Classical and Alternative Activation of Rat Microglia Treated with Ultrapure Porphyromonas gingivalis Lipopolysaccharide In Vitro.

The possible relationship between periodontal disease resulting from the infection of gingival tissue by the Gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) and the development of neuroinflammation remains under investigation. Recently, P. gingivalis lipopolysaccharide (LPS) was reported in the human brain, thus suggesting it might activate brain microglia, a cell type participating in neuroinflammation. We tested the hypothesis of whether in vitro exposure to ultrapure P. gingivalis LPS may result in classical and alternative activation phenotypes of rat microglia, with the concomitant release of cytokines and chemokines, as well as superoxide anion (O2-), thromboxane B2 (TXB2), and matrix metalloprotease-9 (MMP-9). After an 18-h exposure of microglia to P. gingivalis LPS, the concentration-dependent responses were the following: 0.1-100 ng/mL P. gingivalis LPS increased O2- generation, with reduced inflammatory mediator generation; 1000-10,000 ng/mL P. gingivalis LPS generated MMP-9, macrophage inflammatory protein 1α (MIP-1α/CCL3), macrophage inflammatory protein-2 (MIP-2/CXCL2) release and significant O2- generation; 100,000 ng/mL P. gingivalis LPS sustained O2- production, maintained MMP-9, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) release, and triggered elevated levels of MIP-1α/CCL3, MIP-2/CXCL2, and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL-1), with a very low release of lactic dehydrogenase (LDH). Although P. gingivalis LPS was less potent than Escherichia coli (E. coli) LPS in stimulating TXB2, MMP-9, IL-6 and interleukin 10 (IL-10) generation, we observed that it appeared more efficacious in enhancing the release of O2-, TNF-α, MIP-1α/CCL3, MIP-2/CXCL2 and CINC-1/CXCL-1. Our results provide support to our research hypothesis because an 18-h in vitro stimulation with ultrapure P. gingivalis LPS resulted in the classical and alternative activation of rat brain microglia and the concomitant release of cytokines and chemokines.

Sequential feature selection and inference using multi-variate random forests.

Motivation: Random forest (RF) has become a widely popular prediction generating mechanism. Its strength lies in its flexibility, interpretability and ability to handle large number of features, typically larger than the sample size. However, this methodology is of limited use if one wishes to identify statistically significant features. Several ranking schemes are available that provide information on the relative importance of the features, but there is a paucity of general inferential mechanism, particularly in a multi-variate set up. We use the conditional inference tree framework to generate a RF where features are deleted sequentially based on explicit hypothesis testing. The resulting sequential algorithm offers an inferentially justifiable, but model-free, variable selection procedure. Significant features are then used to generate predictive RF. An added advantage of our methodology is that both variable selection and prediction are based on conditional inference framework and hence are coherent. Results: We illustrate the performance of our Sequential Multi-Response Feature Selection approach through simulation studies and finally apply this methodology on Genomics of Drug Sensitivity for Cancer dataset to identify genetic characteristics that significantly impact drug sensitivities. Significant set of predictors obtained from our method are further validated from biological perspective. Availability and implementation: https://github.com/jomayer/SMuRF. Contact: souparno.ghosh@ttu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Modeling the Chronic Loss of Optic Nerve Axons and the Effects on the Retinal Nerve Fiber Layer Structure in Primary Disorder of Myelin.

PURPOSE: We determined whether the chronic lack of optic nerve myelination and subsequent axon loss is associated with optical coherence tomography (OCT) changes in the retinal nerve fiber layer (RNFL), and whether this models what occurs in multiple sclerosis (MS) and confers its use as a surrogate marker for axon degeneration. METHODS: Using an animal model of Pelizaeus-Merzbacher disease (shp) bilateral longitudinal measurements of the peripapillary RNFL (spectral-domain OCT), electroretinograms (ERG), and visual evoked potentials (VEP) were performed in affected and control animals from 5 months to 2 years and in individual animals at single time points. Light and electron microscopy of the optic nerve and retina and histomorphometric measurements of the RNFL were compared to OCT data. RESULTS: Of the shp animals, 17% had an average reduction of OCT RNFL thickness on the superior retinal quadrant compared to controls (P < 0.05). Electroretinograms showed normal photopic A- and B-waves but flash VEPs were disorganized in shp animals. Morphologically, the shp retinas and optic nerves revealed significant RNFL thinning (P < 0.001) without retinal ganglion cell (RGC) loss, decrease total and relative retinal axonal area, and loss of optic nerve axons. There was strong positive correlation between OCT and morphometric RNFL thickness measurements (r = 0.878, P = 0.004). CONCLUSION: The loss of optic nerve axons demonstrated in the shp model resulted in moderate thinning of the RNFL confirmed by OCT and histology. These results indicate that OCT-derived RNFL measurement can be a useful surrogate biomarker of optic nerve axon loss and potentially disease progression in demyelinating diseases.

Myelin and oligodendrocyte development in the canine spinal cord.

We studied the developmental pattern of oligodendrocyte differentiation and myelin formation in the fetal canine spinal cord from E40 to P0. The pattern of development matches what has been described in the spinal cord of humans, rodents, and many other species. Oligodendrocytes were first found at E40, close to the central canal, with their spread in a tangential manner to the ventral and then lateral columns. Myelin development followed the same pattern but was not seen until E46. A clear subpial zone lacking glial cells and myelin was seen in the lateral column in early development, suggesting that there may also be a radial component of migration of oligodendrocyte progenitor cells (OPCs) from a ventral site. This spatial-temporal developmental pattern seen in wild type matches a delay in myelination of the superficial tracts of the spinal cord seen in a canine myelin mutant, suggesting that the mutation prevents the distribution and differentiation of OPCs at an early, but narrow, window of time during fetal development.

Modeling the natural history of Pelizaeus-Merzbacher disease.

Major gaps in our understanding of the leukodystrophies result from their rarity and the lack of tissue for the interdisciplinary studies required to extend our knowledge of the pathophysiology of the diseases. This study details the natural evolution of changes in the CNS of the shaking pup (shp), a model of the classical form of the X-linked disorder Pelizaeus-Merzbacher disease, in particular in glia, myelin, and axons, which is likely representative of what occurs over time in the human disease. The mutation in the proteolipid protein gene, PLP1, leads to a delay in differentiation, increased cell death, and a marked distension of the rough endoplasmic reticulum in oligodendrocytes. However, over time, more oligodendrocytes differentiate and survive in the spinal cord leading to an almost total recovery of myelination, In contrast, the brain remains persistently hypomyelinated. These data suggest that shp oligodendrocytes may be more functional than previously realized and that their early recruitment could have therapeutic value.

A mutation in the canine gene encoding folliculin-interacting protein 2 (FNIP2) associated with a unique disruption in spinal cord myelination.

Novel mutations in myelin and myelin-associated genes have provided important information on oligodendrocytes and myelin and the effects of their disruption on the normal developmental process of myelination of the central nervous system (CNS). We report here a mutation in the folliculin-interacting protein 2 (FNIP2) gene in the Weimaraner dog that results in hypomyelination of the brain and a tract-specific myelin defect in the spinal cord. This myelination disruption results in a notable tremor syndrome from which affected dogs recover with time. In the peripheral tracts of the lateral and ventral columns of the spinal cord, there is a lack of mature oligodendrocytes. A genome-wide association study of DNA from three groups of dogs mapped the gene to canine chromosome 15. Sequencing of all the genes in the candidate region identified a frameshift mutation in the FNIP2 gene that segregated with the phenotype. While the functional role of FNIP2 is not known, our data would suggest that production of truncated protein results in a delay or failure of maturation of a subpopulation of oligodendrocytes.

Autophagy promotes oligodendrocyte survival and function following dysmyelination in a long-lived myelin mutant.

The Long-Evans shaker (les) rat has a mutation in myelin basic protein that results in severe CNS dysmyelination and subsequent demyelination during development. During this time, les oligodendrocytes accumulate cytoplasmic vesicles, including lysosomes and membrane-bound organelles. However, the mechanism and functional relevance behind these oligodendrocyte abnormalities in les have not been investigated. Using high-magnification electron microscopy, we identified the accumulations in les oligodendrocytes as early and late autophagosomes. Additionally, immunohistochemistry and Western blots showed an increase in autophagy markers in les. However, autophagy did not precede the death of les oligodendrocytes. Instead, upregulating autophagy promoted membrane extensions in les oligodendrocytes in vitro. Furthermore, upregulating autophagy in les rats via intermittent fasting increased the proportion of myelinated axons as well as myelin sheath thickness in les and control rats. Overall, this study provides insight into the abnormalities described in les as well as identifying a novel mechanism that promotes the survival and function of oligodendrocytes.

Spontaneous optic nerve compression in the osteopetrotic (op/op) mouse: a novel model of myelination failure.

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.

Characterization of a PLP-overexpressing transgenic rat, a model for the connatal form of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) most frequently results from duplication of the Plp1 gene with a correlation between disease severity and increasing copy number of the gene. Animal models of PMD, in particular those overexpressing the Plp1 gene, have been sought in attempts to provide systems in which potential therapies can be tested. Here we describe a rat model of the severe connatal form of PMD and provide a detailed characterization of its pathology and molecular biology, prior to testing therapeutic approaches. We determined the exact copy number of Plp1, and the resulting effects on RNA and protein expression. Distinct differences in myelin and disparate distributions of myelin protein markers in comparison to wild-type controls were observed. Altered expression of Plp1 also caused an increase in the apoptotic cell death of oligodendrocytes. These results provide the platform from which to test the effectiveness of in vivo therapies.

Interleukin 12 stimulates IFN-gamma-mediated inhibition of tumor-induced regulatory T-cell proliferation and enhances tumor clearance.

To define the factors that modulate regulatory T (Treg) cells in the tumor setting, we cocultured various tumor cells with either purified Treg cells, or with unfractionated splenocytes. We found that Treg expansion occurred only with unfractionated splenocytes, suggesting that accessory cells and/or factors produced by them play an essential role in tumor-induced Treg expansion. We performed gene expression profiling on tumor-associated Treg cells to identify candidate signaling molecules and studied their effects on tumor-induced Treg expansion. We inadvertently discovered that interleukin (IL)-12 treatment blocked Treg expansion in an IL-12 receptor-dependent fashion. Additional studies showed that IL-12 acts by stimulating IFN-gamma mediated inhibition of Treg cell proliferation, which may partially account for the antitumor effects of IL-12. Furthermore, IL-12 treatment was found to decrease IL-2 production, which may lead to IFN-gamma-independent inhibition of Treg cells, as IL-2 is required for their survival and expansion. Mechanistic studies revealed that IFN-gamma signaling directly causes cell cycle arrest in Treg cells. This study shows that an IL-12-IFN-gamma axis can suppress tumor-induced Treg proliferation. This mechanism may counteract the ability of Treg cells to promote tumor growth in vivo.

Differential expression of granzyme B and C in murine cytotoxic lymphocytes.

Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have been extensively characterized (e.g., perforin, granzymes A and B), the role of granzyme C is less clear. We therefore developed a granzyme C-specific mAb and used flow cytometry to examine the expression of granzyme B and C in the lymphocyte compartments of wild-type and mutant GzmB(-/-) cre mice, which have a small deletion in the granzyme B gene. We detected granzyme B and C expression in CD4(+) and CD8(+) T cells activated with CD3/CD28 beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of both granzymes. Granzyme C up-regulation was delayed relative to granzyme B in wild-type lymphocytes, whereas GzmB(-/-) cre cells expressed granzyme C earlier and more abundantly on a per-cell basis, suggesting that the deleted 350-bp region in the granzyme B gene is important for the regulation of both granzymes B and C. Quantitative RT-PCR revealed that granzyme C protein levels were regulated by mRNA abundance. In vivo, a population of wild-type CD8alphaalpha(+) intraepithelial lymphocytes constitutively expressed granzyme B and GzmB(-/-) cre intraepithelial lymphocytes likewise expressed granzyme C. Using a model of a persistent murine CMV infection, we detected delayed expression of granzyme C in NK cells from infected hosts. Taken together, these findings suggest that granzyme C is activated with persistent antigenic stimulation, providing nonredundant backup protection for the host when granzyme B fails.

Assembly properties of the Bacillus subtilis actin, MreB.

The bacterial actin MreB has been implicated in a variety of cellular roles including cell shape determination, cell wall synthesis, chromosome condensation and segregation, and the establishment and maintenance of cell polarity. Toward elucidating a clearer understanding of how MreB functions inside the bacterial cell, we investigated biochemically the polymerization of MreB from Bacillus subtilis. Light scattering and sedimentation assays revealed pH-, ionic-, cationic-, and temperature-dependent behavior. B. subtilis MreB polymerizes in the presence of millimolar divalent cations in a protein concentration-dependent manner. Polymerization is favored by decreasing pH and inhibited by monovalent salts and low temperatures. Although B. subtilis MreB binds and hydrolyzes both ATP and GTP, it does not require a bound nucleotide for assembly and polymerizes indistinguishably regardless of the nucleotide species bound, with a critical concentration of approximately 900 nM. A number of the presently reported properties of B. subtilis MreB differ significantly from those of T. maritima MreB1 (Bean and Amann [2008]: Biochemistry 47: 826-835), including the nucleotide requirements and temperature and ionic effects on polymerization state. These observations collectively suggest that additional factors interact with MreB to account for its complex dynamic behavior in cells.

NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex.

Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNA-binding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.