Contrary to many other viruses, the initial steps of the hepatitis B virus (HBV) infection, including attachment to hepatocytes, specific receptor interactions, and membrane fusion, are unsolved. Using HepaRG cells as an in vitro cell culture system, we here report that HBV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cell-surface-associated heparan sulfate proteoglycans. Binding to GAGs requires the integrity of the pre-S domain as a part of the large (L-) viral envelope protein. HBV infection was abrogated by incubation of virions with heparin, but not the structurally related GAGs chondroitin sulfate A, B, and C. Infection was also abolished by suramin, a known inhibitor of duck hepatitis B virus infection or highly sulfated dextran sulfate. Polycationic substances such as poly-L-lysine, polybrene, and protamine also prevented infection, however, by addressing cellular components. Enzymatic removal of defined acidic carbohydrate structures from the cell surface using heparinase I/III or the obstruction of GAG synthesis by sodium chlorate inhibited HBV infection of HepaRG cells and, moreover, led to a reduction of HBV cell surface binding sites. The biochemical analysis showed selective binding of L-protein-enriched viral particles (virions or filaments) to heparin. GAG-dependent binding of HBV was improved by polyethylene glycol, a substance that specifically enhances HBV infection.

CONCLUSIONS

HBV infection requires the initial attachment to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors. This interaction initializes the multistep entry process of HBV and cannot be bypassed by alternative routes.

Axons fail to regenerate in the central nervous system after injury. Chondroitin sulfate proteoglycans (CSPG) expressed in the scar significantly contribute to the nonpermissive properties of the central nervous system environment. To examine the inhibitory activity of a CSPG mixture on retina ganglion cell (RGC) axon growth, we employed both a stripe assay and a nerve fiber outgrowth assay. We show that the inhibition exerted by CSPGs in vitro can be blocked by application of either C3 transferase, a specific inhibitor of the Rho GTPase, or Y27632, a specific inhibitor of the Rho kinase. These results demonstrate that CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK signaling pathway. Consistent with these results, we found that retina ganglion cell axon growth on glial scar tissue was enhanced in the presence of C3 transferase and Y27632, respectively. In addition, we show that the recently identified inhibitory CSPG Te38 is upregulated in the lesioned spinal cord.

Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.

The disappearance of notochordal cells is correlated with early degenerative changes in the intervertebral disc. With increased disc degeneration there is a marked decrease in proteoglycan synthesis, resulting in loss of mechanical function. One possible mechanism for the decrease in proteoglycan synthesis is the loss of notochordal cells from the tissue. In this study, nucleus pulposus cells cocultured with notochordal cells exhibit an increase in proteoglycan synthesis. Interestingly, purified notochordal cells synthesize little proteoglycan as observed by [35S]sulfate incorporation into proteoglycans. The observed increase in proteoglycan synthesis does not appear to be dependent on cell-cell contact; rather it is the result of soluble factor(s) produced by notochordal cells. Finally, no difference in chondroitin sulfate chain size in notochordal-stimulated nucleus pulposus cells was observed which is consistent with an up-regulation in aggrecan core protein synthesis. These results are consistent with canine breeds where notochordal cells persist into adult age and disc degeneration is not observed. This suggests notochordal cells play a vital role in maintaining disc integrity.

Mouse mast cells were differentiated and grown by culturing bone marrow cells in medium containing 2 X 10(-10) M purified interleukin 3 (IL 3). The cells obtained were similar in ultrastructure, membrane antigen phenotype, proteoglycan type, and lipid products generated upon immunologic activation to mast cells differentiated in culture by WEHI-3-conditioned medium (WEHI-3-CM) and by concanavalin A (Con A) splenocyte-conditioned medium. Phenotypically, these cells expressed IgE receptors and H-2 antigens and were recognized by a monoclonal antibody (B23.1) that did not react with mouse serosal heparin-containing mast cells. The classic phenotypic markers of mouse T cells or macrophages were not detected. The mouse mast cells differentiated with IL 3 as well as those differentiated in WEHI-3-CM incorporated [35S]sulfate into a nonheparin proteoglycan of 150,000 to 200,000 m.w. Most of the 35S-labeled macromolecules were degraded by chondroitinase ABC to yield only two disaccharides, which co-chromatographed on ascending thin layer chromatography with delta Di-4S and delta Di-diSE; thus, the proteoglycan in these cells is composed of chondroitin sulfate E glycosaminoglycans. After sensitization with monoclonal IgE, washing, and antigen activation, the IL 3 differentiated cells released the preformed mediator beta-hexosaminidase and generated and released two major classes of lipid mediators. The quantities of leukotriene C4 (LTC4), leukotriene B4 (LTB4), and platelet-activating factor (PAF-acether) generated/10(6) cells were 17, 3.0, and 3.1 ng, respectively. The ratio of these three lipid mediators was similar to that obtained from mast cells differentiated in WEHI-3-CM and in Con A-conditioned medium. Thus, T cell-derived IL 3 is the component present in the conditioned media that is required for differentiation and growth of the subclass of mast cells containing chondroitin sulfate E proteoglycan, designated E-MC. The IL 3-dependent E-MC may represent the in vitro counterpart of the T-cell-dependent mucosal mast cell, suggesting in turn that the production of LTC4 and LTB4 and of PAF-acether may play a role in adaptive intestinal immunity to helminthic parasites.

A perineuronal net (PNN) is a layer of lattice-like matrix which enwraps the surface of the soma and dendrites, and in some cases the axon initial segments, in sub-populations of neurons in the central nervous system (CNS). First reported by Camillo Golgi more than a century ago, the molecular structure and the potential role of this matrix have only been unraveled in the last few decades. PNNs are mainly composed of hyaluronan, chondroitin sulfate proteoglycans, link proteins, and tenascin R. The interactions between these molecules allow the formation of a stable pericellular complex surrounding synapses on the neuronal surface. PNNs appear late in development co-incident with the closure of critical periods for plasticity. They play a direct role in the control of CNS plasticity, and their removal is one way in which plasticity can be re-activated in the adult CNS. In this review, we examine the molecular components and formation of PNNs, their role in maturation and synaptic plasticity after CNS injury, and the possible mechanisms of PNN action.

Following injury to the adult CNS, the expression of a number of extracellular matrix molecules increases in regions of reactive gliosis. This glial matrix includes certain chondroitin sulfate proteoglycans (CS-PGs) which have been correlated with an inhibition of axon outgrowth. In order to test the influence of glial associated CS-PGs on neurite elongation directly, we sought to determine whether enzymatic modification of injury-induced CS-PGs could enhance neurite outgrowth across the surface of intact glial scars formed in vivo after implanting nitrocellulose filters into the cortex of adult rats. This gliotic tissue was subsequently explanted in vitro and used as a substrate for growing embryonic retinal neurons. Treatment of adult explants with chondroitinase ABC led to a significant increase in mean neurite length over the scar surface. Heparitinase treatment caused a much smaller, although significant, increase in neurite outgrowth. This suggested that more than one type of PG was present or that a single PG with both CS and HS side chains was upregulated. Western analysis revealed that a PG(s) with a core protein between 180 and 400 kDa was found to be relatively more abundant in areas of reactive gliosis induced to form in adult rather than neonatal animals. Simultaneous treatment of adult glial scars with chondroitinase and antibodies to the beta 1, beta 2 chain of laminin partially reversed the growth-enhancing effect of enzymatic digestion alone. These data demonstrate that the increase in neurite outgrowth along the surface of reactive astrocytes following enzymatic modification of injury-induced PGs was due, in part, to the presence of laminin. Thus, in this model of gliosis, particular PGs may act as inhibitors of neurite outgrowth by attenuating the potential for axon elongation that could occur due to the concomitant expression of growth-promoting molecules in regions of reactive gliosis.

Successful axon regeneration in the mammalian central nervous system (CNS) is at least partially compromised due to the inhibitors associated with myelin and glial scar. However, the intracellular signaling mechanisms underlying these inhibitory activities are largely unknown. Here we provide biochemical and functional evidence that conventional isoforms of protein kinase C (PKC) are key components in the signaling pathways that mediate the inhibitory activities of myelin components and chondroitin sulfate proteoglycans (CSPGs), the major class of inhibitors in the glial scar. Both the myelin inhibitors and CSPGs induce PKC activation. Blocking PKC activity pharmacologically and genetically attenuates the ability of CNS myelin and CSPGs to activate Rho and inhibit neurite outgrowth. Intrathecal infusion of a PKC inhibitor, Gö6976, into the site of dorsal hemisection promotes regeneration of dorsal column axons across and beyond the lesion site in adult rats. Thus, perturbing PKC activity could represent a therapeutic approach to stimulating axon regeneration after brain and spinal cord injuries.

Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division, and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane leukocyte common antigen-related phosphatase (LAR). CSPGs bind LAR with high affinity in transfected COS-7 cells and coimmunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knock-out mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.

Specific transfer of (orthodenticle homeobox 2) Otx2 homeoprotein into GABAergic interneurons expressing parvalbumin (PV) is necessary and sufficient to open, then close, a critical period (CP) of plasticity in the developing mouse visual cortex. The accumulation of endogenous Otx2 in PV cells suggests the presence of specific Otx2 binding sites. Here, we find that perineuronal nets (PNNs) on the surfaces of PV cells permit the specific, constitutive capture of Otx2. We identify a 15 aa domain containing an arginine-lysine doublet (RK peptide) within Otx2, bearing prototypic traits of a glycosaminoglycan (GAG) binding sequence that mediates Otx2 binding to PNNs, and specifically to chondroitin sulfate D and E, with high affinity. Accordingly, PNN hydrolysis by chondroitinase ABC reduces the amount of endogenous Otx2 in PV cells. Direct infusion of RK peptide similarly disrupts endogenous Otx2 localization to PV cells, reduces PV and PNN expression, and reopens plasticity in adult mice. The closure of one eye during this transient window reduces cortical acuity and is specific to the RK motif, as an Alanine-Alanine variant or a scrambled peptide fails to reactivate plasticity. Conversely, this transient reopening of plasticity in the adult restores binocular vision in amblyopic mice. Thus, one function of PNNs is to facilitate the persistent internalization of Otx2 by PV cells to maintain CP closure. The pharmacological use of the Otx2 GAG binding domain offers a novel, potent therapeutic tool with which to restore cortical plasticity in the mature brain.

Expression of CXCR3, the receptor for the CXC chemokines IFN-gamma-inducible 10-kDa protein (IP10) and monokine induced by IFN-gamma (Mig), in human T lymphocytes and their responses to IP10 and Mig were analyzed. About 40 % of resting T lymphocytes (and low numbers of B cells and natural killer cells) stained positive for CXCR3 but these cells did not express CXCR3 transcripts and did not respond to these chemokines. However, treatment with IL-2 with or without addition of phytohemagglutinin for 10 or more days resulted in cultures of fully responsive, CXCR3-positive T lymphocytes. Treatment with anti-CD3 antibodies in the presence or absence of soluble anti-CD28 antibodies was inhibitory. Addition of chondroitin sulfate C to CXCR3-expressing murine pre-B cells allowed the determination of high-affinity binding for Mig and IP10 with Kd of 0.9-1.2 nM and 0.2-0.3 nM, respectively, and 1.3 x 10(4) binding sites per cell. The gene for CXCR3 was localized on human chromosome Xq13 which is in clear contrast to all other chemokine receptor genes, suggesting unique function(s) for this receptor and its ligands that may lie beyond their established role in T cell-dependent immunity.

We have previously shown that aggregation of microbeads coated with N-CAM and Ng-CAM is inhibited by incubation with soluble neurocan, a chondroitin sulfate proteoglycan of brain, suggesting that neurocan binds to these cell adhesion molecules (Grumet, M., A. Flaccus, and R. U. Margolis. 1993. J. Cell Biol. 120:815). To investigate these interactions more directly, we have tested binding of soluble 125I-neurocan to microwells coated with different glycoproteins. Neurocan bound at high levels to Ng-CAM and N-CAM, but little or no binding was detected to myelin-associated glycoprotein, EGF receptor, fibronectin, laminin, and collagen IV. The binding to Ng-CAM and N-CAM was saturable and in each case Scatchard plots indicated a high affinity binding site with a dissociation constant of approximately 1 nM. Binding was significantly reduced after treatment of neurocan with chondroitinase, and free chondroitin sulfate inhibited binding of neurocan to Ng-CAM and N-CAM. These results indicate a role for chondroitin sulfate in this process, although the core glycoprotein also has binding activity. The COOH-terminal half of neurocan was shown to have binding properties essentially identical to those of the full-length proteoglycan. To study the potential biological functions of neurocan, its effects on neuronal adhesion and neurite growth were analyzed. When neurons were incubated on dishes coated with different combinations of neurocan and Ng-CAM, neuronal adhesion and neurite extension were inhibited. Experiments using anti-Ng-CAM antibodies as a substrate also indicate that neurocan has a direct inhibitory effect on neuronal adhesion and neurite growth. Immunoperoxidase staining of tissue sections showed that neurocan, Ng-CAM, and N-CAM are all present at highest concentration in the molecular layer and fiber tracts of developing cerebellum. The overlapping localization in vivo, the molecular binding studies, and the striking effects on neuronal adhesion and neurite growth support the view that neurocan may modulate neuronal adhesion and neurite growth during development by binding to neural cell adhesion molecules.

The role of cell-surface proteoglycans in human immunodeficiency virus (HIV) infection of T-cell lines was investigated. HIV-1-susceptible lymphoblastic T-cell lines, MT-4 and H9, were analyzed for proteoglycan synthesis and found to make heparan sulfate (HS) and chondroitin sulfate proteoglycans. Enzymatic treatment of these cells with heparitinase, but not chondroitinase, significantly prevented HIV-1(IIIB) infection as measured by inhibition of cytopathicity, reverse transcriptase production, and syncytia formation. Sulfation of glycosaminoglycans HS chains was critical to viral entry as shown by inhibition of viral infection with sodium chlorate and its specific reversal with exogenous sulfate addition. Quantitation of direct virus binding to cells showed that treatment of cells with heparitinase inhibited HIV-1 binding to the T-cell surface. Exogenous HS added to cultures inhibited virus infection in a manner analogous to dextran sulfate, further supporting a functional role for HS in HIV-1 binding. These results provide evidence for participation of cell-surface HS proteoglycans in HIV-cell attachment and virus entry.

The transplantation of neural stem/progenitor cells (NPCs) is a promising therapeutic strategy for spinal cord injury (SCI). However, to date NPC transplantation has exhibited only limited success in the treatment of chronic SCI. Here, we show that chondroitin sulfate proteoglycans (CSPGs) in the glial scar around the site of chronic SCI negatively influence the long-term survival and integration of transplanted NPCs and their therapeutic potential for promoting functional repair and plasticity. We targeted CSPGs in the chronically injured spinal cord by sustained infusion of chondroitinase ABC (ChABC). One week later, the same rats were treated with transplants of NPCs and transient infusion of growth factors, EGF, bFGF, and PDGF-AA. We demonstrate that perturbing CSPGs dramatically optimizes NPC transplantation in chronic SCI. Engrafted NPCs successfully integrate and extensively migrate within the host spinal cord and principally differentiate into oligodendrocytes. Furthermore, this combined strategy promoted the axonal integrity and plasticity of the corticospinal tract and enhanced the plasticity of descending serotonergic pathways. These neuroanatomical changes were also associated with significantly improved neurobehavioral recovery after chronic SCI. Importantly, this strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. For the first time, we demonstrate key biological and functional benefits for the combined use of ChABC, growth factors, and NPCs to repair the chronically injured spinal cord. These findings could potentially bring us closer to the application of NPCs for patients suffering from chronic SCI or other conditions characterized by the formation of a glial scar.

Rabbit aortic medial cells were grown on purified elastin membranes, which were then subjected to repeated elongation and relaxation or to agitation without stretching. Cells remained attached to the membranes, and cyclic stretching resulted in a two- to fourfold increase in rates of collagen, hyaluronate, and chondroitin 6-sulfate synthesis over those in agitated or stationary preparations. Synthesis of types I and III collagen was increased to the same degree. Stretching did not increase rates of chondroitin 4-sulfate or dermatan sulfate synthesis. Differences were not attributable to differences in cell number, for DNA synthetic rates were not increased by stretching. The model system devised to demonstrate these effects provides a means for relating various modes of mechanical stimulation to cell metabolism.

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPGs) after CNS injuries contributes to the impediment of functional recovery by restricting both axonal regeneration and synaptic plasticity. In the present study, the effect of degrading CSPGs with the application of the bacterial enzyme chondroitinase ABC (chABC) into the cuneate nucleus of rats partially denervated of forepaw dorsal column axons was examined. A dorsal column transection between the C6-C7 dorsal root entry zones was followed immediately by an ipsilateral brainstem injection of either chABC or a bacterial-derived control enzyme [penicillinase (P-ase)] and then subsequently (1 week later) followed with a second brainstem enzyme injection and cholera toxin B subunit (CTB) tracer injection into the ipsilateral forepaw digits and pads. After 1 additional week, the rats underwent electrophysiological receptive field mapping of the cuneate nucleus and/or anatomical evaluation. Examination of the brainstems of rats from each group revealed that CSPGs had been reduced after chABC treatment. Importantly, in the chABC-treated rats (but not in the P-ase controls), a significantly greater area of the cuneate nucleus was occupied by physiologically active CTB traced forepaw afferents that had been spared by the initial cord lesion. These results demonstrate, for the first time, a functional change directly linked to anatomical evidence of sprouting by spinal cord afferents after chABC treatment.

The interaction of interleukin 8 (IL-8) with heparin was studied by using synthetic IL-8 analogs with C- and N-terminal truncations. Elimination of the N-terminal region preceding the first cysteine, which constitutes the IL-8 receptor binding site, did not affect the affinity to heparin-Sepharose. Affinity, however, decreased with progressive truncation at the C terminus, and no binding was observed when the C-terminal alpha-helix was eliminated. The effect of heparin and other glycosaminoglycans on IL-8 activity was also tested. When IL-8 was applied together with heparan sulfate, neutrophil chemotaxis in vitro was enhanced up to 4-fold, and the stimulus-dependent increase in cytosolic free Ca2+ increased markedly in both rate and peak value. Heparin had a similar effect on the Ca2+ response but did not enhance chemotaxis. The glycosaminoglycans by themselves did not elicit neutrophil responses. Their enhancing effect was restricted to stimulation with IL-8 and was not observed when the unrelated chemoattractant fMet-Ile-Phe-Leu was used as the stimulus. Elastase released from stimulated neutrophils was inhibited by heparin, heparan sulfate, and, to a lesser extent, chondroitin sulfate B, confirming previous observations. Taken together, these results suggest that heparan sulfate, which is present on the endothelial cell surface and in the basement membrane, may have a dual function in diapedesis, promotion of IL-8-dependent transmigration of neutrophils, and protection of the tissue microenvironment from damage by lytic enzymes released from the migrating cells.

Respiratory syncytial virus (RSV) is an important human respiratory pathogen, particularly in infants. Glycosaminoglycans (GAGs) have been implicated in the initiation of RSV infection of cultured cells, but it is not clear what type of GAGs and GAG components are involved, whether the important GAGs are on the virus or the cell, or what the magnitude is of their contribution to infection. We constructed and rescued a recombinant green fluorescent protein (GFP)-expressing RSV (rgRSV) and used this virus to develop a sensitive system to assess and quantify infection by flow cytometry. Evaluation of a panel of mutant Chinese hamster ovary cell lines that are genetically deficient in various aspects of GAG synthesis showed that infection was reduced up to 80% depending on the type of GAG deficiency. Enzymatic removal of heparan sulfate and/or chondroitin sulfate from the surface of HEp-2 cells also reduced infection, and the removal of both reduced infection even further. Blocking experiments in which RSV was preincubated with various soluble GAGs revealed the relative blocking order of: heparin>> heparan sulfate>> chondroitin sulfate B. Iduronic acid is a component common to these GAGs. GAGs that do not contain iduronic acid, namely, chondroitin sulfate A and C and hyaluronic acid, did not inhibit infection. A role for iduronic acid-containing GAGs in RSV infection was confirmed by the ability of basic fibroblast growth factor to block infection, because basic fibroblast growth factor binds to GAGs containing iduronic acid. Pretreatment of cells with protamine sulfate, which binds and blocks GAGs, also reduced infection. In these examples, infection was reduced by pretreatment of the virus with soluble GAGs, pretreatment of the cells with GAG-binding molecules, pretreatment of the cells with GAG-destroying enzymes or in cells genetically deficient in GAGs. These results establish that the GAGs involved in RSV infection are present on the cell rather than on the virus particle. Thus, the presence of cell surface GAGs containing iduronic acid, like heparan sulfate and chondroitin sulfate B, is required for efficient RSV infection in cell culture.

The regenerative capacity of injured adult mammalian central nervous system (CNS) tissue is very limited. Disease or injury that causes destruction or damage to neuronal networks typically results in permanent neurological deficits. Injury to the spinal cord, for example, interrupts vital ascending and descending fiber tracts of spinally projecting neurons. Because neuronal structures located proximal or distal to the injury site remain largely intact, a major goal of spinal cord injury research is to develop strategies to reestablish innervation lost as a consequence of injury. The growth inhibitory nature of injured adult CNS tissue is a major barrier to regenerative axonal growth and sprouting. An increasing complexity of molecular players is being recognized. CNS inhibitors fall into three general classes: members of canonical axon guidance molecules (e.g., semaphorins, ephrins, netrins), prototypic myelin inhibitors (Nogo, MAG, and OMgp) and chondroitin sulfate proteoglycans (lecticans, NG2). On the other end of the spectrum are molecules that promote neuronal growth and sprouting. These include growth promoting extracellular matrix molecules, cell adhesion molecules, and neurotrophic factors. In addition to environmental (extrinsic) growth regulatory cues, cell intrinsic regulatory mechanisms exist that greatly influence injury-induced neuronal growth. Various degrees of growth and sprouting of injured CNS neurons have been achieved by lowering extrinsic inhibitory cues, increasing extrinsic growth promoting cues, or by activation of cell intrinsic growth programs. More recently, combination therapies that activate growth promoting programs and at the same time attenuate growth inhibitory pathways have met with some success. In experimental animal models of spinal cord injury (SCI), mono and combination therapies have been shown to promote neuronal growth and sprouting. Anatomical growth often correlates with improved behavioral outcomes. Challenges ahead include testing whether some of the most promising treatment strategies in animal models are also beneficial for human patients suffering from SCI.

The meniscus is characterized at the light microscopic and ultrastructural levels by thick collagen fibers that are predominantly circumferential in orientation. The extracellular matrix of the meniscus is composed mainly of collagen, with smaller quantities of proteoglycans, matrix glycoproteins, and elastin. The collagen is predominantly Type I, with smaller quantities of Types II, III, and V. The proteoglycans are mainly large, aggregating proteoglycans with chondroitin sulfate as their dominant glycosaminoglycan. A small proportion of small dermatan sulfate proteoglycans is probably present. The matrix glycoproteins include the link proteins, the 116-k protein, and a group of adhesive or potentially adhesive proteins that includes Type VI collagen (here classified as a glycoprotein with a collagenous domain), fibronectin, and thrombospondin. The fibrochondrocytes of the meniscus appear to have considerable potential to respond to growth and other modulating factors in the repair or regeneration of the tissue.

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross-linked by antibodies, they initially assimilate into detergent-resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.