Scrapie-associated tubulofilamentous particles in scrapie hamsters.

Examination of thin sections from the cerebral cortex of scrapie-infected hamster brains revealed characteristic circular 26-30 nm diameter tubulofilamentous particles, identical to those previously described in both experimentally induced scrapie in mice, hamsters and natural scrapie of sheep, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease and mice and chimpanzees infected with Creutzfeldt-Jakob disease. Longitudinal forms of tubulofilamentous particles were also observed in dendrites and myelinated axons. Both transverse and longitudinally cut particles were readily distinguished from microtubules and synaptic vesicles, thus there appears to be no relationship between tubulofilamentous particles, and microtubules or synaptic vesicles.     

Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells.

Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.     

Morphological and quantitative comparison between infectious and non-infectious forms of influenza virus

Electron microscopic study has revealed the morphological entity responsible for the rise in viral hemagglutinin observed in brains of mice after intracerebral inoculation of non-neurotropic strains of influenza virus. This rise in hemagglutinin, although dependent on inoculation of fully infectious virus, is not associated with an increase in infectious titer. The hemagglutinating principle is functionally similar to the "incomplete" influenza virus which can be obtained from chick embryos by serial egg-to-egg transfer of undiluted, infected allantoic fluid according to the method of von Magnus. A method has been described which facilitates selective adsorption of viral particles recovered from organ extracts on saponine-lysed ghosts of fowl erythrocytes. This procedure has been utilized in studying the morphology of non-infectious, hemagglutinating virus from chorio-allantoic membranes or mouse brains and in comparing these two forms with each other and with ordinary, infectious (standard) influenza virus. Standard virus isolated from allantoic fluids or membranes of infected eggs was found to contain uniform particles of predominantly spherical shape with smooth surface and even density, resembling those described by others. The appearance of such particles was not affected by the procedure of extraction and concentration used. In contrast, non-infectious, hemagglutinating virus obtained either from allantoic sacs ("undiluted passages") or from mouse brain was pleomorphic and seemed to consist of disintegrating particles. The majority appeared flattened and bag-like and had a rough, granular surface and reduced, uneven density. 37 per cent of the non-infectious particles isolated from mouse brain infected with the non-neurotropic strain WS had diameters in excess of 170 mµ, as compared with only 2 per cent of the particles of the parent strain itself. Regardless of whether or not the contrast in appearance of standard and of non-infectious particles was due to differing resistance to the preparatory treatment, it indicated the existence of basic structural differences between the two types of virus. Correlation of particle counts with hemagglutinin titers has shown that the non-infectious virus obtained from mouse brain is, unit for unit, an equivalent counterpart of standard virus derived from infected eggs. The end-point of hemagglutination in a pattern test corresponds for both forms to that dilution at which the ratio virus particles/red cells approaches one. The quantitative data based on particle counts support the assumption that non-infectious virus arises in mouse brain as a product of viral multiplication.     

In vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii.

The in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against the cyst form of Toxoplasma gondii were evaluated. In vitro treatment (100 micrograms of 566C80 per ml for 3 days) of cysts isolated from brains of mice infected for 1, 2, 3, 4, or 9 months resulted in loss of viability of the cysts and did not reveal any influence of the duration of in vivo infection on sensitivity to the drug. In vivo experiments to determine the effect of prolonged treatment with 200 mg of 566C80 per kg of body weight per day on cysts in brains of CBA/Ca mice infected with strain ME49 revealed a steady and significant decline in the numbers of cysts compared with the numbers in untreated controls. Histopathology of brains from control mice revealed inflammatory infiltrates around capillaries and in the parenchymas and meninges which were consistently less evident in the brains of treated mice. In addition, cysts were rarely observed in treated mice, whereas extensive inflammation and large numbers of cysts were found throughout the entire brain in control mice infected for the same period. The reduction in the numbers of cysts was evident as early as day 5 of treatment but was more marked at 8 weeks of treatment. The numbers of cysts in the brains of Swiss Webster mice infected for 3 or 6 months also significantly decreased following treatment for 15 or 30 days with the same dose of 566C80. Our results indicate that 566C80 has excellent activity against cysts of T. gondii both in vivo and in vitro and that sensitivity of the cysts to 566C80 is not affected by the duration of the infection in vivo.     

Unexpected detection of DNA by nucleic acid sequence-based amplification technique

Nucleic acid sequence-based amplification (NASBA) is a technique that has been previously shown to selectively mediate the detection of RNA in microbial cells. In a series of tests, nucleic acids were extracted from Salmonella enterica serotype Typhimurium and Mycobacterium avium subsp. paratuberculosis, and subjected to four enzymatic treatments prior to NASBA. These enzymatic treatments were DNase, RNase, S1 nuclease, and RNase/S1 nuclease. The results obtained were different for the two bacteria. With S. enterica serotype Typhimurium, RNase and RNase/S1 nuclease abolished the NASBA signal, as expected. But with M. avium subsp. paratuberculosis RNase, S1 nuclease, and RNase/S1 nuclease had no effect on the NASBA signal, whereas DNase treatment abolished it. This indicates that in the latter bacterium, NASBA can detect DNA, and demonstrates the necessity of verifying the nucleic acid origin of a NASBA signal if detection of RNA is objective.     

A NEW MOUSE VIRUS CAUSING NECROSIS OF THE THYMUS IN NEWBORN MICE

A new mouse virus has been recovered from laboratory and wild mice. The agent induces a non-fatal disease in infant mice, characterized by acute massive necrosis of the thymic medulla and cortex, granulomatous reaction, and subsequent restoration of essentially normal architecture with scarring. Intranuclear inclusion bodies are produced. Virus may persist in tissues of convalescent mice for many months. Electron micrographs of acutely infected thymuses showed nuclear and cytoplasmic karyoannular particles and masses of parallel fibrils in nuclei.     

Rescue of the adeno-associated virus 2 genome correlates with alterations in DNA-modifying enzymes in human cells.

The adeno-associated virus 2 (AAV) genome can be rescued either from a recombinant plasmid upon transfection into human cells or from cells latently infected with AAV, following subsequent infection with adenovirus. Using human diploid fibroblasts as a model for a natural AAV infection, we observed increased efficiency of rescue of the AAV genome in these cells as they traversed their limited proliferative life span in vitro. The efficiency of rescue correlated well with the augmented nuclease activity in these cells. Furthermore, rescue of the AAV genome, either from a recombinant plasmid or from the chromosomal DNA, was more efficient in cells from a patient with Bloom's syndrome, a rare autosomal recessive disease associated with increased chromosomal breakage due to DNA ligase I deficiency, as compared with normal human diploid fibroblasts. These studies suggest that alterations in DNA-modifying enzymes may play a role in rescue of the AAV genome in human cells.     

Hepatitis B core particles with endogenous DNA polymerase activity from chimpanzee liver

Hepatitis B core antigen (HBcAg) particles, approximately 27-28 nm in diameter and rho = 1.30-1.35 g/cm3, were purified from the liver of a chimpanzee experimentally infected with hepatitis B virus (HBV) while under cyclophosphamide treatment. The purified HBcAg particles incorporated radioactive deoxythymidine triphosphate. The product was precipitable by trichloroacetic acid and sensitive to DNase, but resistant to digestion by RNase. The reaction required four deoxyribonucleosise triphosphates- dATP, dCTP, dGTP and dTTP. Exogenous template did not enhance the reaction. From these findings, it was suggested that HBcAg particles purified from the HBV-infected chimpanzee liver contained DNA polymerase and endogenous DNA.     

Tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie

Tubulovesicular structures, measuring 20-50 nm in diameter, were found in dilated neuronal processes in brains from mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease virus. These particles were similar to those observed in brains from hamsters infected with scrapie. These structures are consistently present in naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species or virus strain. Their role in pathogenesis is undetermined.     

Physical, chemical, and ultrastructural studies of water-soluble human amyloid fibrils. Comparative analyses of nine amyloid preparations

Amyloid fibrils were isolated from the tissues of nine patients with amyloidosis in a state of high purity by homogenization of the tissue followed by extraction with distilled water. Physical, chemical, and ultrastructural studies suggest that amyloid fibrils from different individuals resemble each other, but are not identical. In tissue sections as well as by negative staining of isolated fibrils, morphologic variations were observed. Among the isolated fibrils at least three types were noted. The majority resembled those described previously. However, one subject had two types of fibrils which differed in size and appearance. Most of the preparations sedimented as a single component with a sedimentation coefficient of 45–50S or as a larger polymer. However, two of the preparations had sedimentation coefficients of 8–9S, and a third one had a major 95S component and a minor 9S fraction. While the preparations of amyloid were not sufficiently pure for amino acid analyses, peptide maps demonstrated differences among amyloid preparations from different subjects. The amyloid fibrils in their native state proved to be remarkably resistant to digestion by a number of proteolytic enzymes. Several chemical methods were tried to produce smaller subunits. Of these, the most successful one was the use of 0.1 M NaOH which yielded a smaller, soluble fraction with sedimentation coefficients ranging from 1.1 to 2.8S. Accompanying this degradation, there was little loss of peptides or carbohydrates. Based on the results of the chemical analyses, it is estimated that the subunit produced by sodium hydroxide had a molecular weight of approximately 35,000–40,000.     

Production and catabolism of amyloid peptides in Alzheimer's disease

Senile plaques that accumulate in cortical and sub-cortical areas of Alzheimer's disease (AD)-affected brains are mainly composed of a set of hydrophobic and aggregated peptides referred to as amyloid β-peptides (Aβ). These peptides derive from the proteolytic processing of a transmembrane precursor, the β-amyloid precursor protein (βAPP) that undergoes subsequent cleavages by β- and γ-secretases, respectively. Another enzyme called α-secretase cleaves βAPP in the middle of its Aβ sequence and thereby, lowers its production. Once produced, Aβ peptides can be cleared off by neuropeptidases, mainly insulin-degrading enzyme and neprilysin, or, alternatively, can be biotransformed upon N-terminal truncation by exopeptidases and subsequent cyclisation of the glutamate in position 3. Here, we will describe the nature of the various proteolytic activities documented above and we will discuss briefly their advantages and drawbacks as putative therapeutic targets in AD.     

Activity of clarithromycin alone or in combination with other drugs for treatment of murine toxoplasmosis.

The activity of the macrolide antibiotic clarithromycin was examined alone or in combination with other drugs for the treatment of acute or chronic infections with Toxoplasma gondii in mice. A dose of 300 mg of clarithromycin per kg per day administered alone for 10 days, beginning 24 hours after infection, protected 10 to 30% of mice infected with lethal inocula of tachyzoites or tissue cysts of different strains of T. gondii, including some strains isolated from patients with both AIDS and toxoplasmosis. Although clarithromycin was protective, a wide variation in its activity against different strains was observed. Survival of infected mice was increased significantly by treatment with clarithromycin in combination with pyrimethamine or with sulfadiazine. Treatment of chronically infected mice with clarithromycin at 300 mg/kg/day administered alone for 8 weeks resulted in significant reduction in the numbers of T. gondii cysts in their brains. The combination of clarithromycin and minocycline resulted in an activity against T. gondii cysts that was significantly greater than the activity of clarithromycin or minocycline administered alone. These results indicate a role for clarithromycin in the treatment of human toxoplasmosis, particularly when this antibiotic is used in combination with other drugs with activity against T. gondii.     

Comparative assessment of physicochemical and antioxidative properties of mung bean protein hydrolysates

Two commercial plant proteases namely ficin and bromelain, were acquired to hydrolyze mung bean protein over 300 min hydrolysis, and the physicochemical and antioxidative properties of the obtained hydrolysates were investigated. Bromelain-treated mung bean protein hydrolysates presented a higher degree of hydrolysis in comparison with ficin-treated hydrolysates, further modifying their physicochemical and emulsifying properties. All mung bean protein hydrolysates exhibited 50% scavenging of DPPH radical (IC₅₀) in the concentration range from 8.67 to 16.22 μg mL⁻¹. Our results also showed that strong metal ion-chelating activity was found in the ficin- (higher activity) and bromelain-treated protein hydrolysates. In addition, oxidative stability of linoleic acid was significantly enhanced by two selected protein hydrolysates, particularly the bromelain-treated hydrolysate with the highest inhibition effect of linoleic acid oxidation (94.55 ± 0.10%). Interestingly, both of these two hydrolysates could effectively retard lipid oxidation of sunflower oil and sunflower oil-in-water emulsion, while the ficin-treated hydrolysate showed slightly better performance. Therefore, mung bean protein hydrolysates showed potential to inhibit lipid oxidation, which could be advantageous in the food industry for producing fortified food.

Two commercial plant proteases namely ficin and bromelain, were acquired to hydrolyze mung bean protein over 300 min hydrolysis, and the physicochemical and antioxidative properties of the obtained hydrolysates were investigated.     

THE RELATION OF STREPTOCOCCI TO HERPES VIRUS ENCEPHALITIS

Cultures of microorganisms similar to those described by Evans have been obtained in media inoculated with suspensions of herpes virus-infected brains prepared by grinding. But they have also been isolated from saline suspensions of uninoculated meat particles ground in a sterile mortar, and from dextrose broth treated in the same way. It is believed that these organisms are contaminants introduced during the process of grinding. Since they enter the material in no great number, one may suppose them to be suppressed by animals inoculated with the ground substance. In artificial media, on the other hand, they find favorable conditions for multiplication. In our experience, no growth of microorganisms is obtained in routine cultures of virus-infected brains, when fragments, instead of ground material, are used—a fact which may be taken to support the explanation just given. The tests of the part played by streptococci in experimental virus encephalitis failed to disclose that the microorganisms have any etiological relationship to the affection. The intracerebral injection of rabbits with the cultures procured in the course of the experiments produces a purulent type of meningoencephalitis which does not resemble virus encephalitis either in its symptom-complex or in its pathology. The same type of meningitis follows the injection of streptococci derived from ground meat particles, from "ground" broth, from normal brains, and those infected with herpes virus. Some rabbits manifested resistance to the streptococci, whereas all that have been inoculated intracerebrally with the three strains of herpes virus used in this study have proved susceptible thereto. Certain of the rabbits just mentioned which had proved resistant to streptococci inoculated into the brain or cornea were injected with herpes virus and reacted typically. Comparative tests have revealed that the streptococci are more sensitive to the destructive effect of 50 per cent glycerol than is herpes virus. From all this, it can be concluded that streptococci are not the visible form of herpes virus, nor do they produce in rabbits effects like those induced in the brain and cornea by the herpes virus.     

Pathological changes in the brains of rabbits experimentally infected with Angiostrongylus cantonensis after albendazole treatment: histopathological and magnetic resonance imaging studies

OBJECTIVES: To determine the effects of albendazole on rabbits infected with larvae of Angiostrongylus cantonensis by histopathological and magnetic resonance imaging (MRI) techniques. METHODS: Male rabbits were infected with 400 A. cantonensis larvae and treated with albendazole (5 mg/kg/day) for 2-14 days on day 5, 10, 15 or 20 post-infection. RESULTS: Although there were pathological changes in the brains, MRI revealed unremarkable findings in the untreated group. However, the treated rabbits exhibited eosinophilic meningitis, choroid plexus inflammation, meningeal congestion, encephalitis, perivascular cuffing and meningitis, and were also found to have abnormal signal intensities on brain MR images in the 20 day post-infection treated group. CONCLUSIONS: Pathological changes in the brains of the treated rabbits are more severe than those without albendazole treatment, suggesting that the drug may not be very suitable for the treatment of cerebral angiostrongyliasis.     

Reversal of shortened platelet survival in rats by the antifibrinolytic agent, epsilon aminocaproic acid.

Platelet survival in rabbits and rats is shortened by placing indwelling catheters in the aorta; this shortening appears to be at least partly related to the extent of vessel wall injury and platelet interaction with the repeatedly damaged wall. Treatment of rabbit platelets with plasmin and other proteolytic enzymes in vitro shortens their survival when they are returned to the circulation. Because platelets may be exposed to plasmin and other proteolytic enzymes in rabbits and rats with indwelling aortic catheters, we examined the effect of epsilon-aminocaproic acid (EACA) on platelet survival in rats. At a dose of 1 g/kg every 4 h, EACA significantly reduced whole blood fibrinolytic activity and prolonged the shortened platelet survival in rats with indwelling aortic catheters. Mean platelet survival for untreated rats with indwelling aortic catheters was 38.6 +/- 1.9 h, and for rats treated with EACA, 53.8 +/- 3.8 h. Scanning electron microscopy showed that the injured vessel wall of these animals was mainly covered with platelets and fibrin, whereas in control animals that did not receive EACA, the injured surface was mainly covered with platelets and little fibrin was observed. Thus shortened platelet survival during continuous vessel wall injury may result from the local generation of plasmin or the release of proteolytic enzymes at sites where platelets (and possibly leukocytes) interact with the vessel wall.     

Cyclophosphamide increases transgene expression mediated by an oncolytic adenovirus in glioma-bearing mice monitored by bioluminescence imaging.

Approaches to improve the oncolytic potency of replication-competent adenoviruses include the insertion of therapeutic transgenes into the viral genome. Little is known about the levels and duration of in vivo transgene expression by cells infected with such "armed" viruses. Using a tumor-selective adenovirus encoding firefly luciferase (AdDelta24CMV-Luc) we investigated these questions in an intracranial mouse model for malignant glioma. Luciferase expression was detected by bioluminescence imaging, and the effect of the immunosuppressive agent cyclophosphamide (CPA) on transgene expression was assessed. Intratumoral AdDelta24CMV-Luc injection led to a localized dose-dependent expression of luciferase. Surprisingly, this expression decreased rapidly during the course of 14 days. In contrast, mice injected with nonreplicating Ad.CMV-Luc demonstrated stable transgene expression. Treatment of mice with CPA in combination with AdDelta24CMV-Luc retarded the loss of transgene expression. Staining of mouse brains for inflammatory cells demonstrated decreased tumor infiltration by immune cells in CPA-treated mice. Moreover, in immunodeficient NOD/SCID mice loss of transgene expression was less rapid and not prevented by CPA treatment. Together, our data demonstrate that transgene expression and viral replication decrease rapidly after intratumoral injection of oncolytic adenovirus in mouse brains and that treatment with the immunomodulator CPA prolongs viral-mediated gene expression.     

Mass isolation and culture of rat kupffer cells

Collagenase perfusion of the liver followed by pronase treatment of the cell suspension thus obtained gave a quantitative recovery of viable nonparenchymal liver cells (NPC). From these NPC, Kupffer (K) cells can be purified by attachment to tissue culture dishes. Tail vein injection of carbon 1-2 h before liver perfusion permitted stepwise calculation as well as visualization of carbon-containing K cells. When these K cells have been put into tissue culture medium with serum and incubated overnight, they exhibit typical macrophage characteristics. Phase- contrast and transmission electron microscopy showed typical macrophage morphology and scanning electron microscopy revealed well-spread cells with cytoplasmic projections and ruffled membranes. Endocytosis studies using radioactive colloidal gold and inert latex particles also indicated that these cells are highly active in pinocytosis and phagocytosis. Further characterization of K cells is the identification of Fc receptor on their membranes. Studies on lysosomal enzymes showed that purified K cells possess higher specific activities in beta- glucuronidase, acid DNase, and cathepsin D than in purified parenchymal cells.     

Serologic analyses of an immunosuppressive cell-free factor from mastocytoma cells and neutralization by antiserum.

In order to elucidate receptors of proteolytic enzyme-treated red cells which react with Phaseolus coccineus L. lectin, the receptors prepared by affinity chromatography were serologically investigated. P. coccineus lectin had high agglutinin activity for bromelin-, papain- and pronase-treated red cells but that for the cells treated with ficin and trypsin was relatively low. Analyses of chemical composition revealed that sialic acid of the receptors from normal red cells was considerably much as compared with that from the treated cells. On the contrary, the enzyme treatment did not affect particularly carbohydrate composition of the receptors. Disc electrophoresis showed that the patterns of receptors from red cells treated with bromelin or papain were different from those from the other cells. On two-dimensional immunoelectrophoresis, the receptor of trypsin-treated cells gave five precipitation lines against anti-stroma and that of papain-treated cells three lines, but any other receptors showed no line. These findings indicate that there are plural receptors for P. coccineus lectin in red cells treated with each of proteolytic enzymes and that the receptors from respective red cells have electrophoretically and serologically different property.     

Inhibition of cytotoxic T lymphocyte-induced target cell DNA fragmentation, but not lysis, by inhibitors of DNA topoisomerases I and II

Cytotoxic T lymphocytes (CTL) kill their target cells via a contact-dependent mechanism that results in the perturbation of the target cell's plasma membrane and the fragmentation of the target cell's DNA into nucleosomal particles. The membrane disruption is presumed to be due to the action of perforin, while the DNA fragmentation is thought to be by the activation of an endogenous nuclease(s). DNA topoisomerases I and II are nuclear enzymes with inherent endonuclease activities. We have investigated their role in the CTL-induced DNA fragmentation process. We report that in CTL killing assays, the treatment of target cells with topoisomerase I and II inhibitors blocks the CTL-induced DNA fragmentation process, but not the lysis of the target cell.