M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

T cell response in acute motor axonal neuropathy.

There is evidence that in the acute axonal motor neuropathy (AMAN) subtype of Guillain-Barré syndrome antibodies to gangliosides, produced through molecular mimicry by antecedent Campylobacter jejuni (C. jejuni) infection, attack gangliosides expressed in human peripheral nerve axolemma, inducing a primary axonal damage. The aim of this study is to investigate whether the T cell response has a role in AMAN pathogenesis. We isolated monocytes from 4 healthy subjects and 5 AMAN patients with antecedent C. jejuni infection and antibodies to GM1 and/or GD1a gangliosides. Immature dendritic cells expressing CD1 molecules cultured with autologous T cells were stimulated with 2 lipopolysaccharides (LPSs) extracted from C. jejuni strains containing GM1 and GD1a-like structures and with GM1 and GD1a. The T cell response to LPSs and to gangliosides was determined by measuring the release of IFN-gamma and TNF-alpha. We observed a T cell response to both LPSs in controls and AMAN patients, whereas only AMAN patients showed T cell reactivity to gangliosides GM1 and GD1a with a tight correlation between T cell reactivity to the ganglioside and individual antibody responses to the same ganglioside. T cells responding to gangliosides were CD1c-restricted CD8 positive and CD27 negative. These findings indicate a contribution of cellular immunity in the pathogenesis of AMAN. A possible role for ganglioside-reactive T cells might be to facilitate the production of antibodies against gangliosides.

Axonal transport of rabies virus in the central nervous system of the rat.

Stereotaxic inoculation of rabies virus into specific nuclei in the central nervous system has been used for the investigation of the central neural transport mechanisms of viral information. The infection was monitored by specific fluorescence and peroxidase studies and the titration of viral infectivity in dissected brain areas. Twenty-four hours after inoculation into the striatum, cortex, or substantia nigra, infected neurons were detected only in cells from areas and nuclei which were related to the site of inoculation. The distribution of infected neurons showed that retrograde axoplasmic flow plays a determining role in the transport of rabies virus 24 hours after delivery of virus to specific target nuclei. Local destruction of neurons by kainic acid at the site of viral inoculation did not prevent the uptake and subsequent retrograde axonal transport of virus. There was an overall correlation between the major neural connections of the inoculated areas (e.g. the striatum) and the infected areas 24 hours later (e.g. the substantia nigra).

Evidence for an intraaxonal transport of fixed and street rabies virus.

Colchicine was used to inhibit axonal transport and to demonstrate that rabies virus spread from the peripheral inoculation site to the CNS by the retrograde axoplasmic flow. Colchicine was applied by the mean of elastomer implants around the sciatic nerve of young rats in order to obtain higher local concentrations of the drug. This procedure avoided the systemic effects of colchicine encountered with the usual treatment. To confirm the efficiency of the axoplasmic flow inhibition by colchicine, 125I-tetanus toxin was used as a marker. Uptake of colchicine by the sciatic nerve was monitored by the use of 3H-labelled colchicine. Interruption of the retrograde axoplasmic flow resulted in prevention of fixed and street rabies virus propagation. Moreover, the centrifugal spread of rabies could be inhibited using this experimental procedure.

Evidence for intraaxonal spread of Listeria monocytogenes from the periphery to the central nervous system.

Rhombencephalitis due to Listeria monocytogenes is characterized by progressive cranial nerve palsies and subacute inflammation in the brain stem. In this paper, we report observations made on mice infected with L. monocytogenes. Unilateral inoculation of bacteria into facial muscle, or peripheral parts of a cranial nerve, induced clinical and histological signs of mainly ipsilateral rhombencephalitis. Similarly, unilateral inoculation of bacteria into lower leg muscle or peripheral parts of sciatic nerve was followed by lumbar myelitis. In these animals, intraaxonal bacteria were seen in the sciatic nerve and its corresponding nerve roots ipsilateral to the bacterial application site. Development of myelitis was prevented by transsection of the sciatic nerve proximally to the hindleg inoculation site. Altogether, our results support the hypothesis that Listeria rhombencephalitis is caused by intraaxonal bacterial spread from peripheral sites to the central nervous system.

Mouse hepatitis virus-induced recurrent demyelination. A preliminary report.

Four-week-old BALB/c mice inoculated intracerebrally with the JHM strain of mouse hepatitis virus developed an acute demyelinating disease followed by apparent recovery with remyelination. When surviving mice were examined 16 months later, small areas of active demyelination were still present. This is the first reported example, to our knowledge, of an experimental viral infection in which acute demyelination with recovery is followed by persisting or recurring demyelination.

Transneuronal transport of herpes simplex virus from the cervical vagus to brain neurons with axonal inputs to central vagal sensory nuclei in the rat.

The recent introduction of live viruses as intra-axonal tracing agents has raised questions concerning which central neurons are transneuronally labelled after application of the virus to peripheral organs or peripheral nerves. Since the central connections of the vagus nerve have been well described using conventional neuronal tracing agents, we chose to inject Herpes Simplex Virus Type 1 into the cervical vagus of the rat. After survival times of up to 3 days the rat brains were processed immunohistochemically using a polyclonal antiserum against herpes simplex virus. Two days after injection of the virus we observed viral antigen in the area postrema and in the nucleus tractus solitarius and the dorsal motor nucleus of the vagus (dorsal vagal complex), principally ipsilaterally. At this survival time the viral antigen in the dorsal vagal complex was largely confined to glial cells. After 3 days the viral antigen was localized both in glia and in nerve cells within the dorsal vagal complex and in brain regions previously demonstrated, using conventional tracing procedures, to contain neurons with axonal projections to the dorsal vagal complex. This was true for medullary, pontine, midbrain and hypothalamic regions and for telencephalic regions including the amygdala, the bed nucleus of the stria terminalis, and the insular and medial frontal cortices. Many of the nerve cells containing viral antigen were displayed in a Golgi-like manner, with excellent visualization of the dendritic tree. Axonal processes, in contrast, were not visualized. We used co-localization studies to confirm previous findings concerning monoamine neurotransmitter-related antigens present in medullary and pontine neurons projecting to the dorsal vagal complex. After 3 days there were many Herpes Simplex Virus Type 1-containing glial cells along the intra-medullary course of the vagal rootlets. However, no viral antigen was found in brain regions containing neurons whose axons pass through the region of glial cell-labelled rootlets. Glial cells containing viral antigen were particularly numerous in brain regions known to receive an input from neurons in the area postrema and the dorsal vagal complex. Taken together with our observation concerning the early appearance of viral antigen within glial cells in the dorsal vagal complex, this suggests that when the virus reaches the axon terminal portion it is transferred to nearby glial cells and possibly enters central neurons by way of these structures.

Axonal and transsynaptic (transneuronal) spread of Herpesvirus simiae (B virus) in experimentally infected mice.

In order to study the pathogenesis of B virus infection of the nervous system, newborn and young mice were inoculated by four different routes: 1. Intramuscular (i.m.) in the forelimb; 2. I.m. in the hindlimb; 3. Subcutaneous (s.c.) in the abdominal wall; 4. Intraperitoneal (i.p.). Spread of virus was followed by immunohistochemical demonstration of viral antigen in tissue sections of the peripheral and central nervous system. Three distinct patterns emerged: 1. After i.m. limb inoculations, virus progressed along the ipsilateral dorsal column, the bilateral spinothalamic and bilateral spinoreticular systems and along central autonomic pathways. 2. After s.c. inoculation, the dorsal column was spared, otherwise the spread was similar to that following i.m. inoculations. 3. After i.p. inoculation, virus spread in the spinal cord bilaterally, mainly along spinothalamic and central autonomic pathways. The peripheral motoneurons were conspicuously spared, even in the i.m. inoculation mode. In the brain stem, B virus antigen appeared bilaterally, at multiple sites. In the cerebrum, virus infected cells appeared first in the thalamus, hypothalamus and the motor cortex. The mode of spread from spinal levels was mainly orthograde along the ascending systems (dorsal columns, spinothalamic, spinoreticular tracts), but also retrograde along descending systems (pyramidal tract, central autonomic pathways). Oligosynaptic systems transmitted virus more quickly than the polysynaptic ones. In the involvement of various neuronal systems in virus spread, a certain selectivity, sparing the peripheral motoneuron and the cerebellar systems, could be assessed.

Binding, uptake and retrograde axonal transport of herpes virus suis in sympathetic neurons.

Newborn rat dissociated sympathetic neurons were grown in a chamber culture system, where a Teflon ring sealed with silicon grease separated the axonal plexus from the corresponding nerve cell bodies. The binding of 35S-labeled herpes virus suis (HVS) to the neurites was partially inhibited by an excess of unlabeled HVS as well as by concanavalin A, indicating the presence of specific binding sites for the virus. Specific binding was a prerequisite for the subsequent uptake and retrograde transport of HVS to the nerve cell bodies. Predominantly free nucleocapsids were detected by electron microscopy in the axons at the time of retrograde transport, both in culture and in vivo, suggesting the possibility that nucleocapsids without lipid membrane and not contained in cellular membrane compartments can be transported by retrograde axonal transport.

Intra-axonal virus in demyelinative lesions of experimental herpes simplex type 2 infection.

Three-week-old mice which had been infected intracerebrally with herpes simplex virus type 2 (HSV-2) were examined electron-microscopically for the presence of intra-axonal virus in or near optic nerve and spinal cord demyelinative lesions. Acute lesions and their margins frequently contained a very small proportion of abnormal axons, and in a few of these mature virus particles, nucleocapsids, or other incomplete forms were found. A similar range of particle morphology was present in the cytoplasm of infected and degenerating glia. Axons containing similar particles were not identified in fibers in normal white matter surrounding demyelinative lesions. It is proposed that neuronal infection and axonal transport of virus may lead to foci of oligodendroglial infection, destruction and central nervous system (CNS) demyelination near to or remote from the cell bodies of infected neurons. In some instances, the topography of lesions could reflect a tract association. Anatomical features of nervous tissue could favor amplification of demyelination from a relatively minimal neuronal infection, with little evidence of tract degeneration. This hypothesis is consistent with the great predominance of demyelination relative to gray matter disease seen experimentally in non-fatal CNS infections with HSV-2. It would also explain the marked tendency for demyelinative lesions in at least certain CNS locations to be greatly elongated in the long axis of fiber tracts. This mechanism could be of importance in other animal models of virus-induced demyelination, and perhaps also in multiple sclerosis.

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis.

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.

Immunomodulation of experimental murine herpes simplex keratitis: I. UV-HSV protection.

A/J mice were immunized subcutaneously with ultraviolet light (UV) inactivated herpes simplex virus type-1, MP strain (HSV-MP). Control A/J mice were immunized subcutaneously either with media (unimmunized controls) or with live HSV-MP (immunized controls). Immunized and control mice were challenged ocularly with either MP or mP strain HSV-1 after corneal scarification and were followed for 3 weeks post corneal challenge. The mice were observed during this time period for signs of herpes simplex keratitis (HSK), lid lesions and encephalitis. At the time of sacrifice, the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these studies demonstrated that immunization with UV inactivated HSV (UV-HSV) gave the same protection against keratitis and encephalitis as immunization with live virus. Furthermore, the cocultivation assays indicated that immunization with either live HSV-1 or UV inactivated HSV-1 protected against the establishment of latency.

Spiroplasma-like inclusions in Creutzfeldt-Jakob disease.

Spiral membranous inclusions were discovered on electron microscopic study of brain biopsy tissues from a 46-year-old man with Creutzfeldt-Jakob disease (CJD). These replicate coiled membranous configurations measured 850 to 1,000 nm in length and 75 to 137.5 nm in width and were located within axoplasm, primarily in presynaptic terminals. These inclusions closely resemble Spiroplasma, a plant pathogen, and the finding of these structures in CJD suggests the concurrence of Spiroplasma infection with a human chronic degenerative brain disease.

Spread of herpes simplex virus (HSV) strains SC16, ANG, ANGpath and its glyC minus and GlyE minus mutants in DBA-2 mice.

Herpes simplex virus type 1 (HSV-1) strains SC16, ANG, its pathogenic variant ANGpath and the mutants ANG-pathgC18 glycoprotein C (glyC) negative and ANGpathI2-4 (glyE negative) were compared for their ability to spread in DBA-2 mice after peripheral inoculation. Virus infectivity assay in 9 organs at days 2, 3, 4, 5, 6, and 10 post-infection (p.i.) and morphologic examinations (immunofluorescence, PAP staining) showed the following: SC16, ANG, and ANGpath spread first (days 2-3 p.i.) by haematogenic route to spleen, liver, and adrenal gland. Since day 4 the invasion of the vegetative and peripheral nervous system took place in SC16 and ANGpath-infected mice, followed by virus spread to the spinal cord and brain stem. In ANG-infected mice the invasion of peripheral nervous system was minimal although both ANG as well as ANGpath spread along the axons. In ANG pathC18-infected mice a relatively prolonged viraemic phase (days 2-4 p.i.) represented with foci of virus antigen-containing cells in spleen, liver, and mesenterial connective tissue was accompanied with a low grade invasion of the peripheral nervous system (days 3-4 p.i.). No spread by any route of ANGpathI2-4 was observed after intraperitoneal inoculation. When comparing ANGpath and SC16, the latter seemed slightly more lethal, since ANGpath killed 67.2% of DBA-2 mice which were given 2 X 10(6) PFU/0.1 ml by i.p. route as compared to the 100% lethality of SC16-infected animals.

Experimental latent herpesvirus infection in rabbits, mice and hamsters: ultrastructure of the virus activation in explanted gasseric ganglia.

The frequency of latent infection as established in trigeminal ganglia of rabbits, mice and hamsters with human herpesvirus type 1 (HVH) was compared using two different virus strains. Explantation proved to be effective in reisolation of HVH from ganglion tissue, which did not yield infectious virus at time of its removal. After healing of acute keratitis, the latent infection in homolateral gasseric ganglia of rabbits was detected at a relatively high frequency (60-80 per cent) up to 120 days post infection (p.i.) in case of both virus strains. The activation rate was a little lower in hamsters. After inoculation of suckling and young mice with a sublethal dose of HVH by oral and nasal routes, approximately 40-100 per cent of the animals had virus in their gasseric ganglia during the acute period; 30-60 days later only 10-25 per cent had virus in the latent form. Immunofluorescent and electron microscopic examination of the explanted ganglion tissue showed the presence of HVH in neurons, neuronal satellites and Schwann cells. The nuclei of noneural cells contained numerous crystalline arrays. The possibility that pseudounipolar neurons of the regional sensoric ganglion are not the exclusive site of HVH latency is discussed.

The continuing problem of herpes simplex virus persistence.

While the main interest in the pathogenesis of herpes simplex virus (HSV) in the sixties had been focussed on acute infections, in the seventies latent infection has become the main foal of investigation. Despite of overwhelming literature, the HSV persistence has remained a continuing problem from the practical as well as theoretical points of view. Nevertheless, the following conclusions can be made: 1) HSV spreads along nerves inside as well as outside axons; 2) it resides in a non-productive form for lifelong in the sensory or vegetative ganglia; and 3) it is intermittently activated when causing peripheral virus shedding or recurrent disease. The persistence of HSV DNA in neurons may be associated with a limited transcription and translation, but the ganglia in a great majority of subjects are uninfectious during the latency period.

Ultrastructure of peripheral nerves of mice inoculated with rabies virus.

Fourty adult female albino mice were inoculated in the right hind leg with rabies viruses of the street type. The mice were sacrificed with an interval of 24 hours each, starting in the next day after inoculation. From the 10th day ownwards the animals started presenting signs of paralysis, first on the leg where the viruses were inoculated anbnormalities were found in peripheral nerves compatible with axonal degeneration with secondary demyelination but the rabies viruses were not found in the axoplasm, myelin sheet, Schwann cell cytoplasm, endoneural or in the epineural structures.

[Neural pathway of Powassan virus spread in the central nervous system of white mice]. / Nevral'nyi put' rasprostraneniia virusa Povassan v tsentral'noi nervnoi sisteme belykh myshei.

Electron microscopic investigation of the brains and lumbar spinal cords of adult albino mice infected with Powassan virus was carried out. Virus particles were found within all parts of neurons (perikarya, dendrites, axon), as well as within synaptic apparatus and intercellular gaps of the central nervous tissue. The possibility of the virus spread both throughout the cytoplasm of nerve cells and their processes and the extracellular spaces of the brain was confirmed. Localization of virions within neurons, synapses and myelinated fibers of the spinal cord after intracerebral inoculation suggests that virus spread in the CNS can occur through the CNS parenchyma and also through the nervous conduction pathways. The possible mechanisms of virus dissemination in the CNS of albino mice with experimental Powassan virus encephalomyelitis are discussed.

The demyelination neurophysiological criteria can be misleading in Campylobacter jejuni-related Guillain-Barré syndrome.

OBJECTIVE: The exclusive association of Campylobacter jejuni infection with the axonal variant of Guillain-Barré syndrome (GBS) is debatable. The current study aims to elucidate the GBS subtypes of patients with an antecedent C. jejuni infection. METHODS: Nerve conduction study results of 73 patients with GBS were reviewed. Patients were defined as having a recent C. jejuni infection when there was a positive stool culture or serological evidence of C. jejuni in the presence of preceding diarrhea. RESULTS: A total of 23 patients had evidence of a recent C. jejuni infection. At the early stage, patients were classified as AMAN (n=9; 39%), AIDP (n=3; 13%) or equivocal (n=9) using existing electrophysiological criteria. Prolonged distal latencies and conduction slowing that were seen in 11 patients rapidly normalized within 3 weeks in seven, whereas four had minor abnormalities throughout the course. Subsequently, all patients showed either acute motor axonal neuropathy pattern or reversible conduction failure. CONCLUSION: Serial neurophysiology suggests that C. jejuni infections are exclusive to axonal GBS. SIGNIFICANCE: Our findings suggest that AMAN can demonstrate the full complement of demyelinating features at the early stages of disease.