t-PA Suppresses the Immune Response and Aggravates Neurological Deficit in a Murine Model of Ischemic Stroke

Introduction: Acute ischemic stroke (AIS) is a potent trigger of immunosuppression, resulting in increased infection risk. While thrombolytic therapy with tissue-type plasminogen activator (t-PA) is still the only pharmacological treatment for AIS, plasmin, the effector protease, has been reported to suppress dendritic cells (DCs), known for their potent antigen-presenting capacity. Accordingly, in the major group of thrombolyzed AIS patients who fail to reanalyze (>60%), t-PA might trigger unintended and potentially harmful immunosuppressive consequences instead of beneficial reperfusion. To test this hypothesis, we performed an exploratory study to investigate the immunomodulatory properties of t-PA treatment in a mouse model of ischemic stroke.Methods: C57Bl/6J wild-type mice and plasminogen-deficient (plg−/−) mice were subjected to middle cerebral artery occlusion (MCAo) for 60 min followed by mouse t-PA treatment (0.9 mg/kg) at reperfusion. Behavioral testing was performed 23 h after occlusion, pursued by determination of blood counts and plasma cytokines at 24 h. Spleens and cervical lymph nodes (cLN) were also harvested and characterized by flow cytometry.Results: MCAo resulted in profound attenuation of immune activation, as anticipated. t-PA treatment not only worsened neurological deficit, but further reduced lymphocyte and monocyte counts in blood, enhanced plasma levels of both IL-10 and TNFα and decreased various conventional DC subsets in the spleen and cLN, consistent with enhanced immunosuppression and systemic inflammation after stroke. Many of these effects were abolished in plg−/− mice, suggesting plasmin as a key mediator of t-PA-induced immunosuppression.Conclusion: t-PA, via plasmin generation, may weaken the immune response post-stroke, potentially enhancing infection risk and impairing neurological recovery. Due to the large number of comparisons performed in this study, additional pre-clinical work is required to confirm these significant possibilities. Future studies will also need to ascertain the functional implications of t-PA-mediated immunosuppression for thrombolyzed AIS patients, particularly for those with failed recanalization

Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of shear rate gradients (SRG) but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create SRG during blood perfusion. VWF-GPIbα interactions were increased at sites of SRG compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRG but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFVA1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRG on VWF dependent platelet aggregation and developed the shear gradient-sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of SRG. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal hemostasis

An engineered nanosugar enables rapid and sustained glucose-responsive insulin delivery in diabetic mice

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are "nanosugars" bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (approximate to 95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL(-1)) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar

Studies into the interaction between the plasminogen activating system and the blood-brain barrier

The serine protease tissue-type plasminogen activator (t-PA) is best recognised for its role in haemostasis, where it is converts the zymogen plasminogen into plasmin – a potent and broad-spectrum protease responsible for degradation of fibrin and removal of blood clots. Next to their vascular roles, t-PA and plasmin also possess endogenous functions in the brain, both as modulators of neuronal activity and t-PA also as a regulator of the blood-brain barrier (BBB). While the action of t-PA and plasmin is associated with normal brain function, it often contributes deleteriously to a variety of brain-related pathophysiological or pathological conditions. This basic fibrinolytic function of t-PA has led to its development for treatment of ischaemic stroke. Since 1995, t-PA is the only FDA-approved therapy for this acute condition; however, t-PA can only be administered within 3-4.5h post stroke onset, as later use has reduced clinical benefits. Unfortunately, t-PA treatment is accompanied by an increased risk for development of symptomatic intracerebral haemorrhage (sICH), a life-threatening complication which impairs the safety of the treatment and prevents any attempts for its implementation in longer therapeutic windows. In recent years, it was postulated that the elevated incidence of sICH associated with t-PA-induced thrombolysis may be directly linked to the ability of t-PA to modulate the BBB and enhance its permeability. According to this rationale, blockade of direct interactions between t-PA and the BBB may reduce the haemorrhagic side-effects of t-PA treatment without affecting its capacity to restore blood-flow. My PhD project examined this concept in vitro and aimed to uncover central mechanisms linking t-PA to BBB modulation with the hope that their antagonism may ultimately improve the safety of t-PA-induced thrombolysis in stroke. During the project both human and mouse in vitro BBB models were developed to serve as authentic research tools (chapter 4). Our human BBB system was established based on immortalised human astrocytes and high-passaged human brain endothelial cells (BECs) co-cultured in contact conditions, allowing rapid accumulation of human-relevant information. To confirm these data and assist in mechanistic determination we also generated mouse primary cultures of well-differentiated astrocytes and BECs and used them for assembly of a non-contact mouse BBB model. Together, our human and mouse in vitro BBB models provided us with potent tools to reliably examine the effect of t-PA and plasminogen on the neurovascular unit. The effect of t-PA, without or with plasminogen, was then tested in the intact BBB models (chapter 5). t-PA caused a concentration-dependent increase in permeability which relied on its proteolytic activity. This effect was fully dependent on plasmin formation and was strongly potentiated in the presence of plasminogen. Strikingly, solution-applied plasmin or other plasminogen activators (PAs; reteplase, desmoteplase but most importantly urokinase (u-PA), with or without plasminogen) could not substitute for in situ t-PA-dependent plasminogen activation, while the close t-PA derivative tenecteplase was only partially effective. Of particular interest was the effect of u-PA, which in fact reduced plasminogen-dependent increase in BBB permeability, possibly by competition with endogenous t-PA over plasminogen. Inhibition of matrix metalloproteinases (MMPs) and receptor tyrosine kinases by broad-spectrum inhibitors (GM6001 and imatinib, respectively) was ineffective at blocking the effect of t-PA, while t-PA:PAI-1 complex could not open the BBB, suggesting that other known mechanisms for BBB opening by t-PA are less likely to play a role in our system. Finally, when t-PA and plasminogen were employed together, the BBB opening effect occurred, and was even potentiated, under oxygen-glucose deprivation (OGD), indicating the applicability of the primary event also during brain ischaemia. Surprisingly, the sensitivity of the BBB to high levels of t-PA alone was diminished under OGD, suggesting that the overall response of the BBB to t-PA during stroke may be of much greater complexity. Collectively, these observations implied that a unique action of t-PA, by localisation and activation of plasminogen at selective sites on the surface of the BBB, synergised with plasmin to create a t-PA-specific opening of the BBB. Lastly, to better understand the mechanism underlying this effect, we stimulated isolated cultures of BECs and astrocytes with t-PA and plasminogen and followed morphological, signalling and functional aspects associated with this stimulation (chapter 6). Both BECs and astrocytes dramatically reacted by morphology changes to t-PA-generated, cell-surface-associated plasmin. The response of BECs to t-PA/plasmin seemed rather complex. It did not involve LDLRs, but potentially included proteolysis of tight junctions and basement membrane as well as initiation of Rho/Rho-kinase (ROCK) signalling, promoting together morphological changes, loss of adhesion and barrier compromise. In astrocytes, morphological changes exerted by t-PA and plasmin fully mirrored the proteases action on the in vitro BBB, strongly suggesting that they played a functional role in BBB opening. The principal event triggered by t-PA, via plasmin, in astrocytes was activation of the Rho/ROCK signalling pathway; changes in astrocyte morphology coincided with increased formation of actin stress-fibres, enlargement of focal adhesion size and induction of phosphorylated myosin and actomyosin-dependent contractility, all being hallmarks of Rho/ROCK activation. Inhibition of ROCK with selective ROCK inhibitors, and especially with the clinically-utilised HA1077 (fasudil), blocked the astrocytic morphology changes and importantly reduced t-PA/plasminogen-mediated increases in permeability both in the human and the mouse BBB models. We conclude that during t-PA-driven thrombolysis in acute ischaemic stroke substantial levels of plasmin, selectively generated by t-PA in situ, may indeed interact with both ischaemic and healthy brain microvessels, leading to alterations in the function of BEC but especially to modulation of the astrocytic cytoskeleton by ROCK activation. The subsequent changes in astrocyte morphology increase the permeability of the BBB, potentially contributing to formation of sICH. Blockade of the Rho/ROCK pathway in astrocytes may effectively antagonise t-PA-induced BBB opening during thrombolysis, reducing the incidence of sICH and increasing the safety of the treatment without affecting its fibrinolytic capacity. Rock inhibition could thus have beneficial consequences during thrombolytic therapy in patients with ischaemic stroke

Studies into the interaction between the plasminogen activating system and the blood-brain barrier

Valid, reliable and standardised assessment formats and procedures, suited to application in the workplace, are important for meaningful and consistent assessment of the clinical performance of physiotherapy students. The choice of clinical assessment instruments for physiotherapy programs in Australia has typically been influenced by historical precedents and the personal experience of assessors rather than by the known strengths and weaknesses of an assessment instrument, a situation common to that observed in medical programs (E. D. Newble, Jolly, & Wakeford, 1994). The Queensland Health Clinical Education Project (2005) acknowledged the variability of procedures and instruments for assessment of physiotherapy practices across different universities in Australia. At that time there were 16 entry-level physiotherapy programs in Australia, all accredited by the Australian Physiotherapy Council (APC). Each physiotherapy program was required to demonstrate that graduates met the performance standards outlined in the Australian Standards for Physiotherapy (2006). Despite each program having curriculum designed to meet the same standards, when this thesis commenced each physiotherapy program used unique clinical assessment forms and assessment criteria. The Queensland Health Clinical Education Project emphasised the diversity of assessment forms and supporting documentation as a substantial and unnecessary burden on assessors who were required to use multiple assessment instruments. In addition, the measurement properties of these assessment instruments were unknown, impacting on confidence in the reliability and validity of decisions based on these assessment approaches. As new physiotherapy programs commenced, this burden multiplied. This thesis describes the development of a standardized assessment instrument to meet the needs of physiotherapy students and educators and provide valid and reliable measurements of clinical performance. The need for this research was identified by university-based physiotherapy programs across Australia and New Zealand, physiotherapy educators and supervisors, and the APC responsible for accreditation of physiotherapy programs within Australian universities. Funding was provided by the Australian Learning and Teaching Council (formerly The Carrick Institute) to commence work on the development of an assessment instrument. The research in this thesis is reported in chronological order with each phase informing subsequent steps. Streiner and Norman (2003) proposed that the first step in the development of a new instrument was to be fully informed of existing scales and the quality of such instruments prior to embarking on the development of a new instrument. This work began with a systematic review of methods used to assess professional competence in physiotherapy practice (Chapter One). The systematic review found a number of reports of research into assessment of competence in physiotherapy practice; these varied in design and method quality (see Chapter One). Eight instruments developed to assess professional competence of physiotherapy students within the clinical environment were located. The review failed to identify convincing evidence sufficient to support the merits of one instrument above others. In addition, investigation of the psychometric properties of these instruments was not performed utilising Item Response Theory (IRT) or the Rasch Measurement Model (RMM), rather employing the Classical Test Theory (CTT) approach. The thesis argues for the need to investigate instrument properties using IRT or RMM; these approaches offer substantial clinical and scientific advantage over traditional psychometric methods in the development and evaluation of rating scales, and in the analysis of rating scale data (Andrich, 1988; J. Hobart & Cano, 2009; Wilson, 2005; Wright, 1996a; Wright & Mok, 2000). Chapter Two describes and defends the plan for instrument development. The first phase (Chapter Three) involved development of the assessment instrument content, format and processes. The research was guided by the Standards for Educational and Psychological Testing, (American Educational Research Association, 1999). The process of test design was based on the ‘four building blocks’ approach outlined by Wilson (2005) which comprised construct mapping, items design, outcome space and measurement model. Once development of the first version of the instrument with the working title Clinical Assessment of Physiotherapy Skills (CAPS) was complete, cycles of action and reflection on outcomes (an action research approach) were utilised. The iterative research cycles included preliminary information gathering, instrument development, pilot trial / field test stages, and continuous refinement of the instrument based on evaluation throughout the different phases following recommendations for best practice in research of this nature (Coghlan & Brannick, 2001). A pilot trial (Chapters 4 and 5) was conducted, using the instrument to assess 295 third and fourth year physiotherapy students. Rasch analysis of outcome data showed an overall fit to the Rasch model. The difficulty of the items was well matched to the abilities of the persons being assessed and the 5-level rating scale performed as intended. The results of the pilot trial supported the continuation of the research into field tests one and two. The results of both field tests (Chapters 7 and 8) supported the findings of the pilot trial demonstrating that the APP data had adequate fit to the chosen measurement model (Rasch Partial Credit Model), the Person Separation Index demonstrated the scale was internally consistent discriminating between four groups of students with different levels of professional competence (0.96), the items were targeting the intended construct (professional competence) and the instrument demonstrated unidimensionality. Additionally differential item function (DIF) studies demonstrated there was no item bias in either field test for the variables: student age, gender and level of clinical experience, clinical educator age, gender and experience as an educator, facility type, and clinical area. Qualitative data (Chapters 6 and 9) provided evidence of the acceptability of the instrument for use within the work place by educators and students. Further research investigating how educators were interpreting and scoring written communication and the impact on student learning of the assessment process was recommended. Ongoing evaluation and refinement of training methods and resources was also advocated. The results of field testing provided data supported the validity of the APP instrument scores and acceptability of the instrument for use within the workplace. These data enabled the final phase of research, investigation of inter-rater reliability, to proceed (Chapter Ten). Thirty pairs of clinical educators (60 independent educators) and 30 third and fourth year physiotherapy students from five universities participated in the reliability trial. Both correlational coefficients and metricated errors were estimated to provide a comprehensive analysis of the likely utility of APP scores and to enable score and change score interpretation. The Intraclass Correlation Coefficient 2,1 (two-way random effects model) for total APPs scores for the two raters was 0.92 (95% CI 0.84 to 0.96) and the ICC 2,1 for the global rating scale scores was 0.72 (95% CI 0.50 – 0.86). The 95% confidence band around a single score for this data was 6.5 APP points. With a scale width of 0 – 80, an error margin of 6.5 (95%CI) was considered acceptable. This error enables a high level of accuracy in ranking student performance as evidenced by test/retest correlation of .92. For the APP the magnitude of change in scores required to conclude that real change has occurred is in the order of 7.8 points which compared favourably to other instruments used to assessment professional competence of physiotherapy students (Coote, et al., 2007; Meldrum, et al., 2008; Task Force for the Development of Student Clinical Performance Instruments, 2002). Overall the physiotherapy clinical educators demonstrated a high level of reliability in the assessment and marking of physiotherapy students’ performance on clinical placements when using the APP. This was found despite the variability anticipated due to different areas of practice, types of facilities and a spectrum of educator experience. The final step in the research was to evaluate the evidence for validity of APP scores. Using the five sources of validity evidence presented in the American Educational Research Association (1999) standards, data from multiple sources was accumulated to establish the likely validity of interpretations made based on the instrument scores. The validity of scores for workplace-based professional competence awarded by educators to pre-entry level physiotherapy students using the APP was evaluated through Rasch analysis, parametric statistical evaluation, and qualitative data obtained from multiple sources. This approach enabled triangulation and reinforcement of decisions based on quantitative and qualitative data obtained from multiple sources. The APP was found to have strong validity characteristics across all five sources of validity evidence as described in Chapter Eleven. The APP was developed and applied within the constraints of a dynamic and unpredictable clinical environment. This is a key strength of the assessment instrument. The research has delivered an important benefit for physiotherapy education in that a single instrument with known validity and reliability is now available to replace the twenty-five distinct assessment forms formerly being used. To date, 17 out of 18 Universities in Australia and New Zealand have adopted the APP as the sole assessment form, and a further three new programs commencing within the next two years are also adopting the instrument