Barbara A. Scofield

Histologic analysis of neural elements in the human sacroiliac joint

Study Design. The posterior ligament of the human sacroiliac joint was examined for nerves and nerve endings using histologic and immunohistochemical techniques. Objective. To identify nerve fibers and mechanoreceptors in the posterior ligament. Summary of Background Data. According to the findings of previous studies, the human sacroiliac joint receives myelinated and unmyelinated axons that presumably conduct pain and proprioceptive impulses derived from mechanoreceptors and free nerve endings in the human sacroiliac joint. Methods. Tissue obtained from six patients was stained with gold chloride and that obtained from six additional patients was stained using antibodies specific for substance P and protein gene product 9.5. Results. The staining of joint tissue using the gold chloride technique showed myelinated and unmyelinated nerve fibers, two morphotypes of paciniform encapsulated mechanoreceptors, and a single nonpaciniform mechanoreceptor. Analysis using immunohistochemical staining for protein gene product 9.5 did not unequivocally show axons, nerve fascicles, or mechanoreceptors. Similarly, analysis based on immunohistochemical staining for substance P, one of several neurotransmitters known to signal pain from the periphery, showed reactive elements that may have been nerves, but because of background staining, could not be positively identified as such. Conclusions. The presence of nerve fibers and mechanoreceptors in the sacroiliac ligament demonstrates that the central nervous system receives information, certainly proprioceptive, and possibly pain from the sacroiliac joint. Although it is not known how the central nervous system uses such information, it seems reasonable to speculate that the proprioceptive information is used to optimize upper body balance at this joint. In addition, because the staining techniques used generally to show nerves and nerve elements in periarticular connective tissue are nonspecific, the distinction between neural and nonneural should be made on the basis of both morphologic and staining characteristics.

Interferon‐β Modulates Inflammatory Response in Cerebral Ischemia

Background Stroke is a leading cause of death in the world. In >80% of strokes, the initial acute phase of ischemic injury is due to the occlusion of a blood vessel resulting in severe focal hypoperfusion, excitotoxicity, and oxidative damage. Interferon‐β (IFNβ), a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for the treatment of relapsing‐remitting multiple sclerosis for more than a decade. Its anti‐inflammatory properties and well‐characterized safety profile suggest that IFNβ has therapeutic potential for the treatment of ischemic stroke. Methods and Results We investigated the therapeutic effect of IFNβ in the mouse model of transient middle cerebral artery occlusion/reperfusion. We found that IFNβ not only reduced infarct size in ischemic brains but also lessened neurological deficits in ischemic stroke animals. Further, multiple molecular mechanisms by which IFNβ modulates ischemic brain inflammation were identified. IFNβ reduced central nervous system infiltration of monocytes/macrophages, neutrophils, CD4+ T cells, and γδ T cells; inhibited the production of inflammatory mediators; suppressed the expression of adhesion molecules on brain endothelial cells; and repressed microglia activation in the ischemic brain. Conclusions Our results demonstrate that IFNβ exerts a protective effect against ischemic stroke through its anti‐inflammatory properties and suggest that IFNβ is a potential therapeutic agent, targeting the reperfusion damage subsequent to the treatment with tissue plasminogen activator.

Differential effects of IFN-β on IL-12, IL-23, and IL-10 expression in TLR-stimulated dendritic cells

MS is an autoimmune disease characterized by immune cell infiltration in the CNS, leading to cumulative disability. IFN‐β, used clinically in RR‐MS reduces lesion formation and rates of relapse. Although the molecular mechanisms are not entirely elucidated, myeloid cells appear to be a major target for the therapeutic effects of IFN‐β. DCs have a critical role in experimental models of MS through their effect on encephalitogenic Th1/Th17 cell differentiation and expansion. Here we focused on the effects of IFN‐β on DC expression of cytokines involved in the control of Th1/Th17 differentiation and expansion. Administration of IFN‐β to mice immunized with MOG35–55 inhibited IL‐12 and IL‐23 expression in splenic DC and reduced in vivo differentiation of Th1/Th17 cells. IFN‐β affected cytokine expression in TLR‐stimulated DC in a similar manner in vitro, inhibiting IL‐12 and IL‐23 and stimulating IL‐10 at both mRNA and protein levels, by signaling through IFNAR. We investigated the role of the signaling molecules STAT1/STAT2, IRF‐1 and IRF‐7, and of the PI3K→GSK3 pathway. IFN‐β inhibition of the IL‐12 subunits p40 and p35 was mediated through STAT1/STAT2, whereas inhibition of IL‐23 was STAT1 dependent, and the stimulatory effect on IL‐10 expression was mediated through STAT2. IFN‐β induces IRF‐7 and, to a lesser degree, IRF‐1. However, neither IRF mediated the effects of IFN‐β on IL‐12, IL‐23, or IL‐10. We found that the PI3K pathway mediated IL‐12 inhibition but did not interfere with the inhibition of IL‐23 or stimulation of IL‐10.

3H-1,2-dithiole-3-thione as a novel therapeutic agent for the treatment of experimental autoimmune encephalomyelitis

3H-1,2-dithiole-3-thione (D3T), the simplest member of the sulfur-containing dithiolethiones, is found in cruciferous vegetables, and has been previously reported to be a potent inducer of antioxidant genes and glutathione biosynthesis by activation of the transcription factor Nrf2. D3T is a cancer chemopreventive agent and possesses anti-inflammatory properties. Although D3T has been shown to protect against neoplasia, the effect of D3T in the autoimmune inflammatory disease multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) is unknown. The present study is the first report of the therapeutic effect of D3T in EAE. Our results show D3T, administered post immunization, not only delays disease onset but also dramatically reduces disease severity in EAE. Strikingly, D3T, administered post disease onset of EAE, effectively prevents disease progression and exacerbation. Mechanistic studies revealed that D3T suppresses dendritic cell activation and cytokine production, inhibits pathogenic Th1 and Th17 differentiation, represses microglia activation and inflammatory cytokine expression, and promotes microglia phase II enzyme induction. In summary, these results indicate that D3T affects both innate and adaptive immune cells, and the protective effect of D3T in EAE might be attributed to its effects on modulating dendritic cell and microglia activation and pathogenic Th1/Th17 cell differentiation.

3H-1,2-Dithiole-3-thione as a novel therapeutic agent for the treatment of ischemic stroke through Nrf2 defense pathway

Cerebral ischemic stroke accounts for more than 80% of all stroke cases. During cerebral ischemia, reactive oxygen species produced in brain tissue induce oxidative stress and inflammatory responses. D3T, the simplest compound of the cyclic, sulfur-containing dithiolethiones, is found in cruciferous vegetables and has been reported to induce antioxidant genes and glutathione biosynthesis through activation of Nrf2. In addition to antioxidant activity, D3T was also reported to possess anti-inflammatory effects. In this study, we evaluated the therapeutic potential of D3T for the treatment of ischemic stroke and investigated the mechanisms underlying the protective effects of D3T in ischemic stroke. Mice subjected to transient middle cerebral artery occlusion/reperfusion (tMCAO/R) were administered with vehicle or D3T to evaluate the effect of D3T in cerebral brain injury. We observed D3T reduced infarct size, decreased brain edema, lessened blood-brain barrier disruption, and ameliorated neurological deficits. Further investigation revealed D3T suppressed microglia (MG) activation and inhibited peripheral inflammatory immune cell infiltration of CNS in the ischemic brain. The protective effect of D3T in ischemic stroke is mediated through Nrf2 induction as D3T-attenuated brain injury was abolished in Nrf2 deficient mice subjected to tMCAO/R. In addition, in vitro results indicate the induction of Nrf2 by D3T is required for its suppressive effect on MG activation and cytokine production. In summary, we demonstrate for the first time that D3T confers protection against ischemic stroke, which is mediated through suppression of MG activation and inhibition of CNS peripheral cell infiltration, and that the protective effect of D3T in ischemic stroke is dependent on the activation of Nrf2.

Interaction of Staphylococcus epidermidis with endothelial cells in vitro

Abstract Staphylococcus epidermidis is a leading cause of nosocomial bacteremia, yet virtually nothing is known about how this pathogen interacts with human endothelial cells. We present evidence here that two biofilm-producing strains of S. epidermidis adhere to two types of endothelial cell lines in vitro and that adherence is significantly increased after briefly heat-treating the bacteria at 40°C in the presence of calcium. This mild heat treatment resulted in bacteria that were 5 to more than 20 times more adherent than untreated controls. While the adherence of bacteria in all phases of growth was increased after heat treatment, heat-treated late stationary phase cells were generally the most adherent. Electron microscopy demonstrated that S. epidermidis was internalized and appeared to exist free in the cytoplasm. Adherence to endothelium, should it occur in vivo during bacteremia, may be a virulence factor associated with this bacteriums pathogenesis.

Characterization of a monoclonal antibody that binds to an epitope on soluble bacterial peptidoglycan fragments.

ABSTRACT We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from Staphylococcus aureus and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.

Dithiolethione ACDT suppresses neuroinflammation and ameliorates disease severity in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disorder characterized by the central nervous system (CNS) infiltration of myelin-specific pathogenic T cells followed by brain inflammation in association with demyelination. Similarly, experimental autoimmune encephalomyelitis (EAE), the animal model of MS, also exhibits increased CNS infiltration of pathogenic T cells, including Th1 and Th17, leading to detrimental effects of neuroinflammation and demyelination. We previously reported that 3H-1,2-dithiole-3-thione (D3T), the structurally-simplest of the sulfur-containing dithiolethiones, exerted a promising therapeutic effect in EAE. In the current study we report that 5-Amino-3-thioxo-3H-(1,2)dithiole-4-carboxylic acid ethyl ester (ACDT), a substituted derivative of D3T, exhibits anti-inflammatory properties in EAE. ACDT, administered post immunization, delayed disease onset and reduced disease severity in chronic C57BL/6 EAE, and ACDT, administered during disease remission, suppressed disease relapse in relapsing-remitting SJL/J EAE. Further analysis of the cellular and molecular mechanisms underlying the protective effects of ACDT in EAE revealed that ACDT inhibited pathogenic T cell infiltration, suppressed microglia activation, repressed neurotoxic A1 astrocyte generation, lessened blood-brain barrier disruption, and diminished MMP3/9 production in the CNS of EAE. In summary, we demonstrate that ACDT suppresses neuroinflammation and ameliorates disease severity in EAE through multiple cellular mechanisms. Our findings suggest the potential of developing ACDT as a novel therapeutic agent for the treatment of MS/EAE.