miR-155 and miR-146a collectively regulate meningitic Escherichia coli infection-mediated neuroinflammatory responses.

BACKGROUND: Escherichia coli is the most common Gram-negative bacterium causing meningitis, and E. coli meningitis is associated with high mortality and morbidity throughout the world. Our previous study showed that E. coli can colonize the brain and cause neuroinflammation. Increasing evidence supports the involvement of miRNAs as key regulators of neuroinflammation. However, it is not clear whether these molecules participate in the regulation of meningitic E. coli-mediated neuroinflammation. METHODS: The levels of miR-155 and miR-146a, as well as their precursors, in E. coli-infected astrocytes were measured using quantitative real-time PCR (qPCR). Overexpression and knockdown studies of miR-155 and miR-146a were performed to observe the effects on bacterial loads, cytokines, chemokines, and NF-κB signaling pathways. Bioinformatics methods were utilized to predict the target genes, and these target genes were validated using qPCR, Western blotting, and luciferase reporter system. In vivo knockdown of miR-155 and miR-146a was carried out to observe the effects on bacterial loads, inflammatory genes, astrocyte activation, microglia activation, and survival in a mouse model. RESULTS: The levels of miR-155, miR-146a, and their precursors were significantly increased in astrocytes during E. coli infection. miR-155 and miR-146a were induced by the NF-κB-p65 signaling pathway upon infection. Overexpressing and inhibiting miR-155 and miR-146a in astrocytes did not affect the bacterial loads. Further, the in vitro overexpression of miR-155 and miR-146a suppressed the E. coli-induced inflammatory response, whereas the inhibition of miR-155 and miR-146a enhanced it. Mechanistically, miR-155 inhibited TAB2, and miR-146a targeted IRAK1 and TRAF6; therefore, they functioned collaboratively to modulate TLR-mediated NF-κB signaling. In addition, both miR-155 and miR-146a could regulate the EGFR-NF-κB signaling pathway. Finally, the in vivo suppression of E. coli-induced miR-155 and miR-146a further promoted the production of inflammatory cytokines, aggravated astrocyte and microglia activation, and decreased mouse survival time, without affecting the bacterial loads in the blood and brain. CONCLUSIONS: E. coli infection induced miR-155 and miR-146a, which collectively regulated bacteria-triggered neuroinflammatory responses through negative feedback regulation involving the TLR-mediated NF-κB and EGFR-NF-κB signaling pathways, thus protecting the central nervous system from further neuroinflammatory damage.

Cryptococcus neoformans-astrocyte interactions: effect on fungal blood brain barrier disruption, brain invasion, and meningitis progression.

Cryptococcus neoformans is an opportunistic, neurotropic, and encapsulated fungus that causes life-threatening cryptococcal meningitis (CM), especially in regions of the world where AIDS is endemic. The polysaccharide capsule of C. neoformans is the fungus major virulent factor, being copiously released during infection and causing immunosuppressive defects in the host. Although the capsular material is commonly associated with reactive astrocytes in fatal CM, little is known about the molecular and cellular interactions among astroglia and C. neoformans. As astrocytes also make up the neurovascular unit at the blood-brain barrier (BBB), which C. neoformans must transverse to colonize the central nervous system and cause CM; these cells may play a significant regulatory role in the prevention and progression of infection. For example, astrocytes are implicated in neurological disease including the regulation of cerebral intracranial pressure, immune function, and water homeostasis. Hence, in this review, we provide a general overview of astroglia biology and discuss the current knowledge on C. neoformans-astrocyte interactions including their involvement in the development of CM. This "gliocentric view" of cerebral cryptococcosis suggests that therapeutic interventions particularly targeting at preserving the neuroprotective function of astrocytes may be used in preventing and managing C. neoformans BBB transmigration, brain invasion, colonization, and meningitis.

Methods for Monitoring Endocytosis in Astrocytes.

Endocytosis is a vesicle-based mechanism by which eukaryotic cells internalize extracellular material. There are several types of this universal mechanism linked to different types of endocytosed cargo, including pathogens; therefore, several approaches can be applied. Here, we describe techniques that are applicable to study the internalization of flaviviruses; dextrans; transporters, such as, glutamate transporter vGlut1; and peptidergic signaling molecules, including atrial natriuretic peptide into astrocytes, the most heterogeneous neuroglial cells, which play a key homeostatic role in the central nervous system.

Human Commensal Prevotella histicola Ameliorates Disease as Effectively as Interferon-Beta in the Experimental Autoimmune Encephalomyelitis.

Gut microbiota has emerged as an important environmental factor in the pathobiology of multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Both genetic and environmental factors have been shown to play an important role in MS. Among genetic factors, the human leukocyte antigen (HLA) class II allele such as HLA-DR2, DR3, DR4, DQ6, and DQ8 show the association with the MS. We have previously used transgenic mice expressing MS susceptible HLA class II allele such as HLA-DR2, DR3, DQ6, and DQ8 to validate significance of HLA alleles in MS. Although environmental factors contribute to 2/3 of MS risk, less is known about them. Gut microbiota is emerging as an imporatnt environmental factor in MS pathogenesis. We and others have shown that MS patients have distinct gut microbiota compared to healthy control (HC) with a lower abundance of Prevotella. Additionally, the abundance of Prevotella increased in patients receiving disease-modifying therapies (DMTs) such as Copaxone and/or Interferon-beta (IFNß). We have previously identified a specific strain of Prevotella (Prevotella histicola), which can suppress experimental autoimmune encephalomyelitis (EAE) disease in HLA-DR3.DQ8 transgenic mice. Since Interferon-ß-1b [IFNß (Betaseron)] is a major DMTs used in MS patients, we hypothesized that treatment with the combination of P. histicola and IFNß would have an additive effect on the disease suppression. We observed that treatment with P. histicola suppressed disease as effectively as IFNß. Surprisingly, the combination of P. histicola and IFNß was not more effective than either treatment alone. P. histicola alone or in combination with IFNß increased the frequency and number of CD4+FoxP3+ regulatory T cells in the gut-associated lymphoid tissue (GALT). Treatment with P. histicola alone, IFNß alone, and in the combination decreased frequency of pro-inflammatory IFN-γ and IL17-producing CD4+ T cells in the CNS. Additionally, P. histicola alone or IFNß alone or the combination treatments decreased CNS pathology, characterized by reduced microglia and astrocytic activation. In conclusion, our study indicates that the human gut commensal P. histicola can suppress disease as effectively as commonly used MS drug IFNß and may provide an alternative treatment option for MS patients.

Holistic insights into meningitic Escherichia coli infection of astrocytes based on whole transcriptome profiling.

Aim: To investigate the mRNAs and noncoding RNAs (ncRNAs) expression in astrocytes upon meningitic-Escherichia coli infection. Materials & methods: The transcription of mRNAs and ncRNAs were fully investigated and profiled by whole transcriptome sequencing and bioinformatic approaches. Whole transcriptome differences between the infected astrocytes and brain microvascular endothelial cells were further compared and characterized. Results: A total of 2045 mRNAs, 74 long noncoding RNAs, 27 miRNAs and 418 circular RNAs were differentially transcribed in astrocytes upon infection. Competing endogenous RNAs regulatory networks were constructed and preliminary validated. Transcriptomic differences between astrocyte and brain microvascular endothelial cells revealed the cell-specific responses against the infection. Conclusion: Our study comprehensively characterized the ncRNAs and mRNAs profiles in astrocytes upon meningitic-E. coli infection, which will facilitate future functional studies.

Herpetosiphon Secondary Metabolites Inhibit Amyloid-ß Toxicity in Human Primary Astrocytes.

BACKGROUND: The accumulation of extracellular plaques containing amyloid-ß protein (Aß) in the brain is one of the main pathological hallmarks of Alzheimer's disease (AD). Aß peptide can promote the production of highly volatile free radicals and reactive oxygen species (ROS) that can induce oxidative damage to neurons and astrocytes. At present, numerous studies have investigated the neuroprotective and glioprotective effects of natural products derived from plants, animals, and microorganisms. OBJECTIVE: We investigated the glioprotective effect of secondary metabolites obtained from Herpetosiphon sp. HM 1988 against Aß40-induced toxicity in human primary astrocytes. METHODS: The protective effect of bacterial secondary metabolites against Aß40-induced inducible nitric oxide synthase (iNOS) activity was evaluated using the citrulline assay. To confirm the iNOS activity, nitrite production was assessed using the fluorometric Griess diazotization assay. Intracellular NAD+ depletion and lactate dehydrogenase (LDH) release in human primary astrocytes were also examined using well-established spectrophotometric assays. RESULTS: Our results indicate that Aß40 can induce elevation in iNOS and LDH activities, nitrite production, and cellular energy depletion. Importantly, extract of Herpetosiphon sp. HM 1988 decreased iNOS activity, nitrite production, and LDH release. In addition, metabolites of the strain were able to restore cellular energy deficits through inhibition of NAD+ depletion mediated by Aß40. CONCLUSION: These findings suggest that Herpetosiphon metabolites may represent a promising, novel source for the prevention of Aß toxicity in AD.

Porphyromonas gingivalis induces depression via downregulating p75NTR-mediated BDNF maturation in astrocytes.

Many cross-sectional epidemiological studies have shown the incidence of periodontitis is positive correlated with that of depression. However, their causal relationship and underlying mechanism are largely unknown. Porphyromonas gingivalis (Pg) is the main pathogen for periodontitis. Employing female mice treated with Pg every other day for 4 weeks, we found that Pg-mice showed obvious depression-like behavior, an increased number of activated astrocytes and decreased levels of mature brain derived neurotrophic factor (BDNF) and astrocytic p75NTR in the hippocampus. Both hippocampal injection of BDNF and overexpression of p75NTR in astrocytes alleviated Pg-induced depression-like behavior in mice. Moreover, Pg-lipopolysaccharides (LPS) generated similar phenotypes, which were reversed by the TLR-4 inhibitor TAK242. Our results suggest that Pg-LPS decreases the level of astrocytic p75NTR and then downregulates BDNF maturation, leading to depression-like behavior in mice. Our study provides the first evidence that Pg is a modifiable risk factor for depression and uncovers a novel therapeutic target for the treatment of depression.

Murine astrocytes are responsive to the pro-inflammatory effects of IL-20.

Glia are key regulators of inflammatory responses within the central nervous system (CNS) following infection or trauma. We have previously demonstrated the ability of activated astrocytes to rapidly produce pro-inflammatory mediators followed by a transition to an anti-inflammatory cytokine production profile that includes the immunosuppressive cytokine interleukin (IL)-10 and the closely related cytokines IL-19 and IL-24. IL-20, another member of the IL-10 family, is known to modulate immune cell activity in the periphery and we have previously demonstrated that astrocytes constitutively express the cognate receptors for this cytokine. However, the ability of glia to produce IL-20 remains unclear and the effects of this pleiotropic cytokine on glial immune functions have not been investigated. In this study, we report that primary murine and human astrocytes are not an appreciable source of IL-20 following challenge with disparate bacterial species or their components. Importantly, we have determined that astrocyte are responsive to the immunomodulatory actions of this cytokine by showing that recombinant IL-20 administration upregulates microbial pattern recognition receptor expression and induces release of the inflammatory mediator IL-6 by these cells. Taken together, these data suggest that IL-20 acts in a dissimilar manner to other IL-10 family members to augment the inflammatory responses of astrocytes.

Microglia and amyloid precursor protein coordinate control of transient Candida cerebritis with memory deficits.

Bloodborne infections with Candida albicans are an increasingly recognized complication of modern medicine. Here, we present a mouse model of low-grade candidemia to determine the effect of disseminated infection on cerebral function and relevant immune determinants. We show that intravenous injection of 25,000 C. albicans cells causes a highly localized cerebritis marked by the accumulation of activated microglial and astroglial cells around yeast aggregates, forming fungal-induced glial granulomas. Amyloid precursor protein accumulates within the periphery of these granulomas, while cleaved amyloid beta (Aß) peptides accumulate around the yeast cells. CNS-localized C. albicans further activate the transcription factor NF-κB and induce production of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF), and Aß peptides enhance both phagocytic and antifungal activity from BV-2 cells. Mice infected with C. albicans display mild memory impairment that resolves with fungal clearance. Our results warrant additional studies to understand the effect of chronic cerebritis on cognitive and immune function.

Burkholderia pseudomallei-loaded cells act as a Trojan horse to invade the brain during endotoxemia.

Neurologic melioidosis occurs in both human and animals; however, the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however, during melioidosis, lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines, chemokines and cell adhesion molecules; the activation of astrocytes, microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei, B. thailandensis, B. cenocepacia and B. multivorans LPS-induced brains. Accordingly, different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice, B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular, these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption.

Human tuberculosis brain promotes neuronal apoptosis but not in astrocytes with high expression of vascular endothelial growth factor.

The present study aimed to investigate the involvement of the angiogenic marker vascular endothelia growth factor (VEGF) and apoptotic markers of Bcl-2 and Bax in the neurons and astrocytes in the brain infected by Mycobacterium tuberculosis. The immunohistochemistry staining was performed to analyze the expression of the VEGF, Bcl-2 and Bax in the astrocytes and neurons. The expression of VEGF was high in neurons and astrocytes in both the infected brain and control tissues with no difference of angiogenic activity (p = 0.40). Higher Bcl-2 expression was seen in astrocytes of infected brain tissues compared to the control tissues (p = 0.004) promoted a higher anti-apoptotic activity in astrocytes. The neurons expressed strong Bax expression in the infected brain tissues compared to the control tissues (p < 0.001), which indicated more apoptosis in neurons. Thus, neuronal death and survival of infected astrocytes together with high expression of VEGF might be associated with formation of brain tuberculosis. In conclusion, neurons could be more vulnerable than astrocytes in human tuberculosis brain with high expression of VEGF.

High levels of antibodies against PtpA and PknG secreted by Mycobacterium avium ssp. paratuberculosis are present in neuromyelitis optica spectrum disorder and multiple sclerosis patients.

Mycobacterium avium ssp. paratuberculosis (Map) is the etiological agent of Paratuberculosis in ruminants. Protein tyrosine phosphatase A (PtpA) and protein kinase G (PknG) are secreted proteins necessary for the survival of the pathogen within macrophages. In this study we analyzed if Map was able to grow within astrocytes and investigated on the presence of antibodies against PtpA and PknG proteins in MS and NMOSD patients by ELISA. Map was unable to proliferate in astrocytes after of 72 h post-infection, but we observed a high level of antibodies against both virulence factors, suggesting that these patients have been exposed/infected with Map.

Infective capacity of Cryptococcus neoformans and Cryptococcus gattii in a human astrocytoma cell line.

Pathogenesis of cryptococcosis in the central nervous system (CNS) is a topic of ongoing research, including the mechanisms by which this fungus invades and infects the brain. Astrocytes, the most common CNS cells, play a fundamental role in the local immune response. Astrocytes might participate in cryptococcosis either as a host or by responding to fungal antigens. To determine the infectivity of Cryptococcus neoformans var. grubii and Cryptococcus gattii in a human astrocytoma cell line and the induction of major histocompatibility complex (MHC) molecules. A glioblastoma cell line was infected with C. neoformans var. grubii and C. gattii blastoconidia labelled with FUN-1 fluorescent stain. The percentage of infection and expression of HLA class I and II molecules were determined by flow cytometry. The interactions between the fungi and cells were observed by fluorescence microscopy. There was no difference between C. neoformans var. grubii and C. gattii in the percentage infection, but C. neoformans var. grubii induced higher expression of HLA class II than C. gattii. More blastoconidia were recovered from C. neoformans-infected cells than from C. gattii infected cells. Cryptococcus neoformans var. grubii may have different virulence mechanisms that allow its survival in human glia-derived cells.

Aprepitant limits in vivo neuroinflammatory responses in a rhesus model of Lyme neuroborreliosis.

BACKGROUND: Substance P (SP) is produced at high levels in the central nervous system (CNS), and its target receptor, neurokinin 1 receptor (NK-1R), is expressed by glia and leukocytes. This tachykinin functions to exacerbate inflammatory responses at peripheral sites. Moreover, SP/NK-1R interactions have recently been associated with severe neuroinflammation and neuronal damage. We have previously demonstrated that NK-1R antagonists can limit neuroinflammatory damage in a mouse model of bacterial meningitis. Furthermore, we have since shown that these agents can attenuate Borrelia burgdorferi-induced neuronal and glial inflammatory mediator production in non-human primate brain explants and isolated neuronal cells. METHODS: In the present study, we have assessed the role played by endogenous SP/NK-1R interactions in damaging CNS inflammation in an established rhesus macaque model that faithfully reproduces the key clinical features of Lyme neuroborreliosis, using the specific NK-1R antagonist, aprepitant. We have utilized multiplex ELISA to quantify immune mediator levels in cerebrospinal fluid, and RT-PCR and immunoblot analyses to quantify cytokine and NK-1R expression, respectively, in brain cortex, dorsal root ganglia, and spinal cord tissues. In addition, we have assessed astrocyte number/activation status in brain cortical tissue by immunofluorescence staining and confocal microscopy. RESULTS: We demonstrate that aprepitant treatment attenuates B. burgdorferi-induced elevations in CCL2, CXCL13, IL-17A, and IL-6 gene expression in dorsal root ganglia, spinal cord, and/or cerebrospinal fluid of rhesus macaques at 2 to 4 weeks following intrathecal infection. In addition, we demonstrate that this selective NK-1R antagonist also prevents increases in total cortical brain NK-1R expression and decreases in the expression of the astrocyte marker, glial fibrillary acidic protein, associated with B. burgdorferi infection. CONCLUSIONS: The ability of a centrally acting NK-1R inhibitor to attenuate B. burgdorferi-associated neuroinflammatory responses and sequelae raises the intriguing possibility that such FDA-approved agents could be repurposed for use as an adjunctive therapy for the treatment of bacterial CNS infections.

MicroRNA and mRNA Transcriptome Profiling in Primary Human Astrocytes Infected with Borrelia burgdorferi.

Lyme disease is caused by infection with the bacterium Borrelia burgdorferi (Bb), which is transmitted to humans by deer ticks. The infection manifests usually as a rash and minor systemic symptoms; however, the bacteria can spread to other tissues, causing joint pain, carditis, and neurological symptoms. Lyme neuroborreliosis presents itself in several ways, such as Bell's palsy, meningitis, and encephalitis. The molecular basis for neuroborreliosis is poorly understood. Analysis of the changes in the expression levels of messenger RNAs and non-coding RNAs, including microRNAs, following Bb infection could therefore provide vital information on the pathogenesis and clinical symptoms of neuroborreliosis. To this end, we used cultured primary human astrocytes, key responders to CNS infection and important components of the blood-brain barrier, as a model system to study RNA and microRNA changes in the CNS caused by Bb. Using whole transcriptome RNA-seq, we found significant changes in 38 microRNAs and 275 mRNAs at 24 and 48 hours following Bb infection. Several of the RNA changes affect pathways involved in immune response, development, chromatin assembly (including histones) and cell adhesion. Further, several of the microRNA predicted target mRNAs were also differentially regulated. Overall, our results indicate that exposure to Bb causes significant changes to the transcriptome and microRNA profile of astrocytes, which has implications in the pathogenesis, and hence potential treatment strategies to combat this disease.

Cellular Microbiology of Mycoplasma canis.

Mycoplasma canis can infect many mammalian hosts but is best known as a commensal or opportunistic pathogen of dogs. The unexpected presence of M. canis in brains of dogs with idiopathic meningoencephalitis prompted new in vitro studies to help fill the void of basic knowledge about the organism's candidate virulence factors, the host responses that it elicits, and its potential roles in pathogenesis. Secretion of reactive oxygen species and sialidase varied quantitatively (P < 0.01) among strains of M. canis isolated from canine brain tissue or mucosal surfaces. All strains colonized the surface of canine MDCK epithelial and DH82 histiocyte cells and murine C8-D1A astrocytes. Transit through MDCK and DH82 cells was demonstrated by gentamicin protection assays and three-dimensional immunofluorescence imaging. Strains further varied (P < 0.01) in the extents to which they influenced the secretion of tumor necrosis factor alpha (TNF-α) and the neuroendocrine regulatory peptide endothelin-1 by DH82 cells. Inoculation with M. canis also decreased major histocompatibility complex class II (MHC-II) antigen expression by DH82 cells (P < 0.01), while secretion of gamma interferon (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and complement factor H was unaffected. The basis for differences in the responses elicited by these strains was not obvious in their genome sequences. No acute cytopathic effects on any homogeneous cell line, or consistent patterns of M. canis polyvalent antigen distribution in canine meningoencephalitis case brain tissues, were apparent. Thus, while it is not likely a primary neuropathogen, M. canis has the capacity to influence meningoencephalitis through complex interactions within the multicellular and neurochemical in vivo milieu.

Interactions of Streptococcus suis serotype 2 with human meningeal cells and astrocytes.

BACKGROUND: Streptococcus suis serotype 2 is an important porcine pathogen and emerging zoonotic agent responsible for meningitis, of which different sequence types predominate worldwide. Though bacterial meningitis is defined as an exacerbated inflammation of the meninges, the underlying astrocytes of the glia limitans superficialis may also be implicated. However, the interactions between this pathogen and human meningeal cells or astrocytes remain unknown. Furthermore, the roles of well-described virulence factors (capsular polysaccharide, suilysin and cell wall modifications) in these interactions have yet to be studied. Consequently, the interactions between S. suis serotype 2 and human meningeal cells or astrocytes were evaluated for the first time in order to better understand their involvement during meningitis in humans. RESULTS: Streptococcus suis serotype 2 adhered to human meningeal cells and astrocytes; invasion of meningeal cells was rare however, whereas invasion of astrocytes was generally more frequent. Regardless of the interaction or cell type, differences were not observed between sequence types. Though the capsular polysaccharide modulated the adhesion to and invasion of meningeal cells and astrocytes, the suilysin and cell wall modifications only influenced astrocyte invasion. Surprising, S. suis serotype 2 induced little or no inflammatory response from both cell types, but this absence of inflammatory response was probably not due to S. suis-induced cell death. CONCLUSIONS: Though S. suis serotype 2 interacted with human meningeal cells and astrocytes, there was no correlation between sequence type and interaction. Consequently, the adhesion to and invasion of human meningeal cells and astrocytes are strain-specific characteristics. As such, the meningeal cells of the leptomeninges and the astrocytes of the glia limitans superficialis may not be directly implicated in the inflammatory response observed during meningitis in humans.

Activation of Sonic Hedgehog Leads to Survival Enhancement of Astrocytes via the GRP78-Dependent Pathway in Mice Infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytes may protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh) via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infected BABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated with corresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP) and Shh were increased in astrocyte cocultured with living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated in cocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higher in astrocytes pretreated with recombinant Shh. These findings suggest that the expression of Shh may inhibit cell death by activating Bcl-2 through a GRP78-dependent pathway.

Glial network responses to polymicrobial invasion of dentin.

This study investigated the distribution patterns of glial networks disclosed by reactivity for glial fibrillary acidic protein (GFAP) and S100B in healthy and carious human teeth. The objective was to determine the assembly and collapse of glial networks in response to encroaching infection. 15 healthy and 37 carious posterior teeth from adults were studied. Immediately after extraction, teeth were cleaned and vertically split and the half with pulp fixed and prepared for resin or frozen sections. Sections were stained with toluidine blue and for immunofluorescence, with observation by confocal laser microscopy and analysis by ImageJ software. Carious teeth were subdivided into three groups according to degree of carious involvement: microbial penetration through enamel (stage A), extension into dentin (stage B) and advanced penetration into dentin but without invasion of underlying pulp tissue (stage C). In stage A lesions there was marked increase in glial networks in dental pulp tissue that extended beyond the zone of microbial invasion. This response was maintained in stage B lesions. In advanced stage C lesions these networks were degraded in the zone of invasion in association with failure to contain infection. Cells expressing the glial markers GFAP and S100B showed a response to initial microbial invasion of dentin by increase in number and altered anatomical arrangement. The late stage of dentinal caries was marked by collapse of these networks in the region adjacent to advancing bacteria. This behaviour is important for understanding and explaining the defensive response of the neurosensory peripheral dental pulp apparatus to infection.