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The International Standards for Neurologic Classification of Spinal Cord Injury (ISNCSCI) are the most widely used classification system in spinal cord injury medicine. The purpose of the ISNCSCI is to ensure accurate and consistent communication among patients, clinicians, and researchers. Since its first publication in 1982, the ISNCSCI has continued to evolve with the latest updates and revisions published in 2015 and 2019. The updates were incorporated into the 2019 ISNCSCI worksheet and booklet, and the International Standards Training e-Learning Program. This article details the ISNCSCI update from 2015 and revision in 2019., Competing Interests: Disclosure Members of the International Standards Committee of American Spinal Injury Association and International Spinal Cord Society: Randal Betz (Institute for Spine and Scoliosis, Lawrenceville, NJ); Fin Biering-Soerensen (Clinic for Spinal Cord Injuries, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark); Stephen P. Burns (Department of Rehabilitation Medicine, University of Washington School of Medicine, Seattle, WA); William H. Donovan (Institute for Rehabilitation and Research, Houston, TX); Daniel E. Graves (Thomas Jefferson University, Philadelphia, PA); James Guest (Department of Neurologic Surgery, University of Miami Miller School of Medicine, Miami, FL); Linda Jones (University of Colorado, Denver, CO); Steven Kirshblum (Kessler Institute for Rehabilitation, Rutgers New Jersey Medical School, West Orange, NJ); Andrei Krassioukov (International Collaboration On Repair Discovery, Vancouver, Canada); Mary-Jane Mulcahey (Thomas Jefferson University, Philadelphia, PA); Gianna Rodriguez (Michigan Medicine, University of Michigan, Ann Arbor, MI); Rüdiger Rupp (Spinal Cord Injury Center, Heidelberg University Hospital, Heidelberg, Germany); Mary Schmidt Read (Magee Rehabilitation Hospital - Jefferson Health, Philadelphia, PA); Christian Schuld (Spinal Cord Injury Center, Heidelberg University Hospital, Heidelberg, Germany); Keith Tansey (Departments of Neurosurgery and Neurobiology, University of Mississippi Medical Center, Jackson, MS); and Kristen Walden (Rick Hansen Institute, Vancouver, Canada)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We investigated the utility of a cell-saver device for processing out-of-date red blood cells, by washing twenty bags of red blood cells that had been stored for between 36 and 55 days. The volume of recovered cells, and the characteristics of the suspension fluid, were measured before and after treatment. The ratio of free haemoglobin to total haemoglobin was up to 0.02 before processing, and up to 0.011 afterwards, changing by between -0.013 and +0.003. This ratio met the current standard for free haemoglobin (less than 0.008 in more than 75% of samples), both before and after processing. Ninety-three percent of red blood cells survived the process. Potassium ion concentration fell from above 15 mmol.l(-1) in all cases, to a mean of 6.4 mmol.l(-1) (p < 0.001). The pH rose to a mean value of 6.44 (p = 0.001). Lactate ion concentration fell to a mean value of 14 mmol.l(-1) (p < 0.001). Sodium ion concentration rose from a mean value of 93 mmol.l(-1) to a mean value of 140 mmol.l(-1) (p < 0.001). A useful proportion of out-of-date red blood cells remained intact after conditioning using a cell-saver, and the process lowered concentrations of potentially toxic solutes in the fluid in which they were suspended., (© 2014 The Association of Anaesthetists of Great Britain and Ireland.)

Background/purpose: We present a retrospective case series of 2 individuals with motor-incomplete spinal cord injury (SCI) to examine differences in lifetime cost estimates before and after participation in an intensive locomotor training (LT) program. Sections of a life care plan (LCP) were used to determine the financial implications associated with equipment, home renovations, and transportation for patients who receive LT. An LCP is a viable method of quantifying outcomes following any therapeutic intervention., Case Description: The LCP cases analyzed were a 61-year-old woman and a 4½-year-old boy with motor-incomplete SCI and impairments classified by the American Spinal Injury Association Impairment Scale (AIS) as AIS D and AIS C, respectively., Interventions: Each patient received an intensive outpatient LT program 3 to 5 days per week. The 61-year-old woman received 198 sessions over 57 weeks and the 4½-year-old boy received 76 sessions over 16 weeks., Outcomes: The equipment, home renovation, and transportation costs of an LCP were calculated before and after LT. Prior to the implementation of LT, the 61-year-old woman had estimated lifetime costs between $150,247.00 and $199,654.00. Following LT, the estimated costs decreased to between $2010.00 and $2446.00 (a decrease of $148,237.00 and $197,208.00). Similarly, the 4-year-old boy had estimated lifetime costs for equipment, home renovation, and transportation between $535,050.00 and $771,665.00 prior to LT. However, the estimated costs decreased to between $97,260.00 and $200,047.00 (a decrease of $437,790.00 and $571,618.00) following LT., Discussion: The lifetime financial costs associated with equipment, home renovations, and transportation following a motor-incomplete SCI were decreased following an intensive LT program for the 2 cases presented in this article. The LCP, including costs of rehabilitation and long-term medical and personal care costs, may be an effective tool to discern cost benefit of rehabilitation interventions.

Study Design: Multi-center, prospective, cohort study., Objectives: To assess the validity and reliability of the Spinal Cord Independence Measure (SCIM III) in measuring functional ability in persons with spinal cord injury (SCI)., Setting: Inpatient rehabilitation hospitals in the United States (US)., Methods: Functional ability was measured with the SCIM III during the first week of admittance into inpatient acute rehabilitation and within one week of discharge from the same rehabilitation program. Motor and sensory neurologic impairment was measured with the American Spinal Injury Association Impairment Scale. The Functional Independence Measure (FIM), the default functional measure currently used in most US hospitals, was used as a comparison standard for the SCIM III. Statistical analyses were used to test the validity and reliability of the SCIM III., Results: Total agreement between raters was above 70% on most SCIM III tasks and all κ-coefficients were statistically significant (P<0.001). The coefficients of Pearson correlation between the paired raters were above 0.81 and intraclass correlation coefficients were above 0.81. Cronbach's-α was above 0.7, with the exception of the respiration task. The coefficient of Pearson correlation between the FIM and SCIM III was 0.8 (P<0.001). For the respiration and sphincter management subscale, the SCIM III was more responsive to change, than the FIM (P<0.0001)., Conclusion: Overall, the SCIM III is a reliable and valid measure of functional change in SCI. However, improved scoring instructions and a few modifications to the scoring categories may reduce variability between raters and enhance clinical utility.

Objective: To quantify wheelchair mobility of persons with a spinal cord injury (SCI), and to assess the relationship between wheelchair mobility and demographics, type of manual wheelchair, and participation., Design: Cross-sectional study., Setting: Six Model Spinal Cord Injury Systems., Participants: People (N=132) with SCI who use a manual wheelchair as their primary means of mobility., Interventions: Not applicable., Main Outcome Measures: Wheelchair-related mobility characteristics measured by a data-logging device, and community participation measured by the short form of the Craig Handicap Assessment Recording Technique (CHART)., Results: Age was found to be significantly (r=-.225, P<.01) related to average speed traveled per day. Whites were found to travel significantly further (P<.01) and accumulate more minutes per day (P<.01) compared with minorities. Participants who were employed traveled significantly further (P<.01), faster (P<.01), and for more minutes per day (P<.01) compared with those who were not employed. A moderate relationship (r=.245-.390) was found between wheelchair mobility data and CHART total score., Conclusions: Results suggest a need for future investigation of the factors that influence wheelchair mobility and community participation of persons with SCI. Findings indicate the efficacy of a quantitative method to track wheelchair mobility in community settings, which could serve as a way of identifying community participation for persons with SCI and possibly uncovering additional aspects of participation., (Copyright © 2011 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.)

A total of 9 participants with right-hemisphere stroke performed a new virtual reality (VR) wheelchair navigation test of lateralized spatial attention and neglect. The test consists of a virtual path along which participants navigate (or are navigated) as they name virtual objects encountered. There are 4 VR conditions, obtained by crossing the factors array complexity and driver. Participants performed the VR task, a real-life wheelchair navigation task, and a battery of attention and neglect tests. The VR test showed sensitivity to both array complexity and driver, exhibited strong correlations with the wheelchair navigation test, and detected lateralized attention deficits in mild patients. The VR task thus shows promise as a sensitive, efficient measure of real-life navigation.

Background: At the 2006 National Institute on Disability and Rehabilitation Research (NIDRR) sponsored pre-conference on spinal cord injury (SCI) outcomes, several gait and ambulation measures were evaluated for utility in clinical practice, validity, and reliability as research measurement tools. The Conference Subcommittee on Gait and Ambulation chose to review the Walking Index for Spinal Cord Injury II (WISCI II), 50-Foot Walk Test (50FTWT), 6-Minute Walk Test (6MWT), 10-Meter Walk Test (10MWT), and Functional Independence Measure-Locomotor (FIM-L)., Methods: A subcommittee of international experts evaluated each instrument for test construct, administration, population applicability, reliability, sensitivity to change, and validity. Evaluations for each outcome measure were compiled, distributed to the whole committee, and then further reviewed with addition of comments and recommendations for consensus. An audience of experts voted on the validity and usefulness of each measure., Results: WISCI II and 10MWT were found to be the most valid and clinically useful tests to measure improvement in gait for patients with SCI. FIM-L had little utility and validity for research in SCI. 6MWT and 50FTWT were found to be useful but in need of further validation or changes for the SCI population., Conclusion: A combination of the 10MWT and WISCI II would provide the most valid measure of improvement in gait and ambulation in as much as objective changes of speed, and functional capacity allow for interval measurement. To provide the most comprehensive battery, however, it will be important to include a measure of endurance such as the 6MWT. Further validation and study should be devoted to WISCI II, 10MWT, and 6MWT as primary outcome measures for gait in SCI.

Lyophilized canine platelets were infused in a single large bolus dose into splenectomized dogs after 2 hours' perfusion on cardiopulmonary bypass to test their possible efficacy in restoring hemostasis after compromise of platelet function. The vessel bleeding time (VBT) was monitored by venipuncture of the exposed jugular vein. During cardiopulmonary bypass, platelet counts fell quickly and the VBTs became prolonged over baseline. Infusion of lyophilized platelets reconstituted in normal saline occurred just before or immediately after weaning from the cardiopulmonary bypass pump. The results showed consistent and persistent lowering of the VBTs by the infused lyophilized platelets. Controls showed continuously prolonged VBTs. The weighted average VBT in infused subjects was significantly lower than the average in controls: 3 minutes 10 seconds versus 6 minutes 59 seconds, respectively (t test, P= .01). These results in this setting indicate the possible effectiveness of similar human lyophilized platelet preparations in reducing postoperative bleeding in open heart surgery.

Background: The rehydrated, lyophilized (RL) platelet (PLT) is being developed as a hemostatic infusion agent for the control of active bleeding. The key to the method for preparing RL PLTs is a mild aldehyde stabilization that allows for freezing and lyophilizing without cellular rupture. RL PLTs have been shown to be effective at rapidly controlling bleeding in animal models of cardiopulmonary bypass induced PLT dysfunction and washout thrombocytopenia, yet the rehydrated cells have proved to be safe with respect to induction of pathologic intravascular coagulation., Study Design and Methods: In vitro and in vivo studies were performed to better understand the differential effect of the RL PLT manufacturing method on primary and secondary hemostatic processes. The functionality of the von Willebrand factor (VWF) receptor (glycoprotein Ib) complex, the PAR receptors, integrin-mediated aggregation (inside-out signaling), and surface membrane prothrombin to thrombin conversion systems were investigated., Results: RL PLTs were found to retain native VWF-mediated adhesion and surface thrombin generation functions. In contrast, the coupling of thrombin receptors to integrin inside-out signaling was largely inhibited., Conclusion: These results suggest that RL PLTs may stop bleeding by forming primary hemostatic plugs and providing a localized source of thrombin for secondary hemostatic processes, yet do not build up occlusive pathologic clots possibly because integrin functions for forming PLT-PLT aggregates are partially inhibited.

Intravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P <.001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P =.0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B.

Stored human platelets are frequently used in hemorrhagic emergencies, but have limited immediate utility for controlling bleeding due to storage lesion and are frequently contaminated with microorganisms. The development of paraformaldehyde-treated, lyophilized and rehydrated (RL) platelets, which are sterile and have a prolonged shelf life (years), ameliorate the efficacy and sterility problems with stored platelets. RL platelets have been shown to have many native functions of fresh platelets in vitro and to mediate hemostasis in vivo in large animal models of hemorrhagic shock and cardiopulmonary bypass induced platelet dysfunction. To further evaluate the functional properties of this transfusion product, we studied the role of RL platelets in three aspects of thrombus formation and lysis. First, the interaction between RL platelets and fibrinogen was investigated. The surface density of unligated GPIIb-IIIa on RL and fresh platelets were, respectively 30000 and 70000 molecules per cell as detected with the monoclonal antibody 10E-5. Freezing, lyophilization and rehydration steps in the preparation of RL platelets resulted in the surface presentation of 120000 molecules of fibrinogen per cell from alpha granule sources. After ADP activation, RL platelets bound exogenous 125I-labeled fibrinogen in a dose-dependent manner with an affinity that is similar to that of fresh platelets and was inhibited by RGD peptides. 125I-Labeled fibrinogen binding to RL and fresh platelets, respectively, saturated at 14000 and 32000 molecules per cell. Scanning electron microscopic ultrastructural analysis showed that fibrin strands interacted with the surface of RL platelets in a normal manner. The second set of studies investigated the ability of RL platelets to catalyze and amplify the clot formation process in an activation-dependent manner. We showed that RL platelets undergo degranulation in fibrin in clots and functioned as thrombogenic surfaces for the generation of activated coagulation factors and fibrin generation. A final set of studies was performed to investigate fibrin of clots that contained RL platelets. RL platelet clots were lysed in the presence of tissue plasminogen activator with a similar time course as clots without platelets, and lysis occurred faster than when fresh platelets were included in the fibrin mass. The results of these three studies demonstrate that RL platelets are capable of mediating thrombus formation and do not inhibit lysis. Our results help explain how RL platelets restore hemostasis in vivo, and indicate that these cells might be a viable alternative to fresh stored platelets in transfusion medicine.

A variety of platelet substitutes (e.g., rehydrated, lyophilized (RL) platelets, thromboerythrocytes, plateletsomes, infusible platelet membranes, synthocytes, fibrinogen-coated microcapules) are potentially useful as hemostatic agents in transfusion medicine. However, as "foreign" particles, platelet substitutes interact to varying extents with elements of the reticulo-endothelial system for clearance, reducing hemostatic efficacy. Experiments were performed to better understand the interaction of RL platelets with elements of the innate and acquired immune systems. The infusion of heterologous RL platelets into rats resulted in rapid clearance from the free circulation with half-life values of minutes. The clearance of RL platelets was inhibited when macrophages were rendered apoptotic with gadolinium. Transmission EM analysis of splenic tissue after infusion of lyophilized cells, as well as in vitro mixing studies with splenic macrophages and RL platelets, indicated that macrophage-mediated phagocytosis mechanisms were operant in RL platelet clearance by the reticulo-endothelial system. Studies with IV IgG, as a competitive inhibitor of the macrophage Fc receptor, provides evidence that RL platelet destruction is in part mediated by platelet surface bound IgG. This hypothesis was further supported by the finding that RL platelets react with IgG class antibodies that are pre-existing in naïve animals. These studies provide a rational basis for prolonging the circulation time of RL platelets and other platelet substitutes.

The purpose of this paper was to establish proof of concept for administration of human recombinant F.IX (rF.IX) by inhalation for therapy of hemophilia B. The pharmacokinetics of intratracheal (IT) administration of rF.IX was studied in nine hemophilia B dogs randomized into 3 groups that received 200 IU/kg IT, 1,000 IU/kg IT, or 200 IU/kg intravenously (IV). IT rF.IX produced therapeutic levels of F.IX antigen and activity and the pharmacokinetic parameters were consistent with a slow release from a depot site within the lungs. Bioavailability compared to IV administration was 11% for 200 IU/kg IT and 4.9% for 1,000 IU/kg. The whole blood clotting time began to shorten at 2 h but F.IX bioactivity was not detected until 8 h post infusion in both IT groups. In all groups, F.IX activity was detected through 72 h post administration. These data demonstrate that biologically active rF.IX can reach the systemic circulation when given IT. Aerosolization of rF.IX may provide a needle-free therapeutic option for delivery of rF.IX to hemophilia B patients.

This study aimed to evaluate the effect of cross-linking and lyophilization on intracellular signalling processes in rehydrated, lyophilized (RL) platelets, which are under development as a platelet substitute for transfusion. Exposure of RL platelets to thrombin resulted in enhanced phosphorylation of several proteins, including 18 kDa and 42 kDa kinase substrates that were shown to be the substrates of myosin light chain and protein kinase C respectively. Cross-linking and lyophilization depleted the platelets of free cytoplasmic ADP and ATP, but had less effect on protein-bound nucleotides. The surface membrane of RL platelets was found to be permeable to poly dT probes less than approximately 3 kDa in size; larger nucleotide probes and proteins did not penetrate the surface membrane. Taken together, our results indicate that RL platelets retain some of the haemostatic stimulus-response functions of fresh platelets and are capable of feedback amplification in coagulation.

We demonstrate that a single intraportal vein injection of a recombinant adeno-associated virus (rAAV) vector encoding canine factor IX (cFIX) cDNA under the control of a liver-specific enhancer/promoter leads to a long-term correction of the bleeding disorder in hemophilia B dogs. Stable expression of the therapeutic level of cFIX (5% of normal level) was detected in the plasma of a dog injected with an AAV vector at a dose of 4.6 x 10(12) particles/kg for over 7 months. Both whole-blood clotting time (WBCT) and activated partial thromboplastin time (aPTT) of the treated dogs have been greatly decreased since the treatment. No anti-canine factor IX antibodies have been detected in the treated animals. Importantly, no bleeding has been observed in the dog that expresses a therapeutic level of cFIX for 7 months following vector administration. Moreover, no persistent significant hepatic enzyme abnormalities were detected in the treated dogs. Thus, a single intraportal injection of a rAAV vector expressing cFIX successfully corrected the bleeding disorder of hemophilia B dogs, supporting the feasibility of using AAV-based vectors for liver-targeted gene therapy of genetic diseases.

In our initial investigation of functionality of platelets freeze-dried after stabilization with 1.8% paraformaldehyde, we found that the rehydrated cells were morphologically intact and retained adhesive and procoagulant properties. Further testing of fixed, washed freeze-dried platelets has demonstrated the physiologic nature of their adhesion in vitro and their hemostatic efficacy in vivo in correcting the bleeding time in thrombocytopenic animal models. Binding studies with monoclonal antibodies and radiolabelled ligands indicate an intact GpIb vonWillebrands factor receptor as on fresh platelets, but a somewhat attenuated GpIIbIIIa fibrinogen receptor. Repeated infusion of canine lyophilized platelet preparations in a single recipient over several months has shown no incipient cytopenia upon infusion of new doses nor accelerated clearance of platelets. These findings suggest minimal risk of systemic thrombosis or severe immunogenic reaction and support the notion of approaching clinical trials as soon as possible.

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.

The role of von Willebrand factor (vWF) in arterial neointimal formation that develops in arteries with altered shear stress was investigated using normal, heterozygous, and homozygous von Willebrand disease pigs (ie, vWD, or lacking vWF) that were fed normal pig chow. Shear stress was applied to carotid and femoral arteries with a Goldblatt clamp for 14 days, producing a > or = 80% stenosis. Neointimal lesion size was measured by computer-assisted morphometry. Expression of proliferative cell nuclear antigen (PCNA) by neointimial and medial cells was used as a relative index of proliferative activity. For shear-stressed arteries, there was no significant difference in the number of smooth muscle cell layers in the lesion, lesion size, and percent of PCNA-positive neointimal or medial cells among normal, heterozygous, and homozygous vWD pigs (P> or =.1, ANOVA). Lesions in pigs that expressed vWF (normals and heterozygotes) contained large amounts of vWF in the neointima, whereas lesions in vWD pigs had no detectable vWF. Moreover, no foam cells were detected in the lesions. Thus, the absence of vWF apparently does not alter the size of lesions in shear-stressed arteries in vWD pigs or the number of neointimal or medial cells expressing PCNA. Mechanism(s) involved with shear-induced modulation of smooth muscle cell proliferation, then, can operate independently of vWF in normolipemic pigs.

Current therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availability of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.) or intravenous (i.v.) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical i.v. dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the i.v. route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the i.m. injection in the canine resulted in a bioavailability of 82.8%, while the s.c. injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with i.v. administration. These data show that significant levels of F.IX may be obtained via s.c. injection in canine and human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

A new hereditary thrombopathy has been identified in a closed colony of Wistar rats. A simple and reproducible cuticle bleeding time test was developed as a rapid screening procedure for the bleeding diathesis. Affected animals exhibit markedly prolonged bleeding times and complete absence of platelet aggregation either with adenosine diphosphate (ADP) or with thrombin. Inheritance data suggest an autosomal dominant inheritance pattern with variable penetrance. Coagulation tests, platelet counts, plasma von Willebrand factor (vWF) activity, and clot retraction are within normal limits in thrombopathic animals. GPIb-dependent botrocetin-induced platelet agglutination was present in washed thrombopathic rat platelets. No discernible abnormality of intraplatelet organelles or granules was seen by transmission electron microscopy of thrombopathic platelets. A qualitative morphologic assessment of intraplatelet fibrinogen in thrombopathic rat platelets showed no discernible difference as compared with control rat platelets. Thrombopathic rat platelets exhibit decreased glycoprotein IIb/IIIa (GPIIb/IIIa) antigen by flow cytometric analysis and markedly decreased iodine 125-labeled fibrinogen binding to platelet GPIIb/IIIa after ADP activation. This rat colony demonstrates a unique thrombopathy, distinct from previously described animal thrombopathies, with some characteristics of variant Glanzmann's thrombasthenia. This animal model may provide further insight into the regulatory mechanisms and pathophysiology of platelet GPIIb/IIIa.

Two recombinant adenoviruses expressing either human alpha 1-antitrypsin (hAAT) or canine factor IX (cFIX) were modified so that they also contained a temperature-sensitive mutation (ts125) in the DNA binding protein encoded within the viral E2A region. The effects of the inclusion of the ts125 mutation on transgene expression in vivo were evaluated in Balb/c mice and hemophilia B dogs by comparison with adenoviral vectors containing the same transgene but lacking the ts125 mutation. No significant differences in the duration of transgene expression were observed in either animal model. Insufficiency of the ts125 mutation in the prolongation of transgene expression in these two animal models suggests that further modification of the vector backbone may be required to achieve long-term gene expression in a wide variety of applications. Additionally, humoral immune response to transgene products has been demonstrated in immunocompetent animal models, which will also need to be surmounted for long-term efficacy in disease treatment by gene therapy.

In transfusion medicine, platelets cannot be replaced by blood substitutes. Circulating platelets must respond quickly to changes in normal blood flow and blood-vessel injury to promote normal hemostasis. Adhesion of platelets at the site of vessel endothelial rupture is mediated through platelet membrane glycoprotein receptors. The integrity of these surface adhesion receptors and the signal-transduction pathways of activation will determine, in large part, how well a platelet functions in hemostasis. The deterioration of these systems during storage leads to a compromise of function known as the 'platelet-storage lesion'.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Thrombotic thrombocytopenia with severe depletion of plasma von Willebrand factor (vWF) was induced in normal large animals (5 dogs and 2 pigs) by botrocetin, a Bothrops factor requiring vWF for platelet agglutination. Botrocetin (90 to 100 U/kg, 2.14 to 2.38 mg/kg, in a single i.v. injection) reduced plasma vWF activity to < 0.1 U/mL for 24 hours. During this period, multimeric analysis of plasma vWF antigen (Ag) revealed the loss of intermediate- and high-molecular-weight forms with a concomitant increase in lower molecular weight forms. A moderate reduction in factor VIII (FVIII) activity was observed. The vWF reduction was accompanied by transient thrombocytopenia and prolonged bleeding times during the deficiency state. Occlusive platelet thrombi were detected by transmission electron microscopy in the microcirculation of lung and spleen but not kidney or brain 30 minutes after the botrocetin injection. Recovery of plasma vWF and platelet count occurred within 48 hours and was associated with the appearance in the plasma of unusually large forms of vWF:Ag multimers. The vWF:Ag multimer distribution was normal at 72 hours. The ultrastructural distribution of vWF in unstimulated normal porcine and canine platelets was examined by using immunogold staining. VWF was detected in the alpha-granules of normal pig platelets but was not observed in platelets from normal dogs. However, both animals developed thrombotic thrombocytopenia when injected with botrocetin. A second group of animals (2 dogs and 3 pigs) with von Willebrand disease (vWD) was given a single botrocetin injection (90 to 100 U/kg). No thrombocytopenia occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

von Willebrand factor (vWF) is essential for the induction of occlusive thrombosis in stenosed and injured pig arteries and for normal hemostasis. To separate the relative contribution of plasma and platelet vWF to arterial thrombosis, we produced chimeric normal and von Willebrand disease pigs by crossed bone marrow transplantation; von Willebrand disease (vWD) pigs were engrafted with normal pig bone marrow and normal pigs were engrafted with vWD bone marrow. Thrombosis developed in the chimeric normal pigs that showed normal levels of plasma vWF and an absence of platelet vWF; but no thrombosis occurred in the chimeric vWD pigs that demonstrated normal platelet vWF and an absence of plasma vWF. The ear bleeding times of the chimeric pigs were partially corrected by endogenous plasma vWF but not by platelet vWF. Our animal model demonstrated that vWF in the plasma compartment is essential for the development of arterial thrombosis and that it also contributes to the maintenance of bleeding time and hemostasis.