HTLV-1 induced molecular mimicry in neurological disease.

As a model for molecular mimicry, we study patients infected with human T-lymphotropic virus type 1 (HTLV-1) who develop a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease with important biological similarities to multiple sclerosis (MS) (Khan et al. 2001; Levin et al. 1998, 2002a; Levin and Jacobson 1997). The study of HAM/TSP, a disease associated with a known environmental agent (HTLV-1), allows for the direct comparison of the infecting agent with host antigens. Neurological disease in HAM/TSP patients is associated with immune responses to HTLV-1-tax (a regulatory and immunodominant protein) and human histocompatibility leukocyte antigen (HLA) DRB1*0101 (Bangham 2000; Jacobson et al. 1990; Jeffery et al. 1999; Lal 1996). Recently, we showed that HAM/TSP patients make antibodies to heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), a neuron-specific autoantigen (Levin et al. 2002a). Monoclonal antibodies to tax cross-reacted with hnRNP A1, indicating molecular mimicry between the two proteins. Infusion of cross-reactive antibodies with an ex vivo system completely inhibited neuronal firing indicative of their pathogenic nature (Kalume et al. 2004; Levin et al. 2002a). These data demonstrate a clear link between chronic viral infection and autoimmune disease of the central nervous system (CNS) in humans and, we believe, in turn will give insight into the pathogenesis of MS.

IgG in brain correlates with clinicopathological damage in HTLV-1 associated neurologic disease.

OBJECTIVE: To test the authors' hypothesis that antibody deposition in autopsy specimens from patients with human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) would correlate with CNS damage. METHODS: Endogenous immunoglobulin G (IgG) was detected using antihuman IgG in autopsy tissues from HAM/TSP and control patients. IgG was isolated from the CSF, CNS, and sera of patients with HAM/TSP and tested for reactivity to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an autoantigen recently associated with molecular mimicry in HAM/TSP. RESULTS: In situ IgG localized to elements of the corticospinal system including neurons of the frontal cortex and precentral gyrus, as well as throughout axons in subcortical white matter, periventricular white matter, posterior limb of the internal capsule, midbrain, pons, and medulla. Similarly, there was IgG deposition within the posterior-column/medial lemniscal sensory system, including the arcuate fibers of the cranial-cervical junction, the nucleus cuneatus, and throughout the course of the medial lemniscus in the medulla, pons, and midbrain. IgG from brain, CSF, and serum of the patients with HAM/TSP showed immunoreactivity with hnRNP A1. CONCLUSION: Patients with HAM/TSP develop antibodies specific for neurons and axons that are preferentially damaged in the CNS.

Immunolocalization of occludin and claudin-1 to tight junctions in intact CNS vessels of mammalian retina.

The distributions of occludin and claudin-1, two tight junction-associated integral membrane proteins were investigated by immunohistochemical analysis of whole-mount preparations of the blood vessels in the myelinated streak of the rabbit retina. Light microscopy revealed that occludin and claudin-1 immunoreactivities were abundant along the interface of adjacent endothelial cells of all blood vessels. Electron microscopy revealed that both proteins were distributed in a regular pattern (at regular intervals of approximately 80 nm) along the length of tight junctions, probably in the regions of tight junction strands. No other structures or cell types expressed either of these two proteins in the myelinated streak. Whereas occludin immunoreactivity was concentrated only at the tight junction interface, claudin-1 immunoreactivity also extended into the cytoplasm of the endothelial cells, suggesting a different structural role for claudin-1 than for occludin at tight junctions. Retinal pigment epithelial cells expressed occludin around their entire circumference, consistent with the function of these cells as a barrier separating the retina from the leaky vessels of the choroid. Also consistent with the association of occludin expression with vessels that exhibit functional tight junctions, this protein was expressed at only a low level in, and showed an irregular distribution along, the vessels of the choroid, a vascular bed that lacks blood-barrier properties. Further, the distribution of occludin was examined during formation and remodelling of the rat retinal vasculature. Occludin expression was evident at the leading edge of vessel formation and was found on all vessels in both the inner and outer vascular plexus. Numerous vascular segments at the early stage of vascular formation and regression lost occludin expression. The biological significance of this transient loss of occludin expression in terms of barrier function remains to be elucidated.

Concentration of astrocytic filaments at the retinal optic nerve junction is coincident with the absence of intra-retinal myelination: comparative and developmental evidence.

The structure of the lamina cribrosa (LC) and astrocytic density were examined in various species with and without intra-retinal myelination. Sections of optic nerve from various species were stained with Milligan's trichrome or antibodies to glial fibrillary acidic protein, myelin basic protein (MBP) and antibody O4. Marmoset, flying fox, cat, and sheep, which lack intraretinal myelination, were shown to possess a well-developed LC as well as a marked concentration of astrocytic filaments distal to the LC. Rat and mouse, which lack intraretinal myelination, lacked a well-developed LC but exhibited a marked concentration of astrocytic filaments in this region. Rabbit and chicken, which exhibit intraretinal myelination, lacked both a well-developed LC and a concentration of astrocytes at the retinal optic nerve junction (ROJ). A marked concentration of astrocytes at the ROJ of human fetuses was also apparent at 13 weeks of gestation, prior to myelination of the optic nerve; in contrast, the LC was not fully developed even at birth. This concentration of astrocytes was located distal to O4 and MBP immunoreactivity in human optic nerve, and coincided with the site of initial myelination of ganglion cell axons in marmoset and rat. Myelination proceeded from the chiasm towards the retinal end of the human optic nerve. Moreover, the outer limit of oligodendrocyte precursor cells (OPC) migration into the rabbit retina was restricted by the outer limit of astrocyte spread. These observations indicate that a concentration of astrocytic filaments at the ROJ is coincident with the absence of intraretinal myelination. Differential expression of tenascin-C by astrocytes at the ROJ appears to contribute to the molecular barrier to OPC migration (see Bartsch et al., 1994), while expression of the homedomain protein Vax 1 by glial cells at the optic nerve head appears to inhibit migration of retinal pigment epithelial cells into the optic nerve (see Bertuzzi et al., 1999). These observations combined with our present comparative and developmental data lead us to suggest that the astrocytes at the ROJ serve to regulate cellular traffic into and out of the retina.

Contribution of O4+ oligodendrocyte precursors and astrocytes to the glial ensheathment of vessels in the rabbit myelinated streak.

The barrier properties and glial ensheathment of blood vessels in the retinal myelinated streak of adult New Zealand White rabbits were characterized at the ultrastructural level by intravascular injection of horseradish peroxidase (HRP) and immuno-electron microscopy with monoclonal antibody O4 and antibodies to glial fibrillary acidic protein (GFAP). Vessels within the myelinated streak did not leak HRP, and they exhibited tight junctions between adjacent endothelial cells. However, unlike their adult counterparts, the retinal blood vessels at postnatal day 18 exhibited substantial endocytotic activity. Both GFAP+ astrocytes and O4+ cells were evident surrounding the preretinal blood vessels of the myelinated streak. Furthermore, O4+ cells exhibited features indicative of high synthetic activity, including a large proportion of extended chromatin and prominent nucleoli within the nucleus, as well as a well-developed Golgi apparatus and numerous mitochondria in the cytoplasm. O4+ cells also exhibited variable quantities of heterochromatin, indicative of early stages of cellular differentiation. These observations are consistent with previous data showing that O4+ cells in the myelinated streak include oligodendrocyte precursor cells, pre-oligodendrocytes, and immature oligodendrocytes (Morcos Y, Chan-Ling T. Glia 21:163-182, 1997). The present data indicate that the preretinal vessels of the myelinated streak possess barrier properties typical of microvasculature in the central nervous system, and that both O4+ cells and astrocytes contribute to the glial ensheathment of these vessels. These vessels thus differ markedly from the leaky preretinal vessels associated with pathological conditions such as retinopathy of prematurity.

Identification of oligodendrocyte precursors in the myelinated streak of the adult rabbit retina in vivo.

Whole-mount techniques have been applied to the myelinated axons of the rabbit retina in order to study oligodendrocytes in the developing and adult central nervous system in vivo. Retinas from rabbits on embryonic day (E) 26 to postnatal day (P) 50 and from adults were subjected to immunohistochemistry with antibodies to glial fibrillary acidic protein (GFAP), vimentin, glycolipid O4, myelin basic protein (MBP), galactocerebroside (Gal-C), 4D6, and Griffonia simplicifolia isolectin, markers chosen for their specificity for astrocytes, cells of the oligodendrocyte lineage, Müller cells, and microglia. The first glial cells labelled within the myelinated streak (MS) were vimentin+ astrocytes. These cells were first apparent near the optic disc at E26. Their numbers and distribution increased markedly between E26 and E30, but between E29 and P3, vimentin expression was replaced by GFAP expression. Between P3 and P4, O4+, Gal-C-, MBP oligodendrocyte precursor cells, O4+, Gal-C-, MBP- pre-oligodendrocytes, and O4+, Gal-C-, MBP- immature oligodendrocytes appeared in low numbers in the region adjacent to the optic nerve head. O4-/+, Gal-C+, MBP+ mature oligodendrocytes appeared soon after at P4 to P5. With age, the outer extent of Gal-C and MBP immunoreactivity expanded, with positive cells forming a continuous sheath around nerve fibre bundles. The sequence of gliogenesis in the (MS) of the rabbit retina thus appears similar to that in the rat optic nerve. In adult rabbits, a large population of O4+ cells was distributed across the MS, the outer limit of the cells coinciding with the outer limit of retinal vessels. These O4+ cells could be classified into three stages of differentiation on the basis of expression of developmental markers and morphology: O4+, Gal-C-, MBP- oligodendrocyte precursor cells with a bipolar, unipolar, or simple morphology; O4+, Gal-C-, MBP- pre-oligodendrocytes with a complex multipolar morphology; and O4+, Gal-C+, MBP- immature oligodendrocytes with a complex multipolar morphology. The final stage of differentiation was characterized as O4-, Gal-C+, MBP+ mature oligodendrocytes with multiple parallel processes aligned along nerve fibre bundles. These results provide in vivo evidence for the existence of a substantial population of oligodendrocyte precursor cells and non-myelin-producing immature oligodendrocytes in the MS of the adult rabbit retina. This preparation makes possible in situ patch clamping and determination of other functional responses of these cell types in vivo.

Reactivated movement of decondensed rat sperm models and a description of their ultrastructure.

This study aimed at finding optimal conditions to decondense rat sperm nuclear chromatin with minimal damage. This was judged by the ability of the sperm-tail axoneme in the partially decondensed sperm models to be reactivated. Decondensation was assessed by phase contrast microscopy. Partial decondensation was judged to occur when the bright refractive appearance of the sperm nucleus turned black, and full decondensation when the nucleus turned pale and increased in volume. Demembranation was shown to have occurred by electron microscopy. With 0.03% Triton X-100 rat caudal epididymal sperm were partially demembranated to produce sperm models. Demembranation using a 0.1% solution of Triton X-100 was complete, but as with the solution of 0.05% Triton X-100, resulted in poorer reactivation of the partially decondensed sperm models. Reactivated movement of decondensed sperm models was used to assess the effect of the decondensing agents DTT and heparin. We were only able to achieve reactivation of sperm models that had undergone partial decondensation. Optimal reactivation was obtained after rat sperm models had decondensed in the decondensation solution containing 5 mM DTT, 6 mM EDTA, and 27.3 or 34.1 USP/ml heparin. Concentrations of heparin above or below these values resulted in a decrease in the number of sperm models reactivated. Ultrastructurally, sperm partially decondensed with 5 mM DTT, 6 mM EDTA, and 34.1 USP/ml heparin had their plasma membrane further extracted compared with sperm treated with 0.03% Triton X-100 alone. Decondensation was greatest in the peripheral regions of the nucleus with extraction of the acrosome but not of the perforatorium. The decondensed regions had a filamentous appearance. This procedure will allow access to sperm nuclear chromatin for experimental manipulation in rat sperm models.