Ultrasound, microbubbles and the blood-brain barrier

The blood–brain barrier (BBB) is a specialized system of capillary endothelial cells that protects the brain from harmful substances in the blood stream, while supplying the brain with the required nutrients for proper function. The BBB controls transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle for delivery of both small molecules and macromolecular agents. Although many drugs could be potentially used to treat brain disease, there has been no method that allows non-invasive-targeted delivery through the BBB. Recently, promising studies indicate that ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. If microbubbles are combined with ultrasound exposure, the effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity needed to produce BBB opening. Several avenues of transcapillary passage after ultrasound sonication have been identified including transcytosis, passage through endothelial cell cytoplasmic openings, opening of tight junctions and free passage through injured endothelium. This article reviews the topic of transient disruption of the BBB with ultrasound and microbubbles and addresses related safety issues. It also discusses possible roles of the BBB in brain disease and potential interactions with ultrasound and microbubbles in such disease states.     

Kinetochore orientation during meiosis is controlled by Aurora B and the monopolin complex

Kinetochores of sister chromatids attach to microtubules emanating from the same pole (coorientation) during meiosis I and microtubules emanating from opposite poles (biorientation) during meiosis II. We find that the Aurora B kinase Ipl1 regulates kinetochore-microtubule attachment during both meiotic divisions and that a complex known as the monopolin complex ensures that the protein kinase coorients sister chromatids during meiosis I. Furthermore, the defining of conditions sufficient to induce sister kinetochore coorientation during mitosis provides insight into monopolin complex function. The monopolin complex joins sister kinetochores independently of cohesins, the proteins that hold sister chromatids together. We propose that this function of the monopolin complex helps Aurora B coorient sister chromatids during meiosis I.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPalpha, espPbeta, espPgamma, and espPdelta), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPbeta, espPgamma, and espPdelta genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPalpha, espPbeta, espPgamma, and espPdelta in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPalpha (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPgamma cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPbeta and EspPdelta either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

R-(+)-alpha-lipoic acid inhibits endothelial cell apoptosis and proliferation: involvement of Akt and retinoblastoma protein/E2F-1

Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.     

Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.     

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin

High-mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac-1-dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1-mediated recruitment was prevented in mice deficient in the beta2-integrin Mac-1 but not in those deficient in LFA-1. As observed by bone marrow chimera experiments, Mac-1-dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac-1 and RAGE. Consistently, HMGB1 activated Mac-1 as well as Mac-1-mediated adhesive and migratory functions of neutrophils in a RAGE-dependent manner. Moreover, HMGB1-induced activation of nuclear factor-kappaB in neutrophils required both Mac-1 and RAGE. Together, a novel HMGB1-dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac-1 and RAGE is described here.     

Increased tumor cell dissemination and cellular senescence in the absence of beta1-integrin function

Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization, as well as transduction of signals into cells, to promote various aspects of cellular behavior, such as proliferation or survival. Integrins participate in many aspects of tumor biology. Here, we have employed the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis to investigate the role of beta(1)-integrin in tumor progression. Specific ablation of beta(1)-integrin function in pancreatic beta cells resulted in a defect in sorting between insulin-expressing beta cells and glucagon-expressing alpha cells in islets of Langerhans. Ablation of beta(1)-integrin in beta tumor cells of Rip1Tag2 mice led to the dissemination of tumor cell emboli into lymphatic blood vessels in the absence of ongoing lymphangiogenesis. Yet, disseminating beta(1)-integrin-deficient beta tumor cells did not elicit metastasis. Rather, primary tumor growth was significantly impaired by reduced tumor cell proliferation and the acquisition of cellular senescence by beta(1)-integrin-deficient beta tumor cells. The results indicate a critical role of beta(1)-integrin function in mediating metastatic dissemination and preventing tumor cell senescence.     

Changing viral tropism using immunoliposomes alters the stability of gene expression: implications for viral vector design

Many strategies for redirecting the tropism of murine Moloney leukemia virus (MMLV) have been described. Preformed virion-liposome complexes, termed virosomes, have been reported to be relatively stable. Virosomes mediate envelope-independent transduction that allows efficient superinfection of resistant cell lines; however, virosome-mediated transduction behaves in a non-target-specific manner. We developed a novel method using antibodies to direct MMLV to vascular endothelium. We have given the term immunovirosomes to the complexes formed between viruses, liposomes, and antibodies. These immunovirosomes improve the transduction efficiency of the viruses and alter their tropism. We have shown improved transduction when immunovirosomes were targeted at the endocytic receptors CD71 and CD62E/P and rather less good delivery when targeted at CD106. The enhancement of the transduction efficiency was transient, however, suggesting that rerouting the entry pathway of viruses alters the expression properties of the viruses.