The use of transplanted glial cells to reconstruct glial environments in the CNS

Transplantation studies have demonstrated that glia-depleted areas of the CNS can be reconstituted by the introduction of cultured cells. Thus, the influx of Schwann cells into glia-free areas of demyelination in the spinal cord can be prevented by the combined introduction of astrocytes and cells of the O-2A lineage. Although Schwann cell invasion of areas of demyelination is associated with destruction of astrocytes, the transplantation of rat tissue culture astrocytes ("type-1") alone cannot suppress this invasion, indicating a role for cells of the O-2A lineage in reconstruction of glial environments. By transplanting different glial cell preparations and manipulating lesions so as to prevent meningeal cell and Schwann cell proliferation it is possible to demonstrate that the behaviour of tissue culture astrocytes ("type-1") and astrocytes derived from O-2A progenitor cells ("type-2") is different. In the presence of meningeal cells, tissue culture astrocytes clump together to form cords of cells. In contrast, type-2 astrocytes spread throughout glia-free areas in a manner unaffected by the presence of meningeal cells or Schwann cells. Thus, progenitor-derived astrocytes show a greater ability to fill glia-free areas than tissue culture astrocytes. Similarly, when introduced into infarcted white matter in the spinal cord, progenitor-derived astrocytes fill the malacic area more effectively than tissue culture astrocytes, although axons do not regenerate into the reconstituted area.     

Studies on local immune response in mouse model of experimental Escherichia coli intrauterine infections

Studies were conducted to elucidate the immune cell response at infection sites by performing immunostaining of immune cells with a monoclonal antibody in an experimental Escherichia coli (E. coli) mouse uterine infection model. 1. The incidence of uterine infection by E. coli decreased with the passage of time: 4/4 on Day 1, 4/6 on Day 3, 2/6 on Day 7, and 1/6 on each of Days 14 and 21. It was surmised that clearance of the bacteria from the infection sites was being carried out by immune cells. 2. Beginning from infection Day 1, the infected uterine tissue was observed to undergo a moderate degree of invasion by neutrophils, macrophages, CD4+ T cells, CD8+ T cells and IgA+ B cells. Then, beginning from infection Day 3, there was a mild degree of invasion of the infected uterine tissue by IgM+ B cells and IgG+ B cells. The number of neutrophils in the tissue decreased beginning from infection Day 14, but the degree of invasion of the infected tissue by the other kinds of immune cells remained almost constant through infection Day 21. 3. A comparison was made of the immune responses to local infection by E. coli, and Chlamydia trachomatis (C. trachomatis), an intracellular parasite. It was found that the invasion of the infection site by immune cells occurred earlier in the case of E. coli infection than C. trachomatis infection. In addition, the C. trachomatis infection site was observed to contain greater numbers of macrophages and CD8+ T cells play important roles in the immune defense at sites of infection by C. trachomatis.     

Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection

The intracellular protozoan Toxoplasma gondii is a widespread opportunistic parasite of humans and animals. Normally, T. gondii establishes itself within brain and skeletal muscle tissues, persisting for the life of the host. Initiating and sustaining strong T-cell-mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. The parasite induces high levels of gamma interferon (IFN-gamma) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFN-gamma synthesis. The latter cytokine, in addition to promoting the differentiation of Th1 effectors, is important in macrophage activation and acquisition of microbicidal functions, such as nitric oxide release. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-gamma, which is required to prevent cyst reactivation. T-cell-mediated cytolytic activity against infected cells, while easily demonstrable, plays a secondary role to inflammatory cytokine production. While part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression.     

Axonal versus dendritic outgrowth is differentially affected by radial glia in discrete layers of the retina

Formation of neural cell polarity defined by oriented extension of axons and dendrites is a crucial event during the development of the nervous system. Ganglion cells of the chicken retina extend axons exclusively into the inner retina, whereas their dendrites grow into the outer retina. To analyze guidance cues for specific neurite extension, novel in vitro systems were established. Ganglion cells were purified by enzymatically facilitated detachment of the ganglion cell layer. A newly developed retrograde labeling technique and the expression analysis of the cell type-specific 2A1 antigen were used to monitor ganglion cell purification. In highly purified ganglion cells explanted onto retinal cryosections (cryoculture), axon formation was induced when the cells were positioned on the inner retina. In contrast, on outer layers of the developing retina dendritic outgrowth was prevalent. Because radial glia have been demonstrated to be instructive in neuritogenesis, distinct glial cell compartments located in inner and outer retina, respectively, were isolated for functional assays. Glial end feet were purified by a physical detachment technique. Glial somata were purified by complement mediated cytolysis of all nonglial cells. When ganglion cells were cultured on different glial compartments, axon formation occurred on end feet but not on glial somata. In striking contrast, on glial somata dendrites were formed. The data support the notion that ganglion cell polarity is affected by the retinal microenvironment, which in turn is possibly influenced by radial glia, being themselves polarized.     

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.     

Internalization of Staphylococcus aureus by endothelial cells induces apoptosis

The ability of Staphylococcus aureus to invade and survive within endothelial cells is believed to contribute to its propensity to cause persistent endovascular infection with endothelial destruction. In the present study, we show that following invasion of human umbilical vein endothelial cells, intracellular S. aureus organisms remain viable over a 72-h period and, as determined by transmission electron microscopic examination, that the bacteria exist within vacuoles and free within the cytoplasm. We also demonstrate that endothelial cell death following S. aureus invasion occurs at least in part by apoptosis as shown by DNA fragmentation and changes in nuclear morphology. Apoptotic changes were evident as early as 1 h after infection of endothelial cells. Internalization of S. aureus rather than adherence appears to be necessary, since use of the phagocytosis inhibitor cytochalasin D prevented apoptosis. UV-killed staphylococci, although retaining the capacity to be internalized, were not capable of inducing apoptosis, suggesting that apoptosis is dependent upon a factor associated with viable organisms. The studies demonstrate that viable intracellular S. aureus induces apoptosis of endothelial cells and that internalized staphylococci can exist free within the cytoplasm.     

Intercellular signaling in neuronal-glial networks

Glial cells have recently been found to exhibit electrophysiological and metabolic responses to many neurotransmitters and neuromodulators. These findings have focused attention on the possibility that active signaling between neurons and glia could represent an important form of intercellular communication within the brain. Since glial and neuronal networks are both physically and metabolically interlinked, such intercellular signaling may represent a mechanism for inducing collective changes in the cellular physiology of neuronal and glial cell populations. Within the nervous tissue of both vertebrate and invertebrate organisms, glial cells are known to secrete extracellular signal molecules, modulate carbohydrate metabolism, and control the volume and ionic composition of extracellular space. In this paper, the roles that cytoplasmic [Ca2+] transients may play in regulating these glial cell functions are reviewed. Mechanisms by which intracellular Ca oscillations and intercellular Ca waves may be generated in neurotransmitter-stimulated glial cells are also discussed. In addition, it is proposed that rhythmic glial cell contractions and shape changes, which have been observed for many decades, are linked to Ca-induced secretion of ions, water, and neuroactive compounds. These activities represent mechanisms by which Ca-induced changes in glial cell physiology could potentially alter the excitability of neuronal networks.     

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.     

Experimental study of cardiac lymph dynamics and edema formation in ischemia/reperfusion injury--with reference to the effect of hyaluronidase

In order to study the interactions of Toxoplasma gondii and neuroepithelial cells morphologically and biochemically we established an easy in vitro model, which simulates cellular contacts in congenital toxoplasmosis. Monolayer cultures of neuroepithelial cells from 13-14-day-old mouse embryos were prepared containing the typical ventricular cell types found in an embryonic brain, such as young neurons, macroglial and microglial cells. Ultrastructural investigations on cultures incubated for 1, 5 and 30 min or 1, 6, 12, 24 and 48 h with T. gondii indicated that all three cell types had been invaded by the parasites, multiplying in parasite vacuoles by means of endodyogeny. Microglial cells had already been penetrated by trophozoites within one minute and showed up to 3 or 5 parasite vacuoles per cell. Neurons and glial cells were invaded within 5 min and contained only one vacuole per host cell. All the parasite vacuoles were bounded by a membrane and bordered by the rough endoplasmic reticulum and mitochondria of the host cell after a few minutes. The vacuoles also contained some membranic tubuli. After 30 min some neuronal neurites were destroyed while the perikarya seemed to be unchanged. After 6 h the cytoplasm of the microglia lost more and more ribosomes and organelles. Neurons and glial cells showed no alterations. After 12h large areas of the vacuole membrane were folded up and lay curled up in the vacuoles. After 24 h incubation T. gondii had destroyed nearly all the microglial cells. The ultrastructure of neurons and glial cells now began to change in the same way as shown for microglial cells. The organelles and cellular membranes disintegrated and after 48 h incubation nearly all the cells in the neuroepithelial cell culture had fallen to pieces. For an identification of T. gondii in vitro by light microscopy or for the characterization of the cell surface we tried to label the parasites with 11 different FITC-stained lectins. None of the tested lectins bound to the parasites. We conclude that our in vitro-model for invasion of T. gondii in neuroepithelial cells opens an opportunity for studying the interaction of these cells or the pharmacological effects on this interaction under defined conditions.     

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: the roles of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Clostridium perfringens infections are characterized by the lack of an inflammatory response at the site of infection and rapidly progressive margins of tissue necrosis. Studies presented here investigated the role of theta toxin from C. perfringens in the pathophysiology of these events. Mice passively immunized with neutralizing monoclonal antibody against theta toxin and challenged with an LD100 of log phase C. perfringens had significantly less mortality than untreated controls. Intramuscular injection of killed, washed C. perfringens in mice induced a massive time-dependent influx of polymorphonuclear leukocytes (PMNL) into tissue; injection of either viable, washed C. perfringens or killed organisms plus theta toxin dramatically attenuated PMNL influx although PMNL accumulated in adjacent vessels. The anti-inflammatory effects could not be attributed to an absence of chemoattractants since C. perfringens proteins had chemotactic factor activity, and killed bacilli generated serum-derived chemotactic factors. Scanning and transmission electron microscopy demonstrated the dramatic leukocidal effects of high doses of theta toxin on PMNL. In contrast, sublethal concentrations of theta toxin primed PMNL chemiluminescence, disrupted PMNL cytoskeletal actin polymerization/disassembly, and stimulated functional upregulation of CD11b/CD18 adherence glycoprotein. In summary, these results demonstrate that theta toxin is an important virulence factor in C. perfringens infection. In a concentration-dependent fashion, theta toxin contributes to the pathogenesis of clostridial gangrene by direct destruction of host inflammatory cells and tissues, and by promoting dysregulated PMNL/endothelial cell adhesive interactions.     

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.     

An increased number of follicles containing activated CD69+ helper T cells and proliferating CD71+ B cells are found in H. pylori-infected gastric mucosa

Multiple sclerosis is characterized by myelin destruction and oligodendrocyte loss. The neuropathological hallmark of the disease is the presence of demyelinated plaques in the central nervous system. We have recently found a gliotoxic factor in MS cerebrospinal fluid which induces programmed cell death in vitro, in glial cells. Here we show DNA fragmentation and glial cell death in biopsy samples, obtained from a patient who underwent surgery with suspicion of tumor, and whose disease record, including brain autopsy, demonstrated an active multiple sclerosis. We used the in situ TUNEL technique, a method which sensitively detects the DNA fragmentation accompanying programmed cell death in tissue sections, and compatible with classical fixation techniques. We found intense DNA fragmentation in nuclei of glial cells at-or very near-to the site of demyelination. A double labeling technique showed that glial fibrillary associated protein positive astrocytes may undergo programmed cell death in multiple sclerosis.     

Distribution of mitochondria within Müller cells--I. Correlation with retinal vascularization in different mammalian species

The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.     

Ultrastructural changes in the central nervous system of mice in tick-borne encephalitis

Tick-borne encephalitis virus is localized in nerve cells and intracellular spaces. According to the nature of ultrastructural changes, the pathological process in nerve cells may be divided into three stages connected with the stages of virus reproduction. In the first stage, the protein-synthesizing systems of the cell are degraded. In the second stage specific virus inclusions form in the cell cytoplasm as aggregates of mature and immature virions alongside with destruction of ultrastructures and growth of smooth membranes. In the third stage degradation of neurons is accompanied by necrobiosis and discomplexation of the surrounding glial elements, processes, cells of the inflammatory infiltrate and capillary walls. A certain combination of nonspecific changes in organelles of various cells with viral inclusions makes the ultrastructural picture of the focus of lesions in tick-borne encephalitis sufficiently characteristic for differential pathological diagnosis.     

Relationship between lipophilicity and binding to human serum albumin of arylpropionic acid non-steroidal anti-inflammatory drugs

In cases of retinal light damage, glaucoma, or senile macula degeneration, the loss of retinal neurons is thought to cause alterations of glial cells. We performed immunocytochemical studies on retinae of (i) healthy rats and human donors, (ii) rats exposed to enhanced illumination for 24 months, a procedure which leads to complete loss of photoreceptor cells, (iii) a human donor who had suffered from senile macula (photoreceptor cell) degeneration, and (iv) human donors who had suffered from glaucoma, known to be accompanied by a loss of ganglion cells and other retinal neurons. Furthermore, Müller cells were enzymatically isolated from human glaucomatous retinae. All preparations were subjected to immunocytochemistry for CD44 antigen and Apolipoprotein E (ApoE). In normal rat and human retinae, CD44 immunoreactivity was observed in the microvillous sclerad processes of Müller cells: in human retinae, perivascular (astro-)glial cell processes were also CD44 immunopositive. ApoE immunoreactivity was only found in some perivascular (astro-)glial cell processes of human retinae. Both rat and human Müller cells respond to photoreceptor cell damage by increased, and ectopic, expression of the CD44 antigen. Increased ApoE immunoreactivity was found in Müller cells from degenerative human retinae, but rarely in light-damaged rat retinae. It is concluded that degeneration-related reorganization involves enhanced expression of the glial cell adhesion molecule CD44 as well as elevated activity of the glial lipid transport molecule ApoE.     

Apoptosis of human intestinal epithelial cells after bacterial invasion

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.     

Analysis of diagnostic errors and recommendations of diagnostic procedures in bacteriologically negative pulmonary tuberculosis

Malaria infection in a vertebrate host is initiated when Plasmodium sporozoites invade hepatocytes after injection by an infected mosquito. In vitro, the parasites invade and develop in HepG2 cells and these cells have been used to study target cell invasion by sporozoites. Previously described in vitro invasion assays involve staining and counting of intracellular sporozoites or exoerythrocytic forms of the parasite. Here we describe an immunoradiometric assay that can quantify sporozoite invasion of HepG2 cells in vitro. The assay relies on the differential detection of intracellular and extracellular circumsporozoite protein (CS; the major surface protein of the sporozoite) which can then be used to calculate the efficiency of invasion. Since this assay can be performed more rapidly than the current assays in which parasites must be counted under a microscope, it enables investigators to more rapidly screen inhibitors of sporozoite invasion.     

Inoculum composition and Salmonella pathogenicity island 1 regulate M-cell invasion and epithelial destruction by Salmonella typhimurium

In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S. typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S. typhimurium. Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S. typhimurium. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems.