Patient compliance with an anticoagulation management system based on a smartphone application.

We developed a novel anticoagulation management system (Anticlot Assistant) based on a smartphone application (App). This study was performed to evaluate patient compliance with Anticlot Assistant. This prospective case series study involved patients receiving warfarin therapy. The eligible patients were managed via Anticlot Assistant, and outcome data were analyzed. Thirty patients were recruited. The mean time within the therapeutic range (TTR) was 56.5% ± 26.2% and the mean patient compliance with Anticlot Assistant was 52.7% ± 40.4%. The patients in good compliance group had higher TTR (65.6 ± 25.0% vs. 40.0 ± 21.0%, P = 0.009), lower time in the extremely low range (9.4 ± 10.6% vs. 27.4 ± 13.2%, P = 0.000) and in the extremely high range (1.3 ± 2.8% vs. 14.1 ± 22.3%, P = 0.004) than those in poor compliance group. Logistic regression analysis revealed that receiving an education of > 6 years was the only independent predictor of good compliance (odds ratio 8.400, 95% confidence interval 1.274-55.394, P = 0.027). Patient compliance is critical important for good outcomes and it might increase with improvements in education and more widespread use of information technology. Although further improvement is needed, Anticlot Assistant is promising and this study offered valuable experiences for further research.

Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H.

BACKGROUND: Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed. METHODS: We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay. RESULTS: Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody. CONCLUSIONS: This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg mL(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation <20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little cross-reactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

A Three-Monoclonal Antibody Combination Potently Neutralizes BoNT/G Toxin in Mice.

Equine-derived antitoxin (BAT®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with KD values ranging from 3.86 nM to 103 nM (median KD 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG KD values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD50s of BoNT/G at a total mAb dose of 6.25 µg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F.

Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT/F1, F3, F4, and F6 and with no detectable binding to BoNT/F2, F5, or F7. Using multicolor labeling of the different BoNT/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT/F subtypes with high affinity (KD 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants.

Endothelial progenitor cell transplantation improves long-term stroke outcome in mice.

OBJECTIVE: Endothelial progenitor cells (EPCs) play an important role in tissue repairing and regeneration in ischemic organs, including the brain. However, the cause of EPC migration and the function of EPCs after ischemia are unclear. In this study, we demonstrated the effects of EPCs on ischemic brain injury in a mouse model of transient middle cerebral artery occlusion (tMCAO). METHODS: Circulating human EPCs were characterized with immunofluorescent staining and flow cytometry. EPCs (1 x 10(6)) were injected into nude mice after 1 hour of tMCAO. Histological analysis and behavioral tests were performed from day 0 to 28 days after tMCAO. RESULTS: EPCs were detected in ischemic brain regions 24 hours after tMCAO. EPC transplantation significantly reduced ischemic infarct volume at 3 days after tMCAO compared with control animals (p < 0.05). CXCR4 was expressed in the majority of EPCs, and stromal-derived factor-1 (SDF-1) induced EPC migration, which was blocked by pretreated EPCs with AMD3100 in vitro. SDF-1 was upregulated in ischemic brain. Compared with control animals, injecting AMD3100-pretreated EPCs resulted in a larger infarct volume 3 days after tMCAO, suggesting that SDF-1-mediated signaling was involved in EPC-mediated neuroprotection. In addition, EPC transplantation reduced mouse cortex atrophy 4 weeks after tMCAO and improved neurobehavioral outcomes (p < 0.05). EPC injection potently increased angiogenesis in the peri-infarction area (p < 0.05). INTERPRETATION: We conclude that systemic delivery of EPCs protects the brain against ischemic injury, promotes neurovascular repair, and improves long-term neurobehavioral outcomes. Our data suggest that SDF-1-mediated signaling plays a critical role in EPC-mediated neuroprotection.

The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA.

BACKGROUND: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. METHODS: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. RESULTS: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. CONCLUSIONS: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Insulin growth factor-1 gene transfer enhances neurovascular remodeling and improves long-term stroke outcome in mice.

BACKGROUND AND PURPOSE: Insulin-like growth factor I (IGF-1) is a pleiotropic growth factor that has been demonstrated to protect against acute ischemic brain injury. Whether IGF-1 improves long-term functional outcome after ischemic stroke is not known. The aim of this study is to examine whether IGF-1 overexpression through adeno-associated virus (AAV) -mediated gene transfer enhances neurovascular remodeling and improves functional outcome in a mouse model of focal cerebral ischemia. METHODS: Long-term cerebral IGF-1 overexpression was achieved with the AAV transduction system through stereotaxic injection. Control mice were injected with AAV-green fluorescent protein or saline. Three weeks after gene transfer, the mice underwent permanent distal middle cerebral artery occlusion. Histological and behavioral analyses were performed at day 21 after middle cerebral artery occlusion. RESULTS: IGF-1 gene transfer compared with control treatment significantly improved motor performance assessed by sensorimotor tests. The functional recovery was accompanied by reduced volume of cerebral infarction. Immunohistochemical analysis with endothelial cell marker CD31 revealed that IGF-1 gene transfer potently increased neovessel formation in the periinfarct and injection needle tract area compared with AAV-green fluorescent protein transduction. Increased vascular density was associated with increased local vascular perfusion. Additionally, AAV-IGF-1 treatment enhanced neurogenesis in the subventricular zone compared with AAV-green fluorescent protein treatment. CONCLUSIONS: These data demonstrate that IGF-1 overexpression promoted long-lasting functional recovery after cerebral infarction. The improved functional performance was paralleled by enhanced neovascularization and neurogenesis.

Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro.

Circulating blood endothelial progenitor cells (EPCs) contribute to postnatal vasculogenesis, providing a novel therapeutic target for vascular diseases. However, the molecular mechanism of EPC-induced vasculogenesis is unknown. Interleukin-6 plays multiple functions in angiogenesis and vascular remodeling. Our previous study demonstrated that the polymorphism (174G>C) in IL-6 gene promoter was associated with brain vascular disease. In this study, we investigated if IL-6 receptor is expressed in human EPCs derived from circulating mononuclear cells, and if interleukin-6 (IL-6) stimulates EPC angiogenesis in vitro. First, we isolated and cultured mononuclear cells from adult human circulating blood. We obtained EPC clones that were further cultured and expended for the angiogenesis study. We found that the EPCs possessed human mature endothelial cell phenotypes; however, they proliferated much faster than mature endothelial cells (P<0.05). We then found that IL-6 receptor (gp-80) was expressed in the EPCs, and that administration of IL-6 could activate receptor gp80/gp130 signaling pathways including downstream extracellular signal-regulated kinase 1/2 and STAT3 phosphorylation in EPCs. Furthermore, IL-6 stimulated EPC proliferation, migration, and matrigel tube formation in a dose-dependent manner (P<0.05); anti-IL-6 antibodies or IL-6 receptor could abolish these effects (P<0.05). These results suggest that IL-6 plays a crucial role in the biologic behavior of blood-derived EPCs, which may help clarify the mechanism of IL-6 inflammatory-related diseases.

Del-1 gene transfer induces cerebral angiogenesis in mice.

Developmental endothelial locus-1 (Del-1) is a novel angiomatrix protein that has been shown to stimulate a potent angiogenic response and promote functional recovery in hind-limb and cardiac ischemia in animal models; however, its impact on cerebral angiogenesis is unknown. In this study, we investigated whether Del-1 overexpression via gene transfer induces cerebral angiogenesis in a murine model, and examined Del-1 expression after ischemic stroke. Cerebral Del-1 overexpression was achieved with AAV (adeno-associated virus) transduction system via stereotactic injection. Control mice were injected with AAV-lacZ. Del-1 gene transduction led to a significant induction of cerebral angiogenesis compared to AAV-lacZ treatment at 4 weeks after gene transfer (Del-1: 97+/-7 vs lacZ: 64+/-28, vessels/field, p<0.05). Mice transduced with AAV-Del-1 showed significantly elevated vascular densities for up to 6 weeks after gene delivery. In addition, double immunofluorescent staining showed co-localization of endothelial cell marker CD31 with BrdU (specific marker for proliferating cells), indicating that Del-1 promoted endogenous endothelial cell proliferation and angiogenesis. Our immunohistochemical staining also showed that Del-1 expression was markedly up-regulated in the peri-infarct area at 3 days after permanent focal cerebral ischemia compared to the sham-operated non-ischemic control. Our data suggest that Del-1 may participate in modulating cerebral angiogenesis, and that gene transfer of Del-1 may provide a novel and potent means for stimulating cerebral angiogenesis.

Sexual reproduction in higher plants I: fertilization and the initiation of zygotic program.

Sexual plant reproduction is a critical developmental step in the life cycle of higher plants, to allow maternal and paternal genes to be transmitted in a highly regulated manner to the next generation. During evolution, a whole set of signal transduction machinery is developed by plants to ensure an error-free recognition between male and female gametes and initiation of zygotic program. In the past few years, the molecular machineries underlying this biological process have been elucidated, particularly on the importance of synergid cells in pollen tube guidance, the Ca(++) spike as the immediate response of fertilization and the epigenetic regulation of parental gene expressions in early zygotic embryogenesis. This review outlines the most recent development in this area.

Interleukin-6 upregulates expression of KDR and stimulates proliferation of human cerebrovascular smooth muscle cells.

Interleukin-6 (IL-6) may play multiple roles in angiogenesis and vascular remodeling. Our previous study showed that a promoter polymorphism (174G>C) in IL-6 is associated with brain arteriovenous malformation hemorrhage; tissue expression is related to genotype. In this study, we investigated the effects of IL-6 on human cerebral smooth muscle cells (HCSMCs) and smooth muscle cells isolated from brain arteriovenous malformation surgical specimens (AVM SMCs) and surgical controls (control HCSMCs--from structurally normal temporal lobe taken during surgical treatment of epilepsy patients). We found that IL-6 (1.1+/-0.27 versus 0.37+/-0.04 pg/mL, n=5, P<0.05) and endogenous vascular endothelial growth factor (VEGF) receptor II (kinase domain-containing receptor (KDR), 15+/-3 versus 1.5+/-3 pg/mL, n=5, P<0.05) were increased in brain AVM SMCs compared with control HCSMCs. Further research revealed that IL-6 could stimulate SMC proliferation, VEGF release, and KDR activation in control HCSMCs. It could also stimulate KDR phosphorylation in control HCSMCs, further confirming a unique role of IL-6 in the triggering of KDR. Interleukin-6 could increase matrix metalloproteinase-9 (MMP-9) secretion through activating KDR in control HCSMCs (P<0.05 versus control). Inhibiting IL-6-induced KDR could reduce MMP-9 activity at least 50% compared with the control group (P<0.05). Increased MMP-9 activity was accompanied by increased control HCSMC proliferation, and blocking MMP-9 activity significantly reduced IL-6-induced control HCSMC proliferation (P<0.05). Collectively, our results show that IL-6 could activate, amplify, and maintain the angiogenic cascade in HCSMCs. A novel role of IL-6 during HCSMC proliferation is upregulating KDR expression and phosphorylation. The results may contribute to the angiogenic phenotype of human brain vascular diseases, such as brain AVM.

Neutrophil depletion decreases VEGF-induced focal angiogenesis in the mature mouse brain.

To explore the role of neutrophil-derived matrix metalloproteinases (MMPs) during angiogenesis in the brain, we hypothesized that transient neutrophil depletion attenuates the angiogenic response to focal hyperstimulation with vascular endothelial growth factor (VEGF). Brain focal angiogenesis was achieved using an adeno-associated virus delivered VEGF (AAV-VEGF) gene transfer in the mature mouse. Four groups of mice underwent AAV vector injection in the brain parenchyma: (1) AAV-LacZ; (2) AAV-VEGF; (3) AAV-VEGF plus anti-polymorphonuclear (PMN) antibody; and (4) AAV-VEGF plus serum. Animals in groups 3 and 4 underwent 4 days of PMN antibody or serum treatment before transfection; treatment was sustained for an additional 14 days. Anti-PMN treatment decreased circulating neutrophils to 9% of baseline (P<0.001). Microvessels in the AAV-VEGF-group increased 25% compared with the AAV-lacZ-transduced group (256+/-15 versus 208+/-16; P<0.05). Anti-PMN treatment attenuated the increase to 10% compared with control serum treatment (234+/-16 versus 255+/-22; P<0.05). Similarly, compared with control serum treatment, anti-PMN treatment also reduced MMP-9 by 50% (2+/-0.9 versus 4+/-1.4; P<0.05) and MPO expression by 25% (2+/-0.8 versus 3+/-0.9; P<0.05); MMP-9 activity correlated with MPO expression (R(2)=0.8, P<0.05). Our study demonstrated that transient depletion of neutrophils suppressed VEGF-induced angiogenesis, indicating that circulating neutrophils contribute to VEGF-induced focal angiogenesis. In addition, brain MMP-9 activity was attenuated after neutrophil depletion, suggesting that neutrophil is an important source of MMP-9.

Pretreatment with PTD-calbindin D 28k alleviates rat brain injury induced by ischemia and reperfusion.

Calcium toxicity remains the central focus of ischemic brain injury. Calcium channel antagonists have been reported to be neuroprotective in ischemic animal models but have failed in clinical trials. Rather than block the calcium channels, calbindin proteins can buffer excessive intracellular Ca2+, and as a result, maintain the calcium homeostasis. In the present study, we investigated the effect of calbindin D 28k (CaBD) in ischemic brain using the novel technique protein transduction domain (PTD)-mediated protein transduction. We generated PTD-CaBD in Escherichia coli, tested its biologic activity in N-methyl-D-aspartate (NMDA)- and oxygen-glucose deprivation (OGD)-induced hippocampal injury models, and examined the protection of the fusion protein using a rat brain focal ischemia model. Infarct volume was determined using 2,3,5-triphenyl-tetrazolium chloride staining; neuronal injury was examined using terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining and cleaved caspase-3 assay. The results showed that the PTD-CaBD was efficiently delivered into Cos7 cells, hippocampal slice cells, and brain tissue. Pretreatment with PTD-CaBD decreased intracellular free calcium concentration and reduced cell death in NMDA- or OGD-exposed hippocampal slices (P<0.05). Intraperitoneal administration of PTD-CaBD before transient middle cerebral artery occlusion decreased brain infarct volume (280+/-47 versus 166+/-70 mm3, P<0.05), and improved neurologic outcomes compared with the control. Further studies showed that, compared with the control animals, PTD-CaBD decreased TUNEL (58%+/-7% versus 29%+/-3%, P<0.05)- and cleaved caspase-3 (62+/-4/field versus 31+/-6/field, P<0.05)-positive cells in the ischemic boundary zone. These results indicate that systemic administration of PTD-CaBD could attenuate ischemic brain injury, suggesting that PTD-mediated protein transduction might provide a promising and effective approach for the therapies of brain diseases, including cerebral ischemia.

A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes.

Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10-9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10-8 M to 1.47×10-12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

Adeno-associated viral-vector-mediated hypoxia-inducible vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Exogenous delivery of vascular endothelial growth factor gene (VEGF) may provide a useful approach to the treatment of brain ischemia. We investigated the use of a hypoxia-responsive element to control VEGF expression given for neuroprotection. METHODS: Three groups (n=36) of mice received AAVH9-VEGF, AAVH9-lacZ, or saline injection. Five days after gene transfer, the mice underwent 45 minutes of transient middle cerebral artery occlusion (tMCAO) followed by 1 to 7 days of reperfusion. Infarct volume was determined using cresyl violet staining; neuronal injury was examined using TUNEL, cleaved caspase-3, and fluoro-Jade B staining. RESULTS: Hypoxia-inducible factor-1 (HIF-1) was overexpressed after tMCAO in the ischemic hemisphere in the brain. Expression of lacZ, mediated by AAV-lacZ, was seen before and after tMCAO; however, AAVH9-lacZ-mediated lacZ expression was detected only after tMCAO. Infarct volume was smaller in the AAVH9-VEGF-transduced group compared with AAVH9-lacZ and saline groups (55% reduction, P<0.05) with reduced TUNEL (29+/-5% and 30+/-7% versus 12+/-3%, P<0.05), cleaved caspase-3 (20+/-3% and 21+/-5% versus 13+/-4%, P<0.05) and fluoro-Jade B (23+/-3% and 24+/-5% versus 12+/-5%, P<0.05) -positive neurons, respectively. CONCLUSIONS: Exogenous expression of VEGF through AAVH9-VEGF gene transfer 5 days before the onset of ischemia provides neuroprotection. Hypoxia-responsive element is a viable strategy of restricting VEGF expression to areas of ischemia to minimize adverse effects of therapy on adjacent normal parenchyma.

MMP-9 expression is associated with leukocytic but not endothelial markers in brain arteriovenous malformations.

Brain arteriovenous malformations (BAVM) have high matrix metalloproteinase-9 (MMP-9) expression, the source of which is unclear. We hypothesized MMP-9 production might be due to inflammation in BAVM. Compared to control brain tissues (n = 5), BAVM tissue (n = 139) had a higher expression (by ELISA) of myeloperoxidase (MPO) (193 +/- 189 vs. 6 +/- 3, ng/mg, P < .001), MMP-9 (28 +/- 32 vs. 0.7 +/- 0.6, ng/mg, P < .001), and IL-6 (102 +/- 218 vs. 0.1 +/- 0.1, pg/mg, P < .001), but not eNOS (114 +/- 87 vs. 65 +/- 9, pg/mg, P = .09). MMP-9 expression in BAVM highly correlated with myeloperoxidase (R2 = .76, P < .001), as well as with IL-6 (R2 = .32, P < .001). In contrast, MMP-9 in BAVM poorly correlated with the endothelial marker, eNOS (R2 = .03, P = .05), and CD31 (R2 = .004, P = .57). Compared to non-embolized patients (n = 46), patients with pre-operative embolization (n = 93) had higher levels of myeloperoxidase (236 +/- 205 vs. 106 +/- 108, ng/mg, P < .001) and MMP-9 (33 +/- 35 vs. 16 +/- 20, ng/mg, P < .001), however the correlation between MMP-9 and myeloperoxidase was equally strong for both groups (R2 = .69, n = 93, P < .001, for both). MMP-9 expression correlated with the lipocalin-MMP-9 complex, suggesting neutrophils as the MMP-9 source. MPO co-localized with majority of MMP-9 signal by immunohistochemistry. Our data suggest that inflammation is a prominent feature of BAVM lesional phenotype, and neutrophils appear to be a major source of MMP-9 in these lesions.

Apoptosis inhibition in ischemic brain by intraperitoneal PTD-BIR3-RING (XIAP).

Anti-apoptotic treatment is a promising strategy for neuroprotection against various brain injuries resulting from ischemia or neuron degeneration. X-linked inhibitor of apoptosis protein (XIAP) is regarded as the most effective apoptosis inhibitor, in which C-terminal structure BIR3-RING mainly inhibits caspase-9-dependent apoptosis. In the present study, we fused XIAP (BIR3-RING) to the protein transduction domain (PTD) of antennapedia homeodomain of Drosophila (Antp HD), and then used the oxygen glucose deprivation (OGD)-induced hippocampal slices injury in vitro, and the rat transient middle cerebral artery ischemia (tMCAO) models in vivo, to explore the anti-apoptotic effect of this recombinant protein. The results showed that the PTD could efficiently mediate the transduction of BIR3-RING into the hippocampal slices and rat brains. PTD-BIR3-RING could decrease OGD-induced cell death in brain slices (p < 0.05). Intraperitoneal injection of PTD-BIR3-RING could attenuate terminal deoynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive cells and decrease cleaved caspase-3 in the ischemic bounder zone compared with the control animals (p < 0.05). Further studies showed that ischemia-induced neurological outcomes were improved in rats with PTD-BIR3-RING treatment (p < 0.05). These results demonstrate that PTD-BIR3-RING could attenuate cell death in OGD hippocampal slices and decrease cell apoptosis in tMCAO brains through inhibiting of caspase-3 cleavage, suggesting that PTD-mediated protein transduction provides a novel and effective approach for the therapies of brain diseases such as cerebral ischemia.

Delayed protective effect of telmisartan on lung ischemia/reperfusion injury in valve replacement operations.

The present study aimed to investigate the delayed protective effect of telmisartan on lung ischemic/reperfusion injury in patients undergoing heart valve replacement operations. In total, 180 patients diagnosed with rheumatic valve diseases were randomly divided into the telmisartan (T), captopril (C) and placebo (P) groups. In the telmisartan group, the patients were pretreated with telmisartan (1 mg/kg/day), at the time period 96-48 h before the operation, whereas in the C group, the patients were treated with captopril (1 mg/kg/day) at the time period 96-48 h prior to the operation control group. Each drug treatment group included a corresponding placebo treatment. The variables pulmonary vascular resistance (PVR) and A-aDO2 were measured prior to CPB and at 1, 3, 6 and 12 h after CPB. Pulmonary neutrophil (PMN) count in the left and right atrium blood as well as SOD malondialdehyde (MDA), NO, angiotensin II (AngII) value in the left atrium blood, were measured 30 min prior to and after CPB. The PVR parameters of the telmisartan and captopril groups were significantly lower than those of the placebo group (P<0.05). The A-aDO2 values in the telmisartan and captopril groups were significantly lower than those in the placebo group at 1, 3 and 6 h following CPB treatment. The difference between the right and left atrium blood PMN was significantly lower in the telmisartan and captopril intervention groups compared to that in the placebo group 30 min following CPB treatment. The left atrium blood SOD and NO values were significantly higher, whereas the MDA value was significantly lower in the telmisartan group compared to the control group 30 min following CPB treatment. As for AngII, there was no difference between the C and T groups, compared with the P group. In the two groups 30 min after treatment with CPB, 24 patients experienced varying degrees of cough, with the telmisartan group showing a significant difference (P<0.05). The hospitalization time was compared in the three groups of patients and it was found to be significantly shorter in the telmisartan group than the captopril and placebo groups (P<0.05). In conclusion, it was found that for the time period 96-48 h before heart valve replacement operations telmisartan (1 mg/kg/day) delayed the protective effect on lung ischemia/reperfusion injury in patients with rheumatic valve diseases. The results of the present study indicated that the protective effect may be associated with the increment of endogenetic NO and the enhanced ability against lipid peroxidation.

[Analysis of CCM1 gene mutations in Chinese patients with intracranial cavernous malformations].

OBJECTIVE: To study the CCM1 gene (7q11.2 - q22) mutations in Chinese patients with intracranial cavernous malformations (ICM), METHODS: Peripheral blood samples were collected from 25 unrelated patients with ICM confirmed by post-operational pathology, 7 being with familial ICM, all of Han nationality, and from 30 healthy people as controls. The genomic DNA was extracted and the exons 8, 9, 11, 12, 13, 15, 16, 17, and 18 of CCM1 gene and part of intervening sequences near both sides of these exons were amplified by PCR. The PCR products were sequenced directly and then compared with the GenBank data. RESULTS: Seven new mutation sites of CCM1 gene were detected from 11 Chinese ICM patients with a total mutation rate of 44%. Of the seven new mutations there were three missense mutations: 1160A-->C (Q387P) and 1172C-->T (S391F) in exon12, and 1405A-->C (N469H) in exon13; two insertion mutations: 704insT (K246stop) in exon8, and 2138insG (T733stop) in exon18; one intervening sequence mutation: IVS12 - 4C-->T; and one synonymous mutation: 1875C-->T (F625F) in exon17. None mutation was detected in the control group. The CCM1 mutation rate of familial ICM was 85.7%, significantly higher than that of sporadic ICM (27.7%, P < 0.05). CONCLUSION: As the genetic basis of ICM, CCM1 gene mutation exists in Chinese ICM patients too, that leads to functional loss or changes of the gene encoding KRIT1 protein.