Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells

<p>Abstract</p> <p>Background</p> <p><it>Cryptococcus neoformans </it>has a predilection for central nervous system infection. <it>C. neoformans </it>traversal of the blood brain barrier, composed of human brain microvascular endothelial cells (HBMEC), is the crucial step in brain infection. However, the molecular mechanism of the interaction between <it>Cryptococcus neoformans </it>and HBMEC, relevant to its brain invasion, is still largely unknown.</p> <p>Methods</p> <p>In this report, we explored several cellular and molecular events involving the membrane lipid rafts and caveolin-1 (Cav1) of HBMEC during <it>C. neoformans </it>infection. Immunofluorescence microscopy was used to examine the roles of Cav1. The knockdown of Cav1 by the siRNA treatment was performed. Phosphorylation of Cav1 relevant to its invasion functions was investigated.</p> <p>Results</p> <p>We found that the host receptor CD44 colocalized with Cav1 on the plasma membrane, and knockdown of Cav1 significantly reduced the fungal ability to invade HBMEC. Although the CD44 molecules were still present, HBMEC membrane organization was distorted by Cav1 knockdown. Concomitantly, knockdown of Cav1 significantly reduced the fungal crossing of the HBMEC monolayer <it>in vitro</it>. Upon <it>C. neoformans </it>engagement, host Cav1 was phosphorylated in a CD44-dependent manner. This phosphorylation was diminished by filipin, a disrupter of lipid raft structure. Furthermore, the phosphorylated Cav1 at the lipid raft migrated inward to the perinuclear localization. Interestingly, the phospho-Cav1 formed a thread-like structure and colocalized with actin filaments but not with the microtubule network.</p> <p>Conclusion</p> <p>These data support that <it>C. neoformans </it>internalization into HBMEC is a lipid raft/caveolae-dependent endocytic process where the actin cytoskeleton is involved, and the Cav1 plays an essential role in <it>C. neoformans </it>traversal of the blood-brain barrier.</p

A randomized controlled trial to prevent glycemic relapse in longitudinal diabetes care: Study protocol (NCT00362193)

BACKGROUND: Diabetes is a common disease with self-management a key aspect of care. Large prospective trials have shown that maintaining glycated hemoglobin less than 7% greatly reduces complications but translating this level of control into everyday clinical practice can be difficult. Intensive improvement programs are successful in attaining control in patients with type 2 diabetes, however, many patients experience glycemic relapse once returned to routine care. This early relapse is, in part, due to decreased adherence in self-management behaviors. OBJECTIVE: This paper describes the design of the Glycemic Relapse Prevention study. The purpose of this study is to determine the optimal frequency of maintenance intervention needed to prevent glycemic relapse. The primary endpoint is glycemic relapse, which is defined as glycated hemoglobin greater than 8% and an increase of 1% from baseline. METHODS: The intervention consists of telephonic contact by a nurse practitioner with a referral to a dietitian if indicated. This intervention was designed to provide early identification of self-care problems, understanding the rationale behind the self-care lapse and problem solve to find a negotiated solution. A total of 164 patients were randomized to routine care (least intensive), routine care with phone contact every three months (moderate intensity) or routine care with phone contact every month (most intensive). CONCLUSION: The baseline patient characteristics are similar across the treatment arms. Intervention fidelity analysis showed excellent reproducibility. This study will provide insight into the important but poorly understood area of glycemic relapse prevention

Mouse Mammary Tumor Virus Carrying a Bacterial supFGene Has Wild-Type Pathogenicity and Enables Rapid Isolation of Proviral Integration Sites

Mouse mammary tumor virus (MMTV) has frequently been used as an insertional mutagen to identify provirally activated mammary proto-oncogenes. To expedite and facilitate the process of cloning MMTV insertion sites, we have introduced a bacterial supF suppressor tRNA gene into the long terminal repeat (LTR) of MMTV, thus allowing selection of clones containing it in lambda vectors bearing amber mutations. The presence of supF in the LTR should circumvent the screening process for proviral insertion sites, since only those lambda clones with supF-containing proviral-cellular junction fragments should be able to form plaques on a lawn of wild-type Escherichia coli (i.e., lacking supF). The resulting virus (MMTVsupF) induced mammary tumors at the expected rate in infected mice, deleted the appropriate T-cell population by virtue of its superantigen gene, and stably retained the supF gene after passage via the milk to female offspring. To test the selective function of the system, size-selected DNA containing two proviral-cellular junction fragments from an MMTV supF-induced mammary tumor was ligated into λgtWES.λB, packaged, and plated on a supF-deficient bacterial host for selection of supF-containing clones. All plaques tested contained the desired cloned fragments, thus demonstrating the utility of this modified provirus for the rapid cloning of MMTV insertion sites