Abstract  For a better performance of nonradioactive in situ hybridization for specific RNA sequences, signal amplification is sometimes required especially in the use of clinical specimens processed under suboptimal conditions and in the analysis of gene expression with very low reiteration. In the present study, we examined the usefulness of catalyzed signal amplification (CSA) or catalyzed reporter deposition system with biotinylated tyramine to amplify colorimetric in situ hybridization signals. Our CSA protocol included the use of biotinylated tyramine after the reaction of thymine-thymine (T-T) dimerized DNA with horseradish peroxidase (HRP) labeled anti-(T-T dimer) antibody, followed by the reaction with HRP-labeled streptavidin. When thymine-thymine dimerized lambda phage DNA was spotted onto a nitrocellulose filter at 1 pg-10 ng and then detected by the direct immunoperoxidase method, the indirect immunoperoxidase method or the CSA method, the CSA method gave the highest sensitivity with about 100-fold Increase compared to that of the direct method. Next, in the frozen sections of rat brain, which was fixed with 4% paraformaldehyde, 28S rRNA staining by in situ hybridization with only 10 ng/ml of T-T dimerized oligonucleotide probe complementary to rat 28S rRNA was compared among the direct method, the HRP-labeled avidin-biotin complex method and the CSA method. Again, intense 28S rRNA signal was obtained only with the CSA method. Therefore, we confirmed the usefulness of the catalyzed deposition system with biotinylated tyramine in nonradioactive colorimetric in situ hybridization for specific RNA sequences in tissue sections.

Abstract  Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A beta 1-42, indicating that PS-1 is involved in amyloidogenesis. However, PS-l transgenic mice have failed to show amyloid plaques in their brains. Because PS-l mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited A beta 42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Abstract  The sedative drug thalidomide ([+]-alpha phthalimidoglutarimide), once abandoned for causing birth defects in humans', has found new therapeutic license in leprosy and other diseases, with renewed teratological consequences(2). Although the mechanism of teratogenesis(3) and determinants of risk remain unclear, related teratogenic xenobiotics are bioactivated by embryonic prostaglandin H synthase (PHS) to a free- radical intermediates that produce reactive oxygen species (ROS), which cause oxidative damage to DNA and other cellular macromolecules(4,5) Similarly, thalidomide is bioactivated by horseradish peroxidase, and oxidizes DNA(6) and glutathione(7), indicating free radical-mediated oxidative stress. Furthermore, thalidomide teratogenicity in rabbits is reduced by the PHS inhibitor acetylsalicylic acid, indicating PHS-catalyzed bioactivation(8). Here, we show in rabbits that thalidomide initiates embryonic DNA oxidation and teratogenicity, both of which are abolished by pre-treatment with the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (PBN). In contrast, in mice, a species resistant to thalidomide teratogenicity, thalidomide does not enhance DNA oxidation, even at a dose 300% higher than that used in rabbits, providing insight into an embryonic determinant of species-dependent susceptibility. In addition to their therapeutic implications, these results constitute direct evidence that the teratogenicity of thalidomide may involve free radical-mediated oxidative damage to embryonic cellular macromolecules.

Abstract  Objectives. This study sought to identify determinants of workplace exposures to ethylene oxide to assess the effect of the Occupational Safety and Health Administration's (OSHA's) 1984 ethylene oxide standard. Methods. An in-depth survey of all hospitals in Massachusetts that used ethylene oxide from 1990 through 1992 (96% participation, N=90) was conducted. Three types of exposure events were modeled with logistic regression: exceeding the 8-hour action level, exceeding the 15-minute excursion limit, and worker exposures during unmeasured accidental releases. Covariates were drawn from data representing an ecologic framework including direct and indirect potential exposure determinants. Results. After adjustment for frequencies of ethylene oxide use and exposure monitoring, a significant inverse relation was observed between exceeding the action level and the use of combined sterilizer- aerators, an engineering control technology developed after the passage of the OSHA standard. Conversely, the use of positive-pressure sterilizers that employ ethylene oxide gas mixtures was strongly related to both exceeding the excursion limit and the occurrence of accidental releases. Conclusions These findings provide evidence of a positive effect of OSHA's ethylene oxide standard and specific targets for future prevention and control efforts.

Abstract  Conditional forebrain-specific presenilin-1 and presenilin-2 double knockout mice (dKO mice) exhibit several neurodegenerative phenotypes of Alzheimer's disease (AD) pathology, such as tau hyperphosphorylation, neuron loss, forebrain cortical shrinkage and memory impairment. By using capillary electrophoresis assay, monoamine neurotransmitters in forebrain cortex, hippocampus and other forebrain region of dKO mice aged at 6, 9 and 12 months were measured to illustrate the relationship among presenilins function deficiency, neurodegenerative phenotypes and monoamine neurotransmitters. Data showed that levels of monoamine neurotransmitters in forebrain cortex of dKO mice were significantly decreased at 6 months when compared to controls, while as mice getting older, levels of monoamine neurotransmitters increased to that of controls, or even higher. In hippocampus, 5- hydroxytryptamin and epinephrine in dKO mice bad a significant increase at 6 months, followed with a significant increase of each monoamine neurotransmitter at 12 months age. In other forebrain region, 5-hydroxytryptamin and dopamine had a similar level between control and dKO mice at 6 and 9 months but a significant decrease at 12 months; however, level of norepinephrine and epinephrine were significantly decreased at 6 and 12 months except epinephrine of 6 months. These results demonstrated that knockout of presenilins genes could lead to the variation of monoamine neurotransmitters, and the variation profiles were different among forebrain cortex, hippocampus and other forebrain region. However, whether presenilins deficiency caused the variation of monoamine neurotransmitter directly or not, and how about the effects of variation of monoamine neurotransmitters on AD-like pathology need to be further analyzed.

Abstract  Abnormal hyperphosphorylation of tau appears to be crucial in neurofibrillary degeneration in Alzheimer's disease (AD). Previous studies suggest that a down-regulation of protein phosphatase 2A (PP2A), the major tau phosphatase in human brain, contributes to tau hyperphosphorylation in AD. However, the effects of PP2A down-regulation on site-specific tau hyperphosphorylation is not well understood. In the present study, we showed that PP2A dephosphorylated tau at several phosphorylation sites with different efficiencies. Among the sites studied, Thr205, Thr212, Ser214, and Ser262 were the most favorable sites, and Ser199 and Ser404 were the least favorable sites for PP2A in vitro. Inhibition of PP2A with okadaic acid in metabolically active rat brain slices caused inhibition of glycogen synthase kinase-3 beta (GSK-3 beta) via an increase in its phosphorylation at Ser9. GSK-3 beta phosphorylated tau at many sites, with Ser199, Thr205, and Ser396 being the most favorable sites in cells. The overall alterations in tau phosphorylation induced by PP2A inhibition were the result of the combined effects of both reduced tau dephosphorylation due to PP2A inhibition directly and reduced phosphorylation by GSK-3 beta due to its inhibition. Because the impacts of tau phosphorylation on its biological activity and on neurofibrillary degeneration are site-specific, this study provides a new insight into the role of PP2A down-regulation in neurofibrillary degeneration in AD.

Abstract  A beta is the major components of amyloid plaque deposition in the brain, and is considered to be the initiating factor for the pathogenesis of Alzheimer's disease(AD). beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is one of the important enzymes in the A beta production. The process of maturation and expression of BACE1 is regulated by a variety of factors. Some of the pathological changes of early onset of AD such as ischemia, hypoxia, inflammation and stress are considered to be BACE1 related. Many BACE1 and A beta related cytokines in the fluids may become potential biomarkers, which would provide ideas for early clinical diagnosis of AD.

Abstract  Quinoxaline 1,4-dioxide was fed to 400 rats at levels of 10 mg or 1 mg/kg body weight for 18 months. It produced a high incidence of nasal and liver tumors only in the group fed 10 mg/kg. Of diverse histologies, the nasal tumors included anaplastic carcinomas, adenocarcinomas, squamous cell carcinomas, neuroepitheliomas, basal cell carcinomas, and a single fibrosarcoma. The nasal epithelium not involved in the neoplastic process showed dysplasia and hyperplasia.

Abstract  Propylene oxide (CAS: 75-56-9) was studied for potential carcinogenicity and chronic toxicity by inhalation in F344/N rats and (C57BL/6 × C3H)F1 mice. Groups of 50 animals of each sex were exposed to 0, 200, or 400 ppm propylene oxide for 6 hours/day, 5 days/week, for up to 103 weeks. Survival decreased in mice exposed to propylene oxide; the decrease was significant (P < .005) in mice exposed to 400 ppm. Survival of exposed rats was comparable to that of controls. Mean body weight of rats and mice exposed to 400 ppm propylene oxide decreased, when compared to that of controls, during the 2d year of exposure. Exposure to propylene oxide for up to 2 years induced inflammatory and proliferative responses in nasal cavity of both species. There was clear evidence of carcinogenicity in mice exposed to 400 ppm propylene oxide; 10 of 50 males and 5 of 50 females had hemangiomas or hemangiosarcomas of the nasal submucosa. Papillary adenomas involving the nasal respiratory epithelium and underlying submucosal glands were observed in 3 female rats and 2 male rats exposed to 400 ppm propylene oxide.

Abstract  The exposure to light (20 mW/cm2, an incandescence lamp) of weakly alkaline protein solutions which contained silver nitrate and formaldehyde initiated reduction of silver ions with the subsequent generation of colored silver colloids. At light intensities lower than 0.2 mW/cm2 the generation of colored silver colloids was delayed. The rate of silver reduction depended on the protein type and on the light spectral structure. In particular, solutions which contained prealbumin, lysozyme, gamma-globulin, and transferrin were more photosensitive than solutions which contained albumin, pepsin, and beta-amylase. The formation of [Ag(NH3)2]+ complex after an addition of ammonium ions into the solutions preferentially suppressed silver reduction in the dark and under exposure to red light, thus resulting in a significant difference in the time of appearance of colored silver colloids when the solutions were exposed to violet or red light. These findings are promising for the elaboration of selective silver development of proteins in polyacrylamide gels.

Abstract  Sinusoidal cells in the rat liver were studied in vivo and in vitro using the original vital staining with lithium carmine, which has contributed much to the development of the concept of the reticulo-endothelial system. Immunohistochemical and electron-microscopic studies revealed that the dye-incorporating cells were sinusoidal endothelial cells, Kupffer cells, and monocytes. The endothelial cells took up much more dye than did the Kupffer cells and bulged largely into the sinusoidal lumen. Electron microscopy revealed that small particles of lithium carmine were associated with coated vesicles of endothelial cells and ruffled membranes of Kupffer cells. In the endothelial cells, these particles were present in various concentrations within vacuolated structures and condensed in the lysosomes forming large aggregates of lithium carmine lumps. These lumps showed crystalline structures, within which the size of the individual particle was up to 30 nm in width and 50 nm in length. A few endothelial cells containing abundant dye underwent degeneration, and some were taken up by Kupffer cells. Liver endothelial cells isolated from lithium carmine-administered rats endocytosed fluorescence-labeled collagen. Isolated endothelial cells from normal rat liver, when cultured with lithium carmine, did not take up any dye, and their endocytosis of formaldehyde-treated albumin was inhibited dose-dependently. We conclude that in the liver, endothelial cells, but not Kupffer cells, predominantly take up lithium carmine. Furthermore, we propose the existence of a generalized cell system based on its vital staining capacity.

Abstract  Nocardia species are ubiquitous in the environment and may be found in the soil. They are generally responsible for sporadic pulmonary diseases acquired by inhalation of spores, with secondary localizations in the central nervous system and subcutaneous tissues. There is no absolute evidence for person to person transmission. Presumptive outbreaks of nocardiosis were observed in immunocompromised patients, more frequently in kidney transplant patients than in cardiac transplant patients. Nocardia spp., being present in dust particles, closure and disinfection of the transplantation unit with formaldehyde arrested the sequence of cases of nocardiosis. The original sources of the Nocardia sp. remain doubtful. Other possible sources of contamination are other patients, medical staff and the hospital environment. The first studies of Nocardia spp. typing were based on the detection of extracellular antigens, on the susceptibility of actinomycete strains to killer yeasts, and on the biochemical profiles with fluorogenic substrate. The use of molecular typing techniques have given very promising results. Analysis of plasmid profiles is an interesting way to compare the identity of isolates, although the reliability of this method depends of the presence of plasmids in the isolates. Other typing methods, including analysis of restriction length fragment polymorphism of total DNA, ribosomal DNA fingerprinting, require further investigations to evaluate their discriminating power or to be easily interpretable, whereas a random amplified polymorphic DNA (RAPD) assay was successful for epidemiological purposes. Progress in epidemiological analysis of cases of nocardiosis will be consistent when an improved diagnosis of this infection (molecular and serological diagnosis) will be available, when the genetic diversity of Nocardia spp. isolates will be better known, and when molecular typing, that hold promise in complementing investigations of outbreak of these infections, will be systematically performed when an abnormal increase of cases of nocardiosis in a population with risk factors is observed.

Abstract  Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamino)pyrido[2,3-d]pyrimidine antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, (tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they contain a reversal of the C9-N10 bridge present in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate 2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido[2,3-d]pyrimidine (8), followed by reductive amination with the appropriate benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3'4'5'-trimethoxy-substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue of the nonclassical series. Compound 22, the N9 methyl 2'5'-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC 50 = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-substituted analogues 23-25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the mammalian reference, under slightly different assay conditions than those employed for rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent potency (28 nM). Analogue 11, also displayed significant selectivity for sfDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo for the inhibition of the growth of T.gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12 and 21-23 were evaluated as antitumor agents in the National Cancer Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI50s for tumor growth inhibition in the 10 -6 - 10 -7 M range.

Abstract  A plasmid associated bacteriocin (pediocin PO2) was isolated by ammonium sulphate precipitation from cell-free growth media and subsequent studies showed that the partially purified pediocin PO2 was most likely identical (molecular mass approximately 3200 daltons in size by SDS-PAGE, stable to low pH and heat at 121 degrees C for 15 min, inactivated by various proteolytic enzymes and resistant to treatment with a range of solvents, except 10% formaldehyde) to other pediocins (PA-1 and AcH) previously reported. The antagonistic spectrum of activity of pediocin PO2 was compared with nisin and showed a narrower host-range, but a much greater activity against Listeria species including strains of Listeria monocytogences, than did nisin. A rapid method of reflectance colorimetry was used to quantitate growth and acid production (as determined by the colour change in bromcresol purple) of Lactobacillus curvatus, added to a meat product model system. The combined effects of refrigeration temperature, microbial load and bacteriocin concentration were determined in the model over 15 days storage. Both nisin and pediocin demonstrated inhibitory activity against Lactobacillus curvatus in the model system. However, when bacteriocins were incorporated into a manufactured cooked meat product only low nisin activity and no pediocin activity was detected, after challenge of vacuum packaged slices of product with Lactobacillus curvatus, over a 21 day storage trial under refrigeration temperatures.

Abstract  The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm2 and higher. This stress level also was the apparent threshold for formation of large platelet aggregates ("large" refers to particles larger than 10 microns in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37 degrees C was less in extent than at 23 degrees C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.

Abstract  Human umbilical vein endothelial cells (HUVEC) were used as an experimental host model to investigate the mechanism(s) of streptococcal adhesion in infective endocarditis. Adhesion activity of Streptococcus gordonii was maximal during the logarithmic phase of growth and was greatly reduced or eliminated by pretreatment of bacteria with heat, formaldehyde, or trypsin. At saturating numbers of streptococci, an average of 81 bacteria were bound per HUVEC. Streptococcal adhesion was inhibited by low-molecular-weight dextran and heparin but not by sucrose, fibronectin, or laminin. Adhesion was also prevented by pretreatment of HUVEC with proteins dissociated from the surface of S. gordonii with 10 mM EDTA or isolated from spent culture medium. Western blot (immunoblot) assays detected a single adhesion protein of 153 kDa (AP153) on HUVEC after incubation with unfractionated extracts of streptococci. The adhesin exhibited glucosyltransferase (GTF) activity when incubated with sucrose and Triton X-100 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AP153 was purified by affinity chromatography on dextran beads and show to have binding activity for HUVEC, GTF activity, an amino acid composition similar to that reported for GTF of S. gordonii, and the ability to inhibit S. gordonii adhesion. Incubation of the streptococci with antibodies to the adhesin inhibited bacterial attachment to HUVEC monolayers. These results indicate that surface-localized GTF mediates adhesion of S. gordonii to HUVEC in vitro and may serve as a mechanism for colonization of the endocardium in infective endocarditis.

Abstract  Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.

Abstract  In third world countries, the agricultural use of treated wastewater represents an interesting solution. A recent report of W.H.O. shows the importance of strict parasitological criteria for such reuse. The aim of this paper is to study the conditions under which Ascaris eggs have been recovered from artificially contaminated soils (sandy, clay or loamy soil, mould garden). The eggs elution has been carried out from soil particles using various solutions (detergents, distilled water, formaldehyde, sodium hydroxide, sodium hypochlorite). The recovery percentage analysis showed the superiority of the sodium hypochlorite solution titrating 10 chlorometric degrees, whatever the soil type. The eggs concentration in the eluates has been carried out through the flotation technique by testing various reagents with densities ranging from 1.16 to 1.44. The zinc sulphate solution at 50%, 55% and 66% prove to be flotation agents that are well adapted to this sampling type. By carrying out the elution with a sodium hypochlorite solution titrating 10 chlorometric degrees and the concentration by flotation with a zinc sulphate solution at 55% the recovery percentages, which are independent of the parasitic load, vary from 66 to 78%.

Abstract  The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.

Abstract  In acquired immunodeficiency syndrome, the lesions of the central nervous system in association with the human immunodeficiency virus are thought to be related to an infection of microglia, although no studies are available in which cultured and physiological characteristics of microglia cells infected in vivo have been examined. In this report, we used brain tissue from a child dying of human immunodeficiency virus infection and show that microglia cells were the main cell population being infected. Moreover, isolated macrophage-like cells from fresh brain material revealed a close resemblance to peripheral blood macrophages in their content of surface and intracellular antigens. No virus particles or viral antigens were produced by these cells during the first week of cultivation. Productive infection was readily apparent, however, by day 30. This finding illustrates the slow nature of the virus life cycle in these cells and the minimal cytopathology that accompanied the infection.

Abstract  There are many reasons for developing in vitro tests of toxicity including cost, speed, studies of mechanisms, and studies utilizing human cells and tissues. The present study focuses on the development of in vitro tests to predict in vivo toxicity by comparing them to data from the literature. A broad spectrum of model toxic compounds was evaluated for toxicity on mouse skin JB6 cells in culture. These included mercuric chloride, sodium lauryl sulfate, formaldehyde, dimethyl sulfoxide, benzoyl peroxide, and ionomycin, all of which have been proven to be positive in the Draize test or in cutaneous toxicity studies. Cell viability was evaluated every 15 min for up to 1 hr, and then after 24 hr of treatment using the Trypan Blue exclusion method; morphological changes were evaluated using phase-contrast and transmission electron microscopy. Dose- and time-dependent cell death and morphological changes were observed at concentrations ranging from 10(-14) to 10(-2) M. Arbitrary rankings were assigned based on 1) IC50 value estimated from the present data, and 2) in vivo toxicity reported in the Registry of Toxic Effects of Chemical Substances. Good correlation between in vitro and in vivo toxicity based on arbitrary rankings was observed. Thus, these findings suggest that the JB6 cell culture model can be used for predicting in vivo toxicity. In the future, it may be possible to utilize this system for the study of intracellular ionized calcium ([Ca2+]i), and the expression of oncogenes as early indicators of toxicity.

Abstract  Two gold immunolabelling techniques using electron microscopy were compared to examine the in situ localization of a luteovirus, potato leafroll virus (PLRV), inside its main aphid vector, Myzus persicae SULZ. With Gildow's technique, virus particles were labelled prior to fixation, embedding by injecting PLRV-specific IgGs into the living aphids. This facilitated the detection of extracellular particles located between the basal lamina and plasmalemma by trapping them in aggregates. The heavy coating of particles by antibodies and gold indicated good labelling sensitivity. Isometric virus-like particles were also observed inside the cytoplasm, but they were non decorated because the cell membrane prevented labelling reagents from entering the cell. With the second technique, ultrathin sections were immunolabelled after fixation-embedding. Since PLRV lost its antigenicity when aphid tissues were normally treated for electron microscopy, the successful application of this technique required fixation in 4% formaldehyde before embedding in Lowicryl at low temperature; it was also necessary to use PLRV-specific monoclonal antibodies to eliminate non-specific reactions. In these conditions, all intra- and extra-cytoplasmic virions present on the surface of sections were surrounded by gold particles, but the antibody coating was not discernible, and, because the resin limited the access of markers to antigens, the inner virus particles were not labelled. In conclusion, both techniques must be applied on the same material to give complementary information.

Abstract  A series of neoglycoproteins was synthesized by coupling of thiophosgene-activated p-aminophenyl derivatives [Biol. Cell. 47:95-110 (1983); J. Histochem. Cytochem. 32:1091-1094 (1984)] of various sugars to human serum albumin. The compounds were evaluated for their in vitro activity against human immunodeficiency virus (HIV). Neoglycoproteins with the highest sugar content were found to be the most potent inhibitors of HIV-1-induced cytopathogenicity. However, this was not due to the nature of the sugar used but, rather, was related to the extra negative charge of the neoglycoproteins. To investigate whether the antiviral activity of the neoglycoproteins exhibited sugar specificity, increased with increasing negative charge, or depended on both sugar specificity and negative charge, we synthesized albumins and neoglycoproteins with an enhanced negative charge, by treatment with formaldehyde or succinic anhydride. Succinylated human serum albumin had the most pronounced net negative charge and had an IC50 of about 1 microgram/ml. No cytotoxicity was observed at concentrations up to 1 mg/ml, implicating a selectivity index (CC50/IC50) of at least 10(3). To elucidate the mechanism of action of these anionic albumins, we investigated whether they interfered with HIV-1 adsorption to the cells, binding of anti-OKT4A monoclonal antibody (mAb) to the CD4 receptor, binding of anti-gp120 mAb to gp120, or inhibition of syncytium formation in co-cultures of HIV-1-infected HUT-78 cells with MOLT-4 cells. From these experiments, we conclude that albumins with an increased negative charge (a) are potent and nontoxic anti-HIV-1 agents, (b) cause a 50% reduction of syncytium formation in the same concentration range as their IC50 in the antiviral assay, and (c) do not bind to the OKT4A epitope of the CD4 receptor and only partly inhibit anti-gp120 mAb-gp120 interaction and virus-cell binding at concentrations that are 100 times higher than their IC50 in the antiviral assay. Therefore, we conclude that the modified albumins interfere with a post-binding event, of which one of the potential mechanisms is an interaction with the gp41 fusion protein, which is necessary for syncytium formation but is not involved in initial virus binding.