Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Effectiveness of SARS-CoV-2 mRNA Vaccines for Preventing Covid-19 Hospitalizations in the United States.

Background: As SARS-CoV-2 vaccination coverage increases in the United States (US), there is a need to understand the real-world effectiveness against severe Covid-19 and among people at increased risk for poor outcomes. Methods: In a multicenter case-control analysis of US adults hospitalized March 11 - May 5, 2021, we evaluated vaccine effectiveness to prevent Covid-19 hospitalizations by comparing odds of prior vaccination with an mRNA vaccine (Pfizer-BioNTech or Moderna) between cases hospitalized with Covid-19 and hospital-based controls who tested negative for SARS-CoV-2. Results: Among 1210 participants, median age was 58 years, 22.8% were Black, 13.8% were Hispanic, and 20.6% had immunosuppression. SARS-CoV-2 lineage B.1.1.7 was most common variant (59.7% of sequenced viruses). Full vaccination (receipt of two vaccine doses ≥14 days before illness onset) had been received by 45/590 (7.6%) cases and 215/620 (34.7%) controls. Overall vaccine effectiveness was 86.9% (95% CI: 80.4 to 91.2%). Vaccine effectiveness was similar for Pfizer-BioNTech and Moderna vaccines, and highest in adults aged 18-49 years (97.3%; 95% CI: 78.9 to 99.7%). Among 45 patients with vaccine-breakthrough Covid hospitalizations, 44 (97.8%) were ≥50 years old and 20 (44.4%) had immunosuppression. Vaccine effectiveness was lower among patients with immunosuppression (59.2%; 95% CI: 11.9 to 81.1%) than without immunosuppression (91.3%; 95% CI: 85.5 to 94.7%). Conclusion: During March-May 2021, SARS-CoV-2 mRNA vaccines were highly effective for preventing Covid-19 hospitalizations among US adults. SARS-CoV-2 vaccination was beneficial for patients with immunosuppression, but effectiveness was lower in the immunosuppressed population.

Neuromuscular blockade in patients with ARDS: a rapid practice guideline.

The aim of this Intensive Care Medicine Rapid Practice Guideline (ICM-RPG) is to formulate an evidence-based guidance for the use of neuromuscular blocking agents (NMBA) in adults with acute respiratory distress syndrome (ARDS). The panel comprised 20 international clinical experts from 12 countries, and 2 patient representatives. We adhered to the methodology for trustworthy clinical practice guidelines and followed a strict conflict of interest policy. We convened panelists through teleconferences and web-based discussions. Guideline experts from the guidelines in intensive care, development, and evaluation Group provided methodological support. Two content experts provided input and shared their expertise with the panel but did not participate in drafting the final recommendations. We followed the Grading of Recommendations Assessment, Development, and Evaluation approach to assess the certainty of evidence and grade recommendations and suggestions. We used the evidence to decision framework to generate recommendations. The panel provided input on guideline implementation and monitoring, and suggested future research priorities. The overall certainty in the evidence was low. The ICM-RPG panel issued one recommendation and two suggestions regarding the use of NMBAs in adults with ARDS. Current evidence does not support the early routine use of an NMBA infusion in adults with ARDS of any severity. It favours avoiding a continuous infusion of NMBA for patients who are ventilated using a lighter sedation strategy. However, for patients who require deep sedation to facilitate lung protective ventilation or prone positioning, and require neuromuscular blockade, an infusion of an NMBA for 48 h is a reasonable option.

Early cellular interactions and immune transcriptome profiles in human factor VIII-exposed hemophilia A mice.

Essentials Initial immune cell interactions leading to factor (F) VIII immunity are not well characterized. We assessed cellular interactions and expression profiles in hemophilia A mice. MARCO+, followed by SIGLEC1+ and SIGNR1+ macrophages co-localize most with human FVIII. The splenic transcriptome highlights potential therapeutic targets to prevent inhibitors. SUMMARY: Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 µg (2000-8000 IU kg-1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 µg (240 IU kg-1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO+ cells compared with SIGLEC1+ and SIGNR1+ in the splenic marginal zone. FVIII exhibited little co-localization with CD11c+ dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO+ ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

Canine models of inherited bleeding disorders in the development of coagulation assays, novel protein replacement and gene therapies.

Animal models of inherited bleeding disorders are important for understanding disease pathophysiology and are required for preclinical assessment of safety prior to testing of novel therapeutics in human and veterinary medicine. Experiments in these animals represent important translational research aimed at developing safer and better treatments, such as plasma-derived and recombinant protein replacement therapies, gene therapies and immune tolerance protocols for antidrug inhibitory antibodies. Ideally, testing is done in animals with the analogous human disease to provide essential safety information, estimates of the correct starting dose and dose response (pharmacokinetics) and measures of efficacy (pharmacodynamics) that guide the design of human trials. For nearly seven decades, canine models of hemophilia, von Willebrand disease and other inherited bleeding disorders have not only informed our understanding of the natural history and pathophysiology of these disorders but also guided the development of novel therapeutics for use in humans and dogs. This has been especially important for the development of gene therapy, in which unique toxicities such as insertional mutagenesis, germ line gene transfer and viral toxicities must be assessed. There are several issues regarding comparative medicine in these species that have a bearing on these studies, including immune reactions to xenoproteins, varied metabolism or clearance of wild-type and modified proteins, and unique tissue tropism of viral vectors. This review focuses on the results of studies that have been performed in dogs with inherited bleeding disorders that closely mirror the human condition to develop safe and effective protein and gene-based therapies that benefit both species.

An official American Thoracic Society Clinical Practice guideline: the diagnosis of intensive care unit-acquired weakness in adults

RATIONALE: Profound muscle weakness during and after critical illness is termed intensive care unit-acquired weakness (ICUAW). OBJECTIVES: To develop diagnostic recommendations for ICUAW. METHODS: A multidisciplinary expert committee generated diagnostic questions. A systematic review was performed, and recommendations were developed using the Grading, Recommendations, Assessment, Development, and Evaluation (GRADE) approach. MEASUREMENT AND MAIN RESULTS: Severe sepsis, difficult ventilator liberation, and prolonged mechanical ventilation are associated with ICUAW. Physical rehabilitation improves outcomes in heterogeneous populations of ICU patients. Because it may not be feasible to provide universal physical rehabilitation, an alternative approach is to identify patients most likely to benefit. Patients with ICUAW may be such a group. Our review identified only one case series of patients with ICUAW who received physical therapy. When compared with a case series of patients with ICUAW who did not receive structured physical therapy, evidence suggested those who receive physical rehabilitation were more frequently discharged home rather than to a rehabilitative facility, although confidence intervals included no difference. Other interventions show promise, but fewer data proving patient benefit existed, thus precluding specific comment. Additionally, prior comorbidity was insufficiently defined to determine its influence on outcome, treatment response, or patient preferences for diagnostic efforts. We recommend controlled clinical trials in patients with ICUAW that compare physical rehabilitation with usual care and further research in understanding risk and patient preferences. CONCLUSIONS: Research that identifies treatments that benefit patients with ICUAW is necessary to determine whether the benefits of diagnostic testing for ICUAW outweigh its burdens.(AU)

Sedation practices in a cohort of critically ill patients receiving prolonged mechanical ventilation.

BACKGROUND: The aim of this paper was to describe type and amount of sedatives, opioid analgesics and anti- psychotics administered to critically ill patients receiving prolonged mechanical ventilation and identify patient-specific factors associated with their administration. METHODS: Retrospective cohort study of adult patients mechanically ventilated for ≥14 days over a two-year period. RESULTS: The majority of patients (88%) received lorazepam for sedation (median 14-day dose 168 mg, IQR 25-606). Morphine (median 14-day dose 125 mg, IQR 0-850) and fentanyl (median 14-day dose 2032 mcg, IQR 175-15346) were administered with relative equal frequency. Only 48% of the cohort received anti-psychotics during the first 14 days. Age was inversely associated with cumulative dose of lorazepam equivalents (ratio 0.97; 95% CI 0.95-0.99), propofol (ratio 0.93; 95% CI 0.89-0.96), and morphine equivalents (ratio 0.97; 95% CI 0.96-0.98). Substance abuse was associated with cumulative dose of lorazepam (ratio 3.37; 95% CI 1.14-8.21) and morphine equivalents (ratio 3.09; 95% CI 1.48-6.44). Ethanol abuse was associated with >50% lower cumulative dose of morphine equivalents (ratio 0.47; 95% CI 0.25-0.87). CONCLUSION: In critically ill patients receiving prolonged ventilation, history of substance abuse predicted a 3-fold increase in 14-day cumulative dose of sedatives and opioids used. Conversely, older age was associated with decreased use of sedatives and opioids and history of alcohol abuse was only associated with decreased opioid use. Overall, patients receiving prolonged mechanical ventilation appeared to consume high cumulative doses of sedatives and opioids, with less frequent use of antipsychotics. Accounting for patient characteristics may help identify individuals with varying sedative needs.

Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice.

Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti-factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16-disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T- and B-cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti-FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII-treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII-treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro-environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain-dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.

An assessment of the pathogenic significance of the R924Q von Willebrand factor substitution.

BACKGROUND: Type 1 VWD is associated with mutational heterogeneity in the VWF gene. The R924Q substitution was the second most frequent sequence variation in the Canadian type 1 VWD study and this variant was also documented in other type 1 VWD studies. In this study, R924Q was detected in a compound heterozygote possessing both type 2N and 924Q substitutions whose VWF:FVIIIB and FVIII levels were disproportionately low for the heterozygous type 2N state. AIM: To determine the role of R924Q variation in the pathogenesis of type 1 VWD. METHODS: The frequency of the R924Q variant in the normal and type 1 VWD populations was ascertained, along with the associated polymorphic VWF haplotype. The effect of the R924Q substitution on the biosynthesis and intracellular trafficking of VWF was explored by in vitro expression studies in COS-7 and AtT-20 cells. Immunofluorescent staining of VWF was performed in transfected AtT-20 cells and BOECs from the patient. RNA analysis was performed to investigate an RNA processing defect in the patient. RESULTS AND CONCLUSIONS: In vitro expression studies demonstrated that the R924Q variation does not affect biosynthesis, intracellular trafficking and storage significantly. Storage of VWF in the patient's endothelial cells was abnormal. Analysis of the patient's VWF mRNA revealed a novel truncated transcript resulting from the activation of a cryptic splice site in exon 28. The presence of a common VWF haplotype in heterozygotes for 924Q with low VWF levels suggests a founder origin for this variant allele that may mark this splicing defect.

Reduction of the immune response to factor VIII mediated through tolerogenic factor VIII presentation by immature dendritic cells.

BACKGROUND: The development of neutralizing antibodies to factor FVIII (FVIII) represents the most serious complication in the treatment of hemophilia A. OBJECTIVE: We have explored the potential of using immature dendritic cells (iDCs) to present FVIII in a tolerogenic manner to T cells. METHODS: The iDCs were isolated from hemophilic murine bone marrow and pulsed with canine cFVIII (cFVIII-iDCs) in the presence or absence of the NFkappaB pathway blocking compound Andrographolide (Andro-cFVIII-iDCs). Three weekly intravenous infusions of one million cFVIII pulsed-iDCs were administered to a group of five hemophilic Balb/c mice. Anti-FVIII antibody levels were monitored by functional Bethesda assay after four weekly intravenous challenges with 2 IU of cFVIII. RESULTS: We have shown that cFVIII in the presence or absence of Andro is efficiently taken up by iDCs and that this process does not result in the maturation of DCs or the activation of co-cultured T cells. Following repeated infusion of the cFVIII-iDCs and Andro-cFVIII-iDCs into hemophilic mice, which were subsequently challenged with cFVIII, long-term reductions of FVIII inhibitors of 25% and 40%, respectively, were documented. Studies of cytokine release and T-cell phenotypes indicate that the mechanisms responsible for reducing immunologic responsiveness to cFVIII appear to involve an expansion of Foxp3 T regulatory cells in the case of cFVIII-iDC infusion and the elaboration of the immunosuppressive cytokines IL-10 and TGF-beta following andrographolide-treated cFVIII-iDCs. CONCLUSIONS: This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.

Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter.

BACKGROUND: Plasma von Willebrand factor (VWF) is mainly derived from endothelial cells, cells that express a large repertoire of genes that are transcriptionally regulated by fluid shear stress. Endothelial VWF expression is not uniform throughout the vasculature, and levels are increased at regions associated with disturbed blood flow and steep gradients of shear stress. It is, however, unknown whether shear stress influences the regulation of VWF gene expression. OBJECTIVES: Our objective was to evaluate the effect of shear stress on endogenous endothelial VWF mRNA expression and VWF promoter (-2722 to -1224) activity and to determine whether genetic elements modulate this flow-induced expression. METHODS: A parallel plate flow chamber was used to expose endothelial cells to a shear level of 15 dynes cm(-2) for 24 or 6 h. VWF mRNA expression was analyzed. Various VWF promoter constructs that each contain either SNP haplotypes 1 or 2 and either a 17-GT or a 23-GT repeat element were transfected into endothelial cells, and flow-induced promoter activation was assessed. RESULTS: When endothelial cells were exposed to shear stress, endogenous VWF mRNA expression increased 1.84-fold and average VWF promoter activity was enhanced 3.4-fold. Single nucleotide polymorphisms at -2708 and -2525, and the shear stress-response element at -1585, are not responsible for the shear stress-induced increase. Rather a GT repeat element at -2124 mediates the increase in activity, and the length of this polymorphic repeat element influences the magnitude of induction. CONCLUSIONS: Shear stress enhances VWF promoter activity and a polymorphic GT repeat element mediates the stress-induced transactivation.

p11 is up-regulated in the forebrain of stressed rats by glucocorticoid acting via two specific glucocorticoid response elements in the p11 promoter.

Posttraumatic stress disorder (PTSD) is one of the most common psychiatric disorders. Despite the extensive study of the neurobiological correlates of this disorder, the underlying mechanisms of PTSD are still poorly understood. Recently, a study demonstrated that dexamethasone (Dex), a synthetic glucocorticoid, can up-regulate p11, known as S100A10-protein which is down-regulated in patients with depression, (Yao et al., 1999; Huang et al., 2003) a common comorbid disorder in PTSD. These observations led to our hypothesis that traumatic stress may alter expression of p11 mediated through a glucocorticoid receptor. Here, we demonstrate that inescapable tail shock increased both prefrontal cortical p11 mRNA levels and plasma corticosterone levels in rats. We also found that Dex up-regulated p11 expression in SH-SY5Y cells through glucocorticoid response elements (GREs) within the p11 promoter. This response was attenuated by either RU486, a glucocorticoid receptor (GR) antagonist or mutating two of three glucocorticoid response elements (GRE2 and GRE3) in the p11 promoter. Finally, we showed that p11 mRNA levels were increased in postmortem prefrontal cortical tissue (area 46) of patients with PTSD. The data obtained from our work in a rat model of inescapable tail shock, a p11-transfected cell line and postmortem brain tissue from PTSD patients outline a possible mechanism by which p11 is regulated by glucocorticoids elevated by traumatic stress.

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain.

BACKGROUND: The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. OBJECTIVE: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. METHODS: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII-C2) to FVIII-deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII-C2, followed by a subcutaneous challenge of either FVIII-C2 or FVIII. The development of anti-FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. RESULTS AND CONCLUSIONS: Mucosal administration of FVIII-C2 decreases the titer of anti-FVIII-C2 inhibitors after FVIII-C2 challenge, and decreases the percentage of FVIII-C2 specific antibodies after challenge with full-length FVIII. Tolerance induction to FVIII-C2 is associated with increased IL-10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII-C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII-specific mucosal tolerance induction as an immunotherapy treatment for anti-FVIII inhibitor development warrants further investigation.

Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: roles of intracellular calcium, GRP78 and Bcl-2.

We investigated the cytoprotective effects of lithium, the mood-stabilizer, on thapsigargin-induced stress on the endoplasmic reticulum (ER) in rat PC12 cells. Protracted lithium pretreatment of PC12 cells elicited cytoprotection against thapsigargin-induced cytotoxicity. Lithium protection was concurrent with inhibition of thapsigargin-induced intracellular calcium increase and with elevated expression of the molecular chaperone GRP78. Moreover, lithium pretreatment upregulated the antiapoptotic protein Bcl-2, and blocked Bcl-2 downregulation elicited by thapsigargin. Prior to the induction of GRP78, lithium treatment alone increased the expression of c-Fos whose induction by ER stress is necessary for GRP78 induction. Curcumin, an inhibitor of transcription factor AP-1, blocked lithium cytoprotection against thapsigargin cytotoxicity. Thus, the induction of GRP78 and Bcl-2, and activation of AP-1 likely contribute to lithium-induced protection against cytotoxicity resulting from ER stress. Additionally, thapsigargin-induced cytotoxicity was suppressed by pretreatment with another mood-stabilizer, valproate, indicating that cytoprotection against ER stress is a common action of mood-stabilizing drugs.

Comparing wound complication rates following closure of hip wounds with metallic skin staples or subcuticular vicryl suture: a prospective randomised trial.

PURPOSE: To compare 2 methods of wound closure-metallic staples or 3-0 undyed vicryl-according to postoperative wound complication rates. METHODS: Patients who underwent surgery for proximal femoral fractures were randomised to have wound closure with metallic staples or with subcuticular vicryl suture. Wounds were regularly examined postoperatively and only those with positive wound swabs were regarded as wound infections. RESULTS: Five infections and one superficial wound dehiscence occurred in the patients who had wound closure with metallic staples. The complication rate was significantly higher for this group compared with the group who had wound closure with subcuticular vicryl suture (p<0.025). CONCLUSION: Superficial wound complication rates are higher for wounds closed with metallic staples compared to wounds closed with subcuticular vicryl.

Fatigue failure of the femoral component of a cementless total hip arthroplasty.

This case report describes a fracture of the neck of a femoral component of a Furlong (JRI Limited, London) hydroxyapatite-coated cementless total hip arthroplasty.

A novel type 2A (Group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF) associated with an absence of high-molecular-weight multimers. In this study, sequence analysis of the VWF gene from a Type 2A VWD patient showed a novel, heterozygous T-->A transversion at nucleotide 4510, resulting in the non-conservative substitution of L1503Q in the mature VWF subunit. This substitution, which was not found in 55 unrelated normal individuals, was reproduced by in vitro site directed mutagenesis of a full-length VWF cDNA and was subsequently expressed in COS-7 cells. The corresponding recombinant mutant VWF protein was partially retained in COS-7 cells yet the full spectrum of multimers was observed, suggesting that the absence of the highest molecular weight multimers results from increased proteolysis. The recombinant mutant VWF protein was digested with the ADAMTS13 protease from VWF-depleted plasma and the aberrant VWF multimer pattern was observed. These results suggest that the L1503Q substitution induces a conformational change in the VWF protein, which increases the protein's susceptibility to proteolysis. A three-dimensional model of the A2 domain demonstrates that the L1503Q mutation and the physiological proteolytic cleavage site for ADAMTS13 (Y(1605)-M(1606)) are localized close together in two adjacent parallel beta-sheets. The mutation L1503Q does not significantly disrupt the conformation of the protein; thus the subtle loss of multimers in this patient may be due to altered interactions with the ADAMTS13 protease.

Characterization of extracellular accumulation of Zn2+ during ischemia and reperfusion of hippocampus slices in rat.

The mammalian CNS contains an abundance of chelatable zinc that is sequestered in the vesicles of glutamatergic presynaptic terminals and co-released with glutamate. Considerable Zn(2+) is also released during cerebral ischemia and reperfusion (I/R) although the mechanism of this release has not been elucidated. We report here the real time observation of increase of the concentration of extracellular Zn(2+) ([Zn(2+)](o)), accompanied by a rapid increase of intracellular free Zn(2+)concentration, in the areas of dentate gyrus (DG), CA1 and CA3 in acute rat hippocampus slices during ischemia simulated by deprivation of oxygen and glucose (OGD) followed by reperfusion with normal artificial cerebrospinal fluid. A brief period of OGD caused a sustained increase of [Zn(2+)](o). Subsequent reperfusion with oxygenated medium containing glucose resulted in a further increase of [Zn(2+)](o). Longer periods of OGD caused greater increases of [Zn(2+)](o,) and subsequent reperfusion caused still further increases of [Zn(2+)](o,) regardless of OGD duration. The Zn(2+) chelator CaEDTA (10 mM) significantly reduced the increase of [Zn(2+)] induced by OGD and reperfusion. Significant regional differences of [Zn(2+)](o) over the areas of the DG, CA1 and CA3 were not observed during I/R. Neither sodium channel blockade by tetrodotoxin (2 microM), perfusion with nominally calcium-free medium nor anatomical disassociation of the DG, CA1 and CA3 regions from one another by lesioning affected the increase of [Zn(2+)](o). The non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine methyl ester (1 mM), however, blocked the increase of [Zn(2+)](o) during ischemia and reperfusion. The data indicate the important role of NO in causing the release of Zn(2+) during I/R and suggest that NOS inhibitors may be used to reduce Zn(2+)-induced neuronal injury.