Viral neuroinvasion and encephalitis induced by lipopolysaccharide and its mediators.

The present study was designed to test the effect of bacterial endotoxin on penetration of viruses into the central nervous system (CNS). As a model we used two neurovirulent viruses that lack neuroinvasive capacity: West Nile virus-25 (WN-25) and neuroadapted Sindbis virus (SVN). Administration of lipopolysaccharide (LPS, 100 micrograms/mouse) to CD-1 mice, followed by WN-25 inoculation resulted in 83% encephalitis and death, compared with less than 5% in controls. The results in SVN-inoculated CD-1 mice were quite similar. LPS-treated mice suffered 62% mortality compared with 6% in the nontreated group. No changes in viral neuroinvasiveness were demonstrated in viruses isolated from brains of encephalitic mice, suggesting that neuroinvasion is not due to a selection process for an invasive variant, but to direct penetration of the viruses through the blood-brain barrier (BBB). LPS did not induce WN-25 encephalitis in LPS-insensitive C3H/HeJ mice, compared with 100% neuroinvasion in C3H/HeB mice. Induction of neuroinvasion could be transferred to C3H/HeJ mice by transfusion with serum obtained from LPS-treated, LPS-responsive mice. Passive immunization of CD-1 mice with anti-mTNF antibodies before LPS administration did not prevent LPS-induced WN-25 encephalitis. Furthermore, neutralization of tumor necrosis factor activity in the serum of LPS-treated mice did not abolish its activity, and transfusion-associated encephalitis was observed after the administration of the neutralized serum with WN-25. We suggest that LPS can contribute to virus penetration from the blood into the CNS, a process which turns a mild viral infection into a severe lethal encephalitis. This effect is mediated by soluble factors, and is probably achieved by injury to cerebral microvascular endothelium and modulation of BBB permeability.

Distribution of an endogenous lectin in the developing chick optic tectum.

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.

vFLIP protects PC-12 cells from apoptosis induced by Sindbis virus: implications for the role of TNF-alpha.

Sindbis virus (SV) is an alphavirus used as a model for studying the pathogenesis of viral encephalitis. In this study we examined the effects and the mechanisms involved in the apoptosis induced by SV in PC-12 cells, and the role of a vFLIP in this process. Infection of PC-12 cells with a neurovirulent strain of SV, SVNI, induced cell apoptosis. Overexpression of vFLIP encoded by the HHV-8 or treatment with a caspase-8 inhibitor inhibited cell apoptosis. SVNI induced an increase in the expression of tumor necrosis factor alpha (TNF-alpha), and pre-treatment of the cells with an anti-TNF-alpha blocking antibody or with soluble TNF-alpha receptor abrogated the apoptotic effect of SVNI. Moreover, TNF-alpha R1 knockout mice were more resistant to the cytopathic effects of the virus as compared to control animals. Our results indicate that the apoptosis induced by SVNI is mediated by activation of caspase-8, and that TNF-alpha plays an important role in the apoptotic response.

Heterogeneity of childhood acute lymphoblastic leukemia (ALL): monoclonal antibodies phenotyping and induction of differentiation in vitro.

Fifty-eight pediatric patients with non-T acute lymphoblastic leukemia (ALL) were diagnosed and evaluated at the Sambur Center of Pediatric Hematology Oncology. At least six subtypes of non-T ALLs were identified, corresponding to the various stages of B-cell differentiation, by utilizing an extensive panel of monoclonal antibodies directed against T- B- and myeloid-cell differentiation antigens. Moreover, leukemic cells expressing the phenotype of early B cells could be driven to differentiate along the B- cell lineage to express CALLA and BL antigens and cytoplasmic and/or surface immunoglobulins (IgM). A unique phenotype of non-T ALL was also identified. These leukemic cells expressed B cell antigen exclusively, i.e., HLA/DR and B4 (CD19). Myeloid-cell antigens, however, were expressed on these cells spontaneously after a 24-hour incubation in culture medium in vitro. In addition, leukemic cells of four patients with a phenotype of HLA/DR, CD19, and CD10 expressed antigens of the T-cell lineage: CD7 (3AI) and CD2 (leu 5), and/or of the myeloid cell lineage (My7). These results provide confirming evidence for the wide scope of the heterogeneity of ALL. It stresses the validity of accurate classification of leukemia to identify biologically and clinically unique subtypes of ALL, which bears specific prognostic parameters; and designates therapeutic protocols.

A live attenuated West Nile virus strain as a potential veterinary vaccine.

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.

Long-term culture of infant leukemia cells: dependence upon stromal cells from the bone marrow and bilineage differentiation.

Infant leukemia cells with 46XY,t(11; 17)(q23; p13) karyotype and a hybrid pre B myeloid phenotype (HLA-DR, (Ia), B4 and My7-positive and CALLA and T11-negative) and immunoglobulin heavy chain gene rearrangement were maintained in long-term culture for over 10 months. The in-vitro survival and growth of the leukemia cells were strictly dependent upon the presence of their autologous marrow stromal cells. The latter could be replaced by the 14F1.1 clone of preadipocytes derived from mouse bone marrow. Neither heterologous human marrow or foreskin fibroblasts nor fibroblast or endothelial like cell lines from mouse stroma could mimic the effect of autologous stroma or 14F1.1 adipocytes. The leukemia cells maintained their original phenotype throughout the 10-month culture period with either their autologous stroma or the 14F1.1 adipocytes. They could be induced to differentiate in two distinct directions. Phorbol myristate acetate induced adherence of the leukemia cells and development of macrophage properties. In contrast, conditioned medium from a hybridoma producing B-cell growth factor caused aggregation of the leukemia cells and expression of CALLA antigen and surface IgM. This bipotency of the leukemia cells and their dependence upon marrow stroma are properties in common with stem cells.

CNS penetration by noninvasive viruses following inhalational anesthetics.

The effects of inhalational anesthetics on brain penetration by the neurovirulent noninvasive West Nile virus (WN-25) were studied in mice. WN-25 injected intracerebrally causes encephalitis and kills adult mice, but when injected intraperitoneally (i.p.) it is unable to invade the brain and kill. Under stress conditions, this strain causes encephalitis and death even after i.p. inoculation. In the study described in this paper, we used two inhalational anesthetics, a single short-term exposure to 2% halothane for 10 min in oxygen, or 70% nitrous oxide (N2O) for 30 min in air. Both inhalational anesthetics induced WN-25 encephalitis and death in 33% and 20% of the tested mice, respectively. Exposure of inoculated mice to halothane for prolonged periods or for repeated exposures (two or three times) markedly increased the mortality rate (up to 75%). Exposure to 30% CO2, a known modulator of blood-brain barrier (BBB) activity, was used as a positive control (80% mortality). No death was observed in the control non-exposed injected mice. Virus levels were found to be more than 10(7) plaque-forming units (PFU)/brain in all moribund mice. Additional parameter demonstrating the "stressor-like" nature of inhalation anesthetics was the induction of a significant decrease in weight of the lymphoid organs of inoculated mice. We suggest that inhalational anesthetics induces BBB breaching with subsequent entrance of the noninvasive WN-25 virus into the brain, causing encephalitis and death.

Lectin and toxin-like activities of Entamoeba histolytica: comparison of properties.

A comparison was made between properties of a recently discovered Entamoeba histolytica lectin which has a carbohydrate specificity for N-acetylglucosamine oligosaccharides and the previously found toxin-like principle of the ameba. A separation between these two activities was achieved upon subcellular fractionation by high speed centrifugation of freeze-thawed disrupted E. histolytica trophozoites (strain HM-1). Practically all of this lectin activity, as determined by hemagglutination of glutaraldehyde-fixed human erythrocytes, was found associated with the sedimented membrane fraction. This fraction did not affect monolayers of tissue-cultured mammalian cells. On the other hand, the soluble supernatant solution caused extensive damage to the tissue-cultured cells (change in morphology and detachment of cells). Both the lectin and toxin activities were heat-labile and their activities were preserved by the presence of reducing agents and proteolytic enzyme inhibitors. In contrast to the toxin, the isolated lectin was inactive at pH 7.2 and active only at pH 5.7-6.0. Both the lectin and toxin were inhibited by a number of macromolecular compounds such as chitin, peptidoglycan, bovine serum and an IgA fraction isolated from human colostrum. Only the lectin activity, however, was inhibited by low molecular weight chitin oligosaccharides (GlcNAc)n=2-6 or by lysozyme-digested peptidoglycan subunits. Moreover, fetuin and a ganglioside mixture extracted from ox brain were found to inhibit only the toxin-like activity. The IgG fraction of sera from patients with invasive amebiasis neutralized both lectin and toxin-like activities, while IgG from normal sera failed to neutralize either activity. Although our results indicate that in E. histolytica, lectin and toxin are two separate activities, both of them share a considerable number of properties which does not exclude the possibility that they may be related.

The effect of antisynaptosomal plasma membrane antibodies on memory.

Antisynaptosomal plasma membrane antibodies were introduced through an infusion cannula into rat brain and their effects on behaviour were tested. Four different learning paradigms were used, two appetitively and two aversively motivated, to show impairment in memory retrieval. No effects were found on aquisition, motor activity, or motivation.

Sodium dodecylsulphate induces a breach in the blood-brain barrier and enables a West Nile virus variant to penetrate into mouse brain.

A novel and convenient assay was used to determine the effect of recombinant Interleukin-2 (IL-2) on the function of the blood-brain barrier (BBB). The assay is based on a variant of the West Nile virus, WN-25, which had lost its neuroinvasiveness but not its neurovirulence. WN-25, when injected intravenously, can cause the death of mice only if the function of the BBB is impaired. Sodium dodecylsulphate (SDS), a component in IL-2 excipient, was found to cause a short term breach in the BBB, enabling the penetration of viruses into the brain. Minimal amounts (30 ng/mouse) can induce a breach of about 10 min, which allows 0.1% of the injected virus to enter the brain. These findings demonstrate the possible use of SDS as a mean for intentional introduction of drugs into the brain, however they also call attention to the danger of using detergents as additives for drugs given intravenously.

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors.

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.

Retrograde amnesia production by the intracisternal injection of 20 micronl of saline in rats.

The intracisternal injection of 20 micronl of saline into a rat within an hour after training it in an active avoidance response was found to induce retrograde amnesia. The results obtained by combining this procedure with the intracisternal injection of 2,6 Diaminopurine (DAP), which inhibits RNA synthesis and prevents long-term memory formation when administered at the proper time and in the correct dosage, suggest the existence of a medium term memory (MTM) mechanism. MTM was normally evident up to 75 min after training but was demonstrable up to 210 min when LTM formation was prevented by DAP.

Entamoeba histolytica: correlation between virulence and content of proteolytic enzymes.

Intact trophozoites of the virulent Entamoeba histolytica strain HM-1:IMSS (HM-1) destroyed a monolayer of baby hamster kidney (BHK) cells at a higher rate and efficiency than trophozoites of the nonvirulent strain HK-9. The destructive effect could be partially attributed to the proteolytic activity of the amoeba, since quantitative differences were found in the enzymatic activity of the two strains tested. Crude extracts or secreted enzymes of HM-1 trophozoites digested Azocoll, as well as the bovine cold-insoluble globulin fraction, at a much higher rate than the corresponding preparations from HK-9. This proteolytic activity was found to be activated by free sulfhydryl groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the BHK cell proteins of pre- and postamoebic activities showed patterns similar to the trypsin effect on the same target cells. These enzymes were found to digest the proteins participating in the attachment of the target cells to the substrate and, consequently, cause detachment of these cells.

Lectin activity in Entamoeba histolytica trophozoites.

A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH, and HM-1:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 x g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.

A lectin activity in Entamoeba histolytica trophozoites.

A lectin (carbohydrate binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in Diamond's TYI-S-33 media were HK-9, 200:NIH and HM-1: IMSS. Strain HU-1: MUSC (HSC) was grown monoxenically in the same medium. The amoeba lectin agglutinates glutaraldehyde-fixed red blood cells. This activity is pH dependent, heat and oxidation sensitive and is destroyed by proteolysis upon auto-incubation. The relative agglutinating potency of the different strains was investigated. Strain HSC had the highest specific activity (210 units/mg protein) and strain HM-1 the lowest (14 units/mg). One unit of haemagglutinating activity is defined as the amount of leetin present in 1 ml of extract which will agglutinate 1 ml of 4 per cent red blood cells. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two thirds of the activity were detected in the soluble, non-sedimentable (100,000 x g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the haemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300 fold by a one step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.

Adhesion properties of Entamoeba histolytica.

Trophozoites of Entamoeba histolytica adhere to and phagocytize red blood cells and bacteria. Furthermore, in the initial step of the amoebic infectious process the parasite attaches to intestinal epithelial cells. A lectin (carbohydrate-binding protein) which apparently has a role in the attachment of the parasite to host cells was found in trophozoites of E. histolytica. When amoeba cells were disrupted by freeze-thawing, the lectin activity, as determined by haemagglutination of human erythrocytes, remained associated with the sedimented membrane fraction. This activity was pH dependent and heat and oxidation-sensitive, and was destroyed by proteolysis and on autoincubation. Moreover, the lectin activity was inhibited by a variety of N-acetylglucosamine-containing compounds such as chitin and chitin oligosaccharides, bacterial peptidoglycan, rabbit colonic mucus, bovine and human serum, an IgA fraction isolated from human colostrum, and IgG from sera of amoebiasis patients. These glycoconjugates also interfered with the adherence of intact radiolabelled amoeba trophozoites to human intestinal epithelial cells as well as their attachment to red blood cells. Although the lectin activity and the toxin-like activity previously found in E. histolytica seem to be two separate substances, they share a number of properties which suggest that they are related and may have a function in pathogenicity.

Adhesion of Entamoeba histolytica trophozoites to monolayers of human cells.

The adhesion of radiolabeled trophozoites of Entamoeba histolytica to monolayers of a human intestinal epithelial cell line was found to be dependent on time, temperature, and concentration. Adherence seems to be mediated by a carbohydrate binding protein (lectin), which was previously found in trophozoites of E. histolytica. The adherence of the trophozoites was found to be pH-dependent and was inhibited by several N-acetyl-glucosamine-containing glycoconjugates, such as bacterial peptidoglycan, chitin, and IgA; similar results were obtained with the isolated lectin. Furthermore, the semipurified amoebic lectin and wheat germ agglutinin, which have similar sugar specificities, competed with the intact amoeba for receptor sites on the epithelial cells. In addition, both sera from patients with amoebiasis and an IgG fraction from these sera inhibited lectin activity and the adherence of the trophozoites.