The gene for a T lymphocyte triggering factor from African trypanosomes.

An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-gamma (IFN-gamma). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-gamma do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8(+) T lymphocytes to proliferate and to secrete IFN-gamma. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8(+), but not CD4(+), T cells to secrete IFN-gamma. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

Differential invasion of Trypanosoma brucei brucei and lymphocytes into the brain of C57BL/6 and 129Sv/Ev mice.

Trypanosoma brucei subspecies invade the brain parenchyma at late stages of human and experimental rodent infections. In this study, we compared the outcome of infection with T. b. brucei in MHC-matched (H-2b) C57BL/6 (B6) and 129Sv/Ev (Sv-129). Sv-129 showed higher parasitaemia and lower specific IgM (but not IgG) antibody levels than B6 mice. The number of trypanosomes, CD4+ and CD8+ T cells in the brain parenchyma was higher in B6 mice. B6 mice lost weight and showed higher cumulative mortality when compared with Sv-129 mice. Higher levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma, ICAM-1 and E-selectin, but low levels of TGF-beta mRNA were present in brains of B6 when compared with Sv-129-infected mice. Thus, host genetics differentially determine the invasion of T. b. brucei into the brain parenchyma, which is paralleled by the severity of inflammation in the brain and course of the disease, but not by parasitaemia nor by antibody titres.

Trypanosoma brucei and the nervous system.

African sleeping sickness, characterized by a peculiar pain syndrome and prominent neuropsychiatric symptoms, is caused by the parasite Trypanosoma brucei (T.b.). In experimental T.b. infections, a molecule released from the trypanosomes has been isolated that binds to the CD8 molecule of T cells, whereby T cells are activated to secrete interferon gamma. This cytokine binds to the parasites and triggers them to proliferate, establishing a peculiar bidirectional activating signal system. The hypothesis is presented that the molecules involved in these bidirectional signals might also interact with neurons, thus causing brain dysfunctions. Studies on the molecular interactions between parasites and the nervous system in sleeping sickness might reveal basic mechanisms underlying other neuropsychiatric diseases.

Sensitization of dorsal horn neurons in a two-compartment cell culture model: wind-up and long-term potentiation-like responses.

One of the main characteristics of central sensitization associated with postinjury pain and chronic pain is increased excitability of the dorsal horn neurons in the spinal cord. Two electrophysiological features associated with the origin and modulation of central sensitization are wind-up of action potential frequency and long-term potentiation (LTP), which have been demonstrated previously in the intact dorsal horn. Here we present evidence for electrically evoked sensitization of dorsal horn neurons in a two-compartment cell culture system of rat dorsal root ganglia (DRGs) and dorsal horn neurons. Whole-cell recordings of dorsal horn neurons showed that repetitive low-frequency stimulation of DRG axons induced a frequency-dependent cumulative depolarization of the membrane potential with a concomitant increase in action potential frequency in a subset of neurons (41%). The characteristics presented here for dissociated cells are in accordance with those ascribed to classical wind-up in the intact dorsal horn. In addition, tetanic stimulation of DRG axons resulted in a significant increase in the number of action potentials in response to test stimuli in 42% of the cells tested. This prolonged potentiation of neuronal excitability in the dorsal horn lasted throughout the recording period (>1 hr) and tended to be voltage dependent in an LTP-like manner. To our knowledge, this is the first time that wind-up and LTP-like responses are reported for dorsal horn neurons in cell culture.

Measles encephalitis in rodents: defective expression of viral proteins.

Synthesis of measles virus proteins in rodent brains and Vero cell cultures infected with the hamster neurotropic (HNT), and for comparison the LEC strain, was studied by use of monoclonal antibodies against five structural components. In the brains of HNT-infected adult BALB/C mice two proteins, the nucleocapsid (NP) and phosphoprotein (P) were detected. Suckling hamster brains in addition expressed demonstrable hemagglutinin (HA) protein. In cell cultures all structural components except the matrix (M) protein were detected. In contrast, all five proteins were found in LEC strain-infected suckling hamster brains and cell cultures. The restriction in HNT viral replication observed may be caused by a primary defectiveness in M-protein expression, but the possibility that this restriction is secondary to cellular suppression remains to be explored. Minimal inflammation was seen in the brains of HNT-infected adult mice and viral antigen was primarily located in the cerebral cortex. A selective necrosis of the pyramidal cell layer of the hippocampus was observed. This change did not seem to correlate with virus replication.

Non-lethal infection of aminergic reticular core neurons: age-dependent spread of ts mutant vesicular stomatitis virus from the nose.

In order to induce a non-lethal infection restricted to central aminergic neurons projecting to the olfactory bulbs a series of temperature sensitive (ts) and G-protein monoclonal antibody escape mutants of vesicular stomatitis virus (VSV) were instilled into the nasal cavity of mice. In three-week (wk)-old NMRI mice four monoclonal antibody escape mutants caused an extensive infection of the olfactory epithelium and, like a wild type strain, a lethal brain infection after spread along olfactory pathways. Three ts mutant strains showed an attenuated pathogenic potential. Strain G31 caused a lethal infection with a somewhat prolonged course while the strain G11 failed to invade the nervous system. Strain G41 showed minimal invasion of central nervous system in three-wk-old mice and caused a lethal infection in newborn and one-wk-old mice. In contrast, two-wk-old mice survived infection with this mutant, which spread along olfactory pathways and rather selectively affected aminergic reticular core neurons in the diagonal band, the locus ceruleus and the raphe nuclei in the brainstem. Thus, an age-dependent virus infection of the olfactory pathways can cause restricted lesions in the brain providing a model for studies of virus-induced changes in aminergic neurotransmission.

Mumps virus infection of the developing mouse brain--appearance of structural virus proteins demonstrated with monoclonal antibodies.

Newborn mice and hamsters were inoculated intracerebrally with mumps virus strains of high and low neurovirulence, Kilham and RW, respectively and with an egg-adapted patient isolate. The presence of viral antigen in brain tissue was analyzed with the immunofluorescence technique employing monoclonal antibodies against nucleoprotein (NP), polymerase (P), matrix (M), hemagglutinin-neuraminidase (HN) and fusion (F) mumps virus components. As expected, hamsters developed a fatal encephalitis eight to nine days after infection with the Kilham strain and synthesis of all five structural viral antigens was identified. In contrast, mice infected with any of the virus strains did not develop signs of disease, but in brain material collected on days nine and 12 after infection viral antigen was present in many neurons. However, only NP and P antigens were demonstrable and no infectious virus was present. The antibody response in mice developed later than in hamsters. Neurons in the mouse brain may exert a host cell restriction on the virus maturation, and mice offer a suitable host for the establishment of defective, persistent mumps virus infections.

Altered neuronal activity rhythm and glutamate receptor expression in the suprachiasmatic nuclei of Trypanosoma brucei-infected rats.

The parasites Trypanosoma brucei cause African trypanosomiasis (sleeping sickness), a severe neuropsychiatric disease with marked disturbances of sleep-wake alternation. The sites of brain lesions are not well characterized. The present experimental investigation is focused on the hypothalamic suprachiasmatic nuclei, which play a role of a biological clock entraining endogenous rhythms in the mammalian brain. The electrophysiological properties of these neurons were analyzed in slice preparations from trypanosome-infected rats. The neuronal spontaneous activity, which shows a circadian oscillation, was markedly altered in the infected animals, displaying a reduced firing rate and phase advance of its circadian peak. The direct retinal fibers, which play a pivotal role in entrainment of the circadian pacemaker, displayed a normal density and distribution in the suprachiasmatic nuclei of infected animals after intraocular tracer injections in vivo. At the postsynaptic level, immunohistochemistry and Western blotting revealed in the suprachiasmatic nuclei of infected rats a selective decrease of the expression of glutamate AMPA GluR2/3 and NMDAR1 receptor subunits that gate retinal afferents. These data disclose an impairment of the neuronal functions in the biological clock in African trypanosomiasis, and may serve to unravel functional and molecular mechanisms behind endogenous rhythm disturbances.

The lipid compositions of different regions of rat brain during development and aging.

The lipid contents in different regions of the rat brain were analyzed from birth to the age of 2 years. The total brain phospholipid content increased threefold during the first 20 postnatal days. The cholesterol content elevated extensively during the first 2 months of life and, after this period, remained unchanged. The level of dolichol increased almost 100-fold during the first 10 months of life and continued to increase thereafter. Some modifications in the dolichol isoprenoid pattern were also observed. An increase in the brain ubiquinone level occurred during the first few months of life, but no further change was observed after this period. Ubiquinone-9 and -10 constituted 70 and 30%, respectively, of the total ubiquinone in all regions and all subcellular fractions. The alpha-tocopherol content increased during the first 3 weeks of life and was unchanged thereafter. These results demonstrate characteristic changes in the lipid contents of various regions of rat brain during development and aging.

Persistent changes in behaviour and brain serotonin during ageing in rats subjected to infant nasal virus infection.

Suckling rats were infected intranasally with the temperature-sensitive mutant G41 strain of vesicular stomatitis virus. The rats survived but demonstrated lifelong learning deficits in the Morris maze and impaired exploratory behaviour in the open field test. When examined at 18 months of age they had a severe loss of neurons in the medial and dorsal raphe nuclei in the brain stem and reduced levels of serotonin and its metabolite 5-hydroxyindole acetic acid in the cerebral neocortex and hippocampus. The levels of noradrenaline, dopamine, homovanillic acid, 3,4-dihydroxyphenylacetic acid, choline acetyltransferase and glutamate decarboxylase were largely unaffected. The permanent disturbance in brain serotonin metabolism did not cause any histological changes in the cerebral cortex. Thus there were no neurofibrillary tangles or amyloid plaques as has been reported as a late effect of chemically induced lesion to the cholinergic system in the rat brain. It is concluded that the brain serotonergic system is especially vulnerable to an episode of virus attack along olfactory pathways and that the neurochemical and behavioural alterations caused by such an episode persist during a major part of the animal's life span.

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices.

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.

Chronic overexpression of proinflammatory cytokines and histopathology in the brains of rats infected with Trypanosoma brucei.

Overproduction of proinflammatory cytokines in the brains of transgenic animals causes brain pathology. To investigate the relationship between brain cytokines and pathology in the brains of animals with adult-onset, pathophysiologically induced brain cytokine expression, we studied rats infected with the parasite Trypanosoma brucei. Several weeks after infection, in situ hybridization histochemistry showed a pattern of chronic overexpression of the mRNAs for proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha in the brains of the animals. Similar spatiotemporal inductions of mRNAs for inhibitory factor kappaBalpha and interleukin-1beta converting enzyme were found and quantified. The mRNAs for inducible nitric oxide synthase and interleukin-1 receptor antagonist were highly localized to the choroid plexus, which showed evidence of structural abnormalities associated with the parasites' presence there. The mRNAs for interleukin-6, interferon-gamma, and inducible cyclooxygenase showed restricted induction patterns. Another set of animals was processed for degeneration-induced silver staining, TdT-mediated dUTP-digoxigenin nick end-labeling (TUNEL) staining, glial fibrillary acidic protein (GFAP) immunohistochemistry, and several other histological markers. Apoptosis of scattered small cells and degeneration of certain nerve fibers was found in patterns spatially related to the cytokine mRNA patterns and to cerebrospinal fluid diffusion pathways. Furthermore, striking cytoarchitectonically defined clusters of degenerating non-neuronal cells, probably astrocytes, were found. The results reveal chronic overexpression of potentially cytotoxic cytokines in the brain and selective histopathology patterns in this natural disease model. J. Comp. Neurol. 414:114-130, 1999. Published 1999 Wiley-Liss, Inc.

Induction of interferon-gamma, transforming growth factor-beta, and interleukin-4 in mouse strains with different susceptibilities to Trypanosoma brucei brucei.

A Trypanosoma brucei brucei-derived lymphocyte triggering factor (TLTF) induced CD8+ T cells to produce IFN-gamma, which in turn stimulates parasite growth. This parasite-host interaction was studied in mouse strains that are either relatively susceptible (C3H/He) or resistant (C57Bl/6J) to infection, as well as in athymic nude mice. In all mouse strains, T. b. brucei infection caused a strong induction of IFN-gamma production by spleen mononuclear cells (MNC). In vivo blocking of IFN-gamma by intraperitoneal injection of mouse monoclonal anti-IFN-gamma antibody suppressed parasite growth and increased survival of both C3H/H3 and C57Bl/6J animals, suggesting that, irrespective of strain-related disease susceptibility, IFN-gamma is a growth-promoting stimulus for T. b. brucei. Spleen MNC from noninfected mice of all strains were in vitro like-wise strongly induced to IFN-gamma production when exposed to TLTF. This suggests that CD8+ expressing T cell receptor (TCR) alpha/beta, gamma/delta-bearing T cells and NK cells may all be triggered to IFN-gamma production by TLTF. In all mouse strains, TLTF also caused an increase in the number of cells expressing mRNA for TGF-beta in vitro. However, significant triggering to IL-4 mRNA expression only occurred in the relatively disease-resistant C57Bl/6J strain. As IL-4 is required for the synthesis and class switches of immunoglobulins, which are essential host immune defenses against T. b. brucei, the degree of resistance may be related to inherent strain ability to produce IL-4 in response to TLTF.

Viruses and behavioural changes: a review of clinical and experimental findings.

This review focuses on behavioural neurovirology. Profound changes in behaviour are observed following infection of the central nervous system by some viruses. Irritability, insomnia, hyperactivity and learning disability are some of the behavioural disturbances that have been described in both humans and animals with central nervous system infection. The reticular core neurons which innervate the entire brain play an important role in regulating behaviour. Some of these neurons--locus coeruleus, raphe and diagonal bands--send projections to the olfactory bulbs and can be targets for exogenous agents attacking the olfactory epithelium. In infant rats, vesicular stomatitis virus is transported along the olfactory pathway by retrograde transport and reaches the reticular core neurons causing destruction of raphe, diagonal bands and, to a lesser extent, the locus coeruleus. As the neurons degenerate, the viral antigens disappear and the animals sustain severe deficits in neurotransmitter levels and behaviour. Such a "hit and run" effect of the virus suggests the possibility that a similar mechanism may be operating in some human disorders. Apart from their intrinsic interest as possible aetiological factors, viruses may provide valuable tools in experimental work seeking to correlate behaviour, morphology and neurotransmitter function.

Myelinotoxic activity on tadpole optic nerve of IgG isolated from CSF and serum of patients with multiple sclerosis.

IgG from individual cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis and from controls was isolated by protein A-Sepharose column chromatography and tested in the tadpole optic nerve system for myelinotoxic activity. Oligoclonal IgG was present in all multiple sclerosis CSF specimens used. All IgG preparations yielded optic nerve myelin lesions, indicating that IgG has myelinotoxic activity. Significantly higher numbers of myelin lesions were obtained with IgG prepared from multiple sclerosis CSF compared to control CSF IgG.

Myelinotoxic activity on tadpole optic nerve of cerebrospinal fluid from patients with optic neuritis.

The myelinotoxic activity of unconcentrated cerebrospinal fluid (CSF) from eight optic neuritis (ON) and five multiple sclerosis (MS) patients with oligoclonal IgG, and from five ON patients without oligoclonal IgG, was tested in the tadpole optic nerve system. CSF from ON or MS patients with oligoclonal CSF IgG gave a significantly greater number of myelinotoxic lesions than did CSF from ON patients without oligoclonal CSF IgG, CSF from control patients, or physiologic saline. Induction of myelinotoxic lesions may be coupled with the presence of oligoclonal IgG. The findings support the hypothesis that there are two different forms of ON, of which one, characterized by oligoclonal IgG in the CSF, is more closely related to MS.

Scrapie prions alter receptor-mediated calcium responses in cultured cells.

The molecular basis of neurologic dysfunction in prion diseases is unknown. Spongiform degeneration of neurons is the most characteristic neuropathologic change which raises the possibility of abnormal ion channel function. Here we examined the regulation of Ca2+ fluxes in two cell lines chronically infected with scrapie prions, designated ScN2a (scrapie-infected mouse neuroblatoma) and ScHaB (scrapie-infected hamster brain) cells. In uninfected HaB cells, bradykinin caused increases in intracellular Ca2+ concentration ([Ca2+]i) by release of Ca2+ from internal stores and influx of extracellular Ca2+ whereas, in N2a cells, bradykinin increased [Ca2+]i exclusively from internal stores. Prion infection of both cell lines markedly reduced or eliminated bradykinin-activated increases in [Ca2+]i, whether driven by internal or extracellular sources. Stressing the cells with high extracellular [Ca2+], 8 to 20 mM, led to cytopathologic changes in ScHaB but not in ScN2a cells. Cytopathology was not preceded by an increase in [Ca2+]i. These findings indicate that scrapie infection induces abnormalities in receptor-mediated Ca2+ responses and raise the possibility that nerve cell dysfunction and degeneration in prion diseases is related to ion channel aberrations.

Autotomy in rats after nerve section compared with nerve degeneration following intraneural injection of Ricinus communis agglutinin I.

Partial unilateral deafferentation of the hind limb of rats was carried out by section of the sciatic nerve or the intraneural injection of Ricinus communis agglutinin 1 (RCA I). The development of autotomy was observed over a 6 week period. The axotomized animals autotomized more than those injected with RCA I. A neuroma developed on the proximal stump of the axotomized nerves. Within 7 days the axons of the RCA I-injected nerve degenerated and the cell bodies in dorsal root ganglia L4 and L5 were destroyed. Since the RCA I-injected animals autotomized, it is concluded that purely central factors have a role in the generation of this abnormal behavior. As the axotomized animals autotomized more than the RCA I-treated ones it is further concluded that abnormal impulse activity arising from a neuroma may be an additional factor in causing autotomy.

IgM and IgG responses during chronic relapsing experimental allergic encephalomyelitis (r-EAE).

During chronic relapsing experimental allergic encephalomyelitis (r-EAE) in guinea pigs, serum IgM and IgG concentrations increased markedly early in disease. Serum IgM and IgG increased similarly in control animals immunized with Freund's incomplete adjuvant (FIA) and Mycobacterium tuberculosis (MT). In the chronic phase of r-EAE but not in control animals, elevated IgM was also found in central nervous system (CNS) extracts, suggesting intrathecal IgM synthesis. IgG antibodies against myelin and myelin basic protein (MBP) were regularly detected in r-EAE sera from day 21 post inoculation (p.i.), reaching maximum levels in the early chronic phase. IgG antibodies against galactocerebroside (GC) and galactose appeared in some r-EAE sera. Oligoclonal IgG bands were demonstrated in all r-EAE guinea pig sera 21-26 days p.i. The bands in serum decreased in number and strength in the chronic phase. They could be traced to antibodies against MT in 4 of 10 animals, but not to antibodies against myelin, MBP, GC or galactose. Oligoclonal IgG bands were also regularly visualized in r-EAE CNS 124 days p.i., suggesting persistent intrathecal IgG synthesis. They varied in number and migration between different regions of individual CNS. Oligoclonal CNS IgG was related to antibodies against MT in only one of 7 animals, and in no case to antibodies against myelin.

Early major histocompatibility complex (MHC) class I antigen induction in hypothalamic supraoptic and paraventricular nuclei in trypanosome-infected rats.

Sprague-Dawley rats were injected intraperitoneally with a suspension of Trypanosoma brucei brucei. An early induction of major histocompatibility complex (MHC) class I antigens as well as an infiltration of macrophage-like cells and cytotoxic T-cells was detected with immunohistochemical techniques in circumventricular organs, such as the median eminence, neurohypophysis, subfornical organ, pineal gland and area postrema. These areas, which lack a blood-brain barrier, correspond to those showing early invasion of trypanosomes. In addition, there was a marked induction of MHC class I in neurons in two hypothalamic nuclei, the paraventricular and supraoptic nuclei. Neurons in these two nuclei are located behind the blood-brain barrier, but project to the neurohypophysis and to the median eminence, thereby exposing their axon terminals to factors circulating in the blood or released locally from invading trypanosomes or from macrophages or cytotoxic T-cells. It is suggested that the alteration in the nerve cell bodies in the hypothalamic nuclei is caused by retrograde axonal signals from these target areas.