No abstract

Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection.

A method for isolating adult human bone marrow mesenchymal stem cells (MSCs) was established, and the ability of human MSCs to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated. Selected MSC surface antigens were analyzed by flow cytometry. The MSCs at Passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. Characteristics of the putative myogenic cells were determined by immunohistochemistry and transmission electron and confocal microscopies. The expression of myogenic specific genes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and DNA sequencing. The MSCs were spindle-shaped with irregular processes and were respectively positive for CD(13), CD(29), CD(44), CD(71) and negative for CD(3), CD(14), CD(15), CD(33), CD(34), CD(38), CD(45), and HLA-DR. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin, and alpha-cardiac actin. When the myogenic cells were stimulated with low concentration of K(+) (5.0 mM), an increase in intracellular calcium fluorescence was observed. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. The mRNAs of beta-MHC, desmin, alpha-cardiac actin, and cardiac troponin T were highly expressed in the myogenic cells. These results indicate that 5-azacytidine can induce human MSCs to differentiate in vitro into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocytes cultured from bone marrow sources are potentially valuable for repairing injured myocardium.

Rationale Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. Objective Here we addressed the role of platelets in mediating CNS inflammation in EAE. Results We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control non-diseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression upon platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ib alpha (GPIbα) promotes both platelet adhesion as well as inflammatory actions of platelets, and, targeting of GPIbα attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa) also reduced EAE severity in mice. Conclusions Thus, platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

To study the mechanisms involved in leukocyte recruitment induced by local bacterial infection within the CNS, we used intravital microscopy to visualize the interaction between leukocytes and the microvasculature in the brain. First, we showed that intracerebroventricular injection of LPS could cause significant rolling and adhesion of leukocytes in the brain postcapillary venules of wild-type mice, while negligible recruitment was observed in TLR4-deficient C57BL/10ScCr mice and CD14 knockout mice, suggesting recruitment is mediated by TLR4/CD14-bearing cells. Moreover, we observed reduced but not complete inhibition of recruitment in MyD88 knockout mice, indicating both MyD88-dependent and -independent pathways are involved. The leukocyte recruitment responses in chimeric mice with TLR4-positive microglia and endothelium, but TLR4-negative leukocytes, were comparable to normal wild-type mice, suggesting either endothelium or microglia play a crucial role in the induction of leukocyte recruitment. LPS injection induced both microglial and endothelial activation in the CNS. Furthermore, minocycline, an effective inhibitor of microglial activation, completely blocked the rolling and adhesion of leukocytes in the brain and blocked TNF-α production in response to LPS in vivo. Minocycline did not affect activation of endothelium by LPS in vitro. TNFR p55/p75 double knockout mice also exhibited significant reductions in both rolling and adhesion in response to LPS, indicating TNF-α signaling is critical for the leukocyte recruitment. Our results identify a TLR4 detection system within the blood-brain barrier. The microglia play the role of sentinel cells detecting LPS thereby inducing endothelial activation and leading to efficient leukocyte recruitment to the CNS.