Innate immune cells express toll-like receptor-9 (TLR9) and respond to unmethylated, CG dinucleotide motif-rich DNA released from bacteria during infection or endogenous cells during autoimmune tissue injury. Oligonucleotides containing CG dinucleotide (CpG-DNA) mimic the effect of unmethylated DNA and stimulate TLR9. CpG-DNA was cytotoxic to neurons in organotypic brain cultures. Neurotoxicity of CpG-DNA was mediated via microglial cells and started primarily from neurites as determined by time-lapse imaging of enhanced green fluorescent protein (EGFP)-transfected neurons. Cultured brain microglial cells expressed TLR9 and responded to CpG-DNA by production of the inflammatory mediators nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). Blockade of NO synthase and TNF-alpha prevented damage of neurites and neurotoxicity of CpG-DNA. The data suggest that stimulation of microglia via TLR9 and subsequent release of NO and TNF-alpha is a major source of neurotoxicity in bacterial and autoimmune brain tissue injury.

The Streptococcus pneumoniae toxin pneumolysin belongs to the group of cholesterol-dependent cytolysins. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. In cell membranes, it forms prepores and pores. Here, we show that sublytic concentrations of pneumolysin produce rapid activation of Rho and Rac GTPases and formation of actin stress fibers, filopodia, and lamellipodia. That Rac1-specific and Rho-associated kinase ( cholesterol-binding cytolysin ͉ small GTPase

The interaction of endogenous and exogenous stimulators of innate immunity was examined in primary cultures of mouse microglial cells and macrophages after application of defined Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) (TLR4), the synthetic lipopeptide Pam3Cys-Ser-Lys4 (Pam3-Cys) (TLR2) and single-stranded unmethylated CpG-DNA (CpG) (TLR9)] alone and in combination with amyloid beta peptide (Abeta) 1-40. Abeta 1-40 stimulated microglial cells and macrophages primed by interferon-c in a dose-dependent manner. Co-administration of Abeta1-40 with LPS or Pam3-Cys led to an additive release of nitric oxide (NO) and tumour necrosis factor alpha (TNF-a). This may be one reason for the clinical deterioration frequently observed in patients with Alzheimer's disease during infections. In contrast, co-application of Abeta1-40 with CpG led to a substantial decrease of NO and TNF-a release compared with stimulation with CpG alone. Abeta 1-40 and CpG did not co-localize within the same subcellular compartment, making a direct physicochemical interaction as the cause of the observed antagonism very unlikely. This suggests that not all TLR agonists enhance the stimulatory effect of Abeta on innate immunity.

The mechanism of axonal injury in inflammatory brain diseases is still unclear. Increased microglial production of nitric oxide (NO) is a common early sign in neuroinflammatory diseases. We found by fluorescence correlation spectroscopy that synaptophysin tagged with enhanced green fluorescence protein (synaptophysin-EGFP) moves anterogradely in axons of cultured neurons. Activated microglia focally inhibited the axonal movement of synaptophysin-EGFP in a NO synthase-dependent manner. Direct application of a NO donor to neurons resulted in inhibition of axonal transport of synaptophysin-EGFP and synaptotagmin I tagged with EGFP, mediated via phosphorylation of c-jun NH(2)-terminal kinase (JNK). Thus, overt production of reactive NO by activated microglia blocks the axonal transport of synaptic vesicle precursors via phosphorylation of JNK and could cause axonal and synaptic dysfunction.

Astrocytes represent a major component of brain tissue and play a critical role in the proper functioning and protection of the brain. Streptococcus pneumoniae, the most common cause of bacterial meningitis, has a high lethality and causes serious disabilities in survivors. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin group and a major S. pneumoniae neurotoxin, causes deterioration over the course of experimental S. pneumoniae meningitis. At disease-relevant sub-lytic concentrations, PLY produces actin and tubulin reorganization and astrocyte cell shape changes in vitro. In this article, we show that sub-lytic amounts of PLY remodel brain tissue and produce astrocytic process retraction, cortical astroglial reorganization and increased interstitial fluid retention, which is manifested as tissue edema. These changes caused increased tissue permeability to macromolecules and bacteria. The pore-forming capacity of PLY remained necessary for these changes because none of the nonpore-forming mutants were capable of producing similar effects. We suggest that PLY can increase the permeability of brain tissue toward pathogenic factors and bacteria in the course of meningitis, thus contributing to the deterioration caused by the disease.

Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.

Background: The palmaris longus muscle is one of the most variable muscles in the human body and there have been numerous variations reported. The diff erent palmaris longus variations are interesting not only from an anatomical point of view, but they could also have defi nite clinical signifi cance. Aim: The aim of this study was to examine the diff erent types of variations of palmaris longus muscle in the Bulgarian population. Materials and methods: Over a period of 15 years, 56 formol-carbol fixed human cadavers were studied to investigate the diff erent variations of palmaris longus muscle (PLM). Results: Various anatomical variations of PLM have been reported: absence (2.68%); reversed palmaris longus coexisting with an additional abductor digiti minimi muscle (0.89%); digastric (0.89%); palmaris longus with intermediate muscle belly (1.79%) and duplication (1.79%). Conclusions: To reveal the wide variety of the types of palmaris longus muscle and their importance for clinical practice, we make a brief literature review concerning the diff erent types of variations, their role in the median and ulnar neuropathy in the wrist or as structures simulating a soft tissue tumour and the application of palmaris longus tendon in plastic and reconstructive surgery as grafting material. We also present new systematic anatomical and clinical classifi cations of palmaris longus variations by dividing them into two simple groups. BACKGROUND

AIMTo examine the normal morphology of the epiligament tissue of the knee medial collateral ligament (MCL) in humans.METHODSSeveral samples of the mid-substance of the MCL of the knee joint from 7 fresh human cadavers (3 females and 4 males) were taken. Examination of the epiligament tissue was conducted by light microscopy and photomicrography on semi-thin sections of formalin fixed paraffin-embedded blocks that were routinely stained with haematoxylin and eosin, Mallory stain and Van Gieson’s stain. Electron microscopy of the epiligament tissue was performed on ultra-thin sections incubated in 1% osmium tetroxide and contrasted with 2.5% uranyl acetate, lead nitrate, and sodium citrate.RESULTSThe current light microscopic study demonstrated that the epiligament of the MCL consisted of fibroblasts, fibrocytes, adipocytes, neuro-vascular bundles and numerous multidirectional collagen fibers. In contrast, the ligament body was poorly vascularised, composed of hypo-cellular fascicles which were formed of longitudinal groups of collagen fibers. Moreover, most of the vessels of the epiligament-ligament complex were situated in the epiligament tissue. The electron microscopic study revealed fibroblasts with various shapes in the epiligament substance. All of them had the ultrastructural characteristics of active cells with large nuclei, well developed rough endoplasmic reticulum, multiple ribosomes, poorly developed Golgi apparatus, elliptical mitochondria and oval lysosomes. The electron microscopy also confirmed the presence of adipocytes, mast cells, myelinated and unmyelinated nerve fibers and chaotically oriented collagen fibers.CONCLUSIONSignificant differences exist between the normal structure of the ligament and the epiligament whose morphology and function is to be studied further.