to N. fowleri and EMC virus, and exposure to DES also had a neglible or slight effect. There were, however, two model systems in which the effects of CPS and DES diverged. Whereas treatment with DES produced no significant effect on resistance to C. neoformans, acute treatment with CPS prior to the fungal infection produced a marked increase in resistance. Also, while treatment with CPS markedly increased B16F10 lung metastases, treatment with DES significantly decreased the incidence and number of lung metastases. The data support the general validity of host resistance assays, particularly with models of short disease course, for measuring immunosuppression. However, the results also emphasize the complexity of interpreting effects of environmental chemicals on host resistance, because of the interplay of such factors as relative times of exposure to the chemical in relation to pathogenesis of infection, the length of the disease course, the nature of the operative host defense mechanisms, and the compensatory recovery of these mechanisms.

Elevations of thyroid-stimulating hormone during acute nonthyroidal illness.

To determine whether the incidence of unrecognized thyroid disorders is high enough to warrant screening, we studied a group of 95 elderly hospitalized women with the use of clinical examinations, along with thyroid function tests. We found that 13 (13.7%) of the 95 women had elevated serum thyroid-stimulating hormone (TSH) levels and mean serum thyroxine (T4) levels, and their free T4 indexes and triiodothyronine levels (measured by radioimmunoassay) were lower than the levels in the normal control group. In four (4.2%) of the 95 patients, the elevation of TSH levels was transient. In two patients (2.1%), the elevation of TSH levels was caused by underlying hypothyroidism. The results of thyroid function tests, including serum TSH, obtained during acute nonthyroidal illness should be interpreted cautiously, and screening for thyroid disorders among elderly patients with acute illnesses is not warranted. While primary hypothyroidism may be regularly associated with an elevated TSH level, an elevated TSH level may be the result of an acute illness and not associated with clinical hypothyroidism.

Approaches to assessing host resistance.

There is increasing evidence that chronic, subclinical exposure to certain environmental pollutants may upset immune responsiveness and alter susceptibility of animals to infectious agents. Environmental chemicals or drugs may affect diverse aspects of the immune system, leading to immunosuppression, immunopotentiation, hypersensitivity or perturbed innate host resistance. A variety of infectious models is available that involves relatively well defined target organs and host defense mechanisms; for example, infections with encephalomyocarditis virus, Herpesvirus simplex, Listeria monocytogenes, Streptococcus pneumoniae, Escherichia coli or Plasmodium berghei. Important variables in infectious models used to assess immunotoxicity include species and strain of animal used, their age and sex, the route of exposure, and dose of the chemical. No one infectious model has yet emerged as a routine screening tool to detect and assess the subtle effects that may occur in immune responses when animals are exposed to doses of environmental pollutants that cause no adverse effect at a gross level. The selection of useful test systems is complicated because it is difficult to measure the effects of chronic, subclinical exposure to chemicals and sublethal challenges of microorganisms.

Enhanced cytotoxicity in mice of combinations of concanavalin A and selected antitumor drugs.

Concanavalin A (Con A) injected intraperitoneally at a dose of 50 mg per kg was not lethal for male BALB/c mice. Six hours after administration of 5 mg Con A/kg, the proportioy 24 hr, the proportion of granulocytes had decreased to 56%. Adiministration of 5 mg Con A/kg 24 hr before 200 mg of 5[3,3-bis(2-chloroethyl)-triazeno]-imidazole-4-carboxamide per kg, or 100 mg of 5-fluorouracil per kg resulted in a significant enhancement of lethality. Simulatenous administration of 5 mg Con A/gm and 10 mg of daunomycin per kg also resulted in enhanced lethality. Administration of 5 mg Con A/kg 24 hr before 40 mg of 1,3-bis(2-chloroethyl)-1-nitrosourea per kg, 200 mg of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea per kg, 1000 mg of cytosine arabinoside per kg, 0.1 mg of mithramycin per kg, 2 mg of pactamycin per kg or 1 mg of vincristine per kg did not result in enhanced lethality. Lipid A prepared from Escherichia coli 0127:B8 Boivin lipopolysaccharide has been complexed to Con A. The lipid A-Con A complex (5mg/kg) was no more, or less effective in enhancing the lethality of 5-fluorouracil than 2.5 mg Con A/kg. The lipid A-Con A complex (40 mg/kg), given simultaneously with drug, enhanced lethality per kg. In this regard, the lipid A-Con A complex had vincristine per kg. In this regard, the lipid A-Con A complex had activity comparable to the complex formed between lipid A and bovine serum albumin. Conceivably, Con A can be used to enhance the susceptibility of neoplastic cells to phase-specific antitumor drugs, especially those acting on deoxyribonucleic acid synthesis.

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.

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.

Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures.

Amebae of Naegleria fowleri and Naegleria gruberi were cytopathic for nine established mammalian cell cultures, including mouse and human fibroblasts, rabbit and monkey kidney cells, rat and mouse neuroblastoma cells, baby hamster kidney cells, and human epithelioma and carcinoma cells. Nine strains of N. fowleri were equally cytopathic for rodent neuroblastoma cells. As few as one ameba per million neuroblastoma cells destroyed the mammalian target cells after 9 days. The N. fowleri grew and destroyed rat neuroblastoma cells at 30 to 37 C whereas N. gruberi grew and destroyed the target cells at 25 to 30 C. Both N. fowleri and N. gruberi attached efficiently to the target cells at 30 to 37 C; N. gruberi but not N. fowleri attached efficiently at 25 C. Electron microscopic observations of mixed cultures of N. fowleri and neuroblastoma cells established that the amebae, after 12 hr, had ingested portions of the neuroblastoma target cells without causing cell lysis. Conversely, N. gruberi amebae, after attaching to target cells, disrupted the plasma membrane and cytoplasm of the target cells although the target cell nucleus remained intact. The amebae then ingested the target cell debris.

Endotoxic activity of complexes of myristic acid and proteins.

Complexes of myristic acid and bovine serum albumin, myristic acid and concanavalin A, beta-hydroxymyristic acid and concanavalin A, or dimethyl myristamide and concanavalin A are lethal for male BALB/c mice treated with mithramycin. Prior treatment of mice with myristic acid-protein complexes renders the animals resistant to a dose of bacterial endotoxin that is lethal for untreated animals. Prior treatment of mice with bacterial endotoxin renders them resistant to a combination of mithramycin and a complex of myristic acid and bovine serum albumin or dimethyl myristamide and concanvalin A that is lethal for untreated animals. These data indicate that a fatty acid is an important functional component of the endotoxin toxophore.

Relationships among mycobacteria and nocardiae based upon deoxyribonucleic acid reassociation.

The degree of renaturation between Nocardia farcinica 330 deoxyribonucleic acid (DNA) and DNA from 17 nocardial strains and 11 mycobacterial strains, and between Mycobacterium smegmatis 405 DNA and DNA from 11 mycobacterial strains and 14 nocardial strains was determined by using the nitrocellulose membrane filter technique. These results indicated that some cultures designated N. farcinica were identical to some cultures designated N. asteroides but that other strains called N. asteroides were distinctly different. The species M. smegmatis was homogeneous and distinct from the other species examined. Data comparing the extent of nucleotide sequences shared by M. smegmatis 405 DNA and DNA from nine other mycobacterial strains, as determined by the membrane filter technique and by monitoring DNA reassociation optically, were similar. The extent of reassociation between M. tuberculosis H37Ra DNA and DNA from 14 mycobacterial strains was determined optically. DNA from M. bovis BCG was 86% homologous with M. tuberculosis H37Ra DNA. The other mycobacterial DNA preparations examined were less than 40% homologous with M. tuberculosis H37Ra DNA. The genome sizes of the mycobacteria examined ranged between 2.5 x 10(9) daltons and 4.5 x 10(9) daltons.

Composition and ultrastructure of Streptomyces venezuelae.

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.

Characterization of deoxyribonucleic acids from streptomycetes and nocardiae.

The relationships among selected streptomycetes, nocardiae, and mycobacteria have been determined, based upon the base composition of their deoxyribonucleic acid (DNA) and upon the ability of their denatured DNA to anneal with single-stranded reference DNA. The streptomycetes constituted a homogeneous group whose DNA contained between 69 and 73 mole% guanine + cytosine (% GC). Moreover, the streptomycetes examined showed 37 to 88% homology with the Streptomyces venezuelae and S. rimosus reference DNA. The nocardial and mycobacterial DNA both contained 62 to 69% GC. The nocardial strains studied fell into either a 62 to 64% GC group or a 68 to 69% GC group, indicating that they should not be assigned to a single species. The nocardiae having 68 to 69% GC showed 24 to 44% homology with S. venezuelae reference DNA. In competition experiments, wherein unlabeled heterologous DNA interfered with binding of labeled homologous DNA, the nocardial DNA with 68 to 69% GC showed a greater degree of homology with the streptomycetes than did the nocardial DNA with 62 to 64% GC. In addition, the DNA from spores of S. venezuelae was cursorily examined, and interactions between S. venezuelae denatured DNA and polyribonucleotides were sought. The buoyant density of the DNA from S. venezuelae spores was distinctly less than that from mycelia. Moreover, denatured S. venezuelae DNA formed a dense complex with polyriboguanylate.

Action of bacterial endotoxin and lipid A on mitochondrial enzyme activities of cells in culture and subcellular fractions.

Escherichia coli O127:B8 lipopolysaccharide (LPS), prepared by the Westphal procedure, caused a marked decrease in the activities of mitochondrial malate dehydrogenase, succinate dehydrogenase, and adenylate kinase in African green monkey kidney (Vero) cells and primary cultures of mouse liver cells within 2 h after exposure to 10 micrograms of LPS/ml of culture medium. These three enzyme activities leaked into the supernatant fraction, and cytochrome oxidase activity was lost from the mouse liver mitochondrial particulate fraction within 45 min after exposure to 10 micrograms of LPS/mg of protein. Loss malate dehydrogenase activity from isolated mitochondria was also accelerated by LPS from E. coli O26:B6 (Boivin preparation) or Salmonella typhosa O901 (Westphal preparation), and by lipid A from Salmonella minnesota or Shigella sonnei. In addition, LPS and lipid A inhibited state 3 respiration by isolated mitochondria with attendant loss of respiratory control, but adenosine 5'-diphosphate/O ratios were relatively unchanged. Impaired mitochondrial function is an early event after exposure to biologically relevant amounts of LPS or lipid A.