Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE)

The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.     

Phosphorylation of claudin-5 and occludin by rho kinase in brain endothelial cells

Critical to the proper maintenance of blood-brain-barrier (BBB) integrity are the endothelial tight junctions (TJs). Posttranslational modifications of essential endothelial TJ proteins, occludin and claudin-5, contribute and possibly disrupt BBB integrity. Our previous work has shown that Rho kinase (RhoK) activation mediates occludin and claudin-5 phosphorylation resulting in diminished barrier tightness and enhanced monocyte migration across BBB in the setting of human immunodeficiency virus-1 encephalitis (HIVE). To determine whether RhoK can directly phosphorylate TJ proteins, we examined phosphorylation of cytoplasmic domains of recombinant claudin-5 and occludin by RhoK. We found that RhoK predominately phosphorylated two sites on occludin (T382 and S507) and one site on claudin-5 (T207). Specific anti-phosphopeptide antibodies were developed for these sites, allowing the detection of phosphorylated occludin at T382 and S507, and claudin-5 at T207 from full-length recombinant occludin and claudin-5 transiently expressed in COS-7 cells and mouse brain microvascular endothelial cells. Finally, these phosphospecific antibodies demonstrated enhanced staining of brain endothelial cells in the mouse model for HIVE and human HIVE brains featuring mononuclear cell infiltration across disrupted BBB. Our results demonstrated the direct phosphorylation of occludin and claudin-5 by RhoK at specific sites, which was increased in encephalitic brain tissue. These antibodies could be useful reagents for monitoring BBB dysfunction in vivo.     

Crohn’s and Parkinson’s Disease-Associated LRRK2 Mutations Alter Type II Interferon Responses in Human CD14+ Blood Monocytes Ex Vivo

Journal of Neuroimmune Pharmacology - The Leucine Rich Repeat Kinase 2 (LRRK2) is one of causative genes of familial Parkinson’s disease (PD). The M2397T polymorphism in LRRK2 is genetically...     

Clinical trial of in-situ hybridization method for the rapid diagnosis of sepsis

We evaluated the utility of in-situ hybridization (ISH) for the rapid diagnosis of sepsis. We applied this approach to polymorphonuclear neutrophil (PMN)-rich smears from patients with suspected bacterial infection. Positive results by ISH were obtained in the smears of 123 of 292 patients (42%), while only 32 of the 292 (11%) were positive by blood culture. These findings indicate that ISH is almost four times more sensitive than the culture method for the detection of sepsis. ISH results are obtained within 1 day, while 1 day to 2 weeks is required for the results of blood culture. Blood culture and ISH methods detected the same bacteria in two patients. ISH also successfully identified the same bacteria in blood and PMN-rich body fluid (bronchoalveolar lavage samples) in 6 patients. In 19 patients, ISH of blood detected the same bacteria as those found in subcultures from other sources (e.g., stool, sputum, nasal cavity). We discuss these results in comparison with blood culture results in terms of evaluating a rapid approach to the management of patients with sepsis. Received: February 9, 1998 / Accepted: September 14, 1998     

Caveolin-3 upregulation activates beta-secretase-mediated cleavage of the amyloid precursor protein in Alzheimer's disease.

Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD. In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro. Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated beta-secretase-mediated processing of APP. Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes. Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes. In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites.