Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia.

The application of immunotherapies such as chimeric antigen receptor (CAR) T therapy or bi-specific T cell engager (BiTE) therapy to manage myeloid malignancies has proven more challenging than for B-cell malignancies. This is attributed to a shortage of leukemia-specific cell-surface antigens that distinguish healthy from malignant myeloid populations, and the inability to manage myeloid depletion unlike B-cell aplasia. Therefore, the development of targeted therapeutics for myeloid malignancies, such as acute myeloid leukemia (AML), requires new approaches. Herein, we developed a ligand-based CAR and secreted bi-specific T cell engager (sBite) to target c-kit using its cognate ligand, stem cell factor (SCF). c-kit is highly expressed on AML blasts and correlates with resistance to chemotherapy and poor prognosis, making it an ideal candidate for which to develop targeted therapeutics. We utilize γδ T cells as a cytotoxic alternative to αß T cells and a transient transfection system as both a safety precaution and switch to remove alloreactive modified cells that may hinder successful transplant. Additionally, the use of γδ T cells permits its use as an allogeneic, off-the-shelf therapeutic. To this end, we show mSCF CAR- and hSCF sBite-modified γδ T cells are proficient in killing c-kit+ AML cell lines and sca-1+ murine bone marrow cells in vitro. In vivo, hSCF sBite-modified γδ T cells moderately extend survival of NSG mice engrafted with disseminated AML, but therapeutic efficacy is limited by lack of γδ T-cell homing to murine bone marrow. Together, these data demonstrate preclinical efficacy and support further investigation of SCF-based γδ T-cell therapeutics for the treatment of myeloid malignancies.

Invasive Assessment of Microvascular Resistance in Hypertrophic Cardiomyopathy With Echocardiographic Correlates.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is often associated with ischaemia despite lack of focal epicardial coronary stenosis. Our aim was to assess invasive coronary microvascular circulation and correlate findings with echocardiography. METHODS: We prospectively enrolled patients with HCM and controls who were referred for diagnostic coronary angiography. A pressure-temperature sensor coronary guidewire was used with intracoronary injections of room-temperature saline to measure mean coronary transit time during rest and hyperaemia induced with intravenous adenosine. The index of microvascular resistance (IMR) was calculated. Left ventricular mass was calculated during echocardiographic studies. RESULTS: Patients with HCM (n=12) and controls (n=7), had similar demographics. Left ventricular ejection fraction was higher in HCM (76.7%±11.0% vs 55.0%±15.9%, p=0.003). IMR was non-significantly higher in HCM (21.7±10.2 vs 15.3±4.8, p=0.16). Only patients with HCM had abnormal IMR (>25). Coronary flow reserve was non-significantly higher in HCM (2.7±1.6 vs 2.1±1.2, p=0.34). IMR correlated with left ventricular mass in hypertrophic cardiomyopathy subjects (Pearson r=0.68, p=0.02). CONCLUSIONS: Microvascular dysfunction as assessed by IMR may be abnormal in HCM and is correlated with left ventricular mass.

Novel galeterone analogs act independently of AR and AR-V7 for the activation of the unfolded protein response and induction of apoptosis in the CWR22Rv1 prostate cancer cell model.

The androgen receptor (AR) has long been the primary target for the treatment of prostate cancer (PC). Despite continuous efforts to block AR activity through ligand depletion, AR antagonism, AR depletion and combinations thereof, advanced PC tumors remain resilient. Herein, we evaluate two galeterone analogs, VNPT-178 and VNLG-74A, in PC cell models of diverse androgen and AR dependence attempting to delineate their mechanisms of action and potential clinical utility. Employing basic biochemical techniques, we determined that both analogs have improved antiproliferative and anti-AR activities compared to FDA-approved abiraterone and enzalutamide. However, induction of apoptosis in these models is independent of the AR and its truncated variant, AR-V7, and instead likely results from sustained endoplasmic reticulum stress and deregulated calcium homeostasis. Using in silico molecular docking, we predict VNPT-178 and VNLG-74A bind the ATPase domain of BiP/Grp78 and Hsp70-1A with greater affinity than the AR. Disruption of 70 kDa heat shock protein function may be the underlying mechanism of action for these galeterone analogs. Therefore, despite simultaneously antagonizing AR activity, AR and/or AR-V7 expression alone may inadequately predict a patient's response to treatment with VNPT-178 or VNLG-74A. Future studies evaluating the context-specific limitations of these compounds may provide clarity for their clinical application.

Bioengineering and serum free expansion of blood-derived γδ T cells.

BACKGROUND AIMS: Cellular immunotherapy relies on several highly variable patient-specific parameters, such as (i) cell number before and after expansion, (ii) targeting of cells to tumors, (iii) cell survival and function after infusion, and (iv) on- and off-target adverse events. Cellular approaches such as the specific expansion of γδ T cells as opposed to αß T cells are being pursued. γδ T cells are reasonable candidates for immunotherapy because they (i) possess intrinsic anti-tumorigenicity, (ii) require no priming, (iii) direct tumor killing via recognition of stress-responsive ligands, and (iv), as we now show, can be expanded to clinical cell doses in current Good Manufacturing Practice serum-free media (SFM). METHODS: γδ T-cell expansion was evaluated in several SFMs. Additionally, the expanded γδ T cells were evaluated for their transduction efficiency using lentiviral vectors (LV). RESULTS: Of the SFM cultures, robust expansion was only observed in OpTmizer supplemented with high-dose interleukin-2. γδ T-cell percentages and numbers were sufficient for clinical use. Using cells from several donors, transduction efficiencies ranged from 13 to 33%, which is similar to transduction levels observed using αß T cells with similar multiplicity of infection. DISCUSSION: An optimized method of γδT-cell expansion and transduction was developed that can be tested in early-phase clinical trials. With appropriate elimination of the αßT cell-component, the absence of MHC-restriction affords the opportunity for use in the allogeneic setting with limited risk of graft versus host disease. Finally, the use of SFM provides clinically safer, widely applicable and potentially more efficacious cellular immunotherapy.

Effect of growth hormone treatment on diastolic function in patients who have developed growth hormone deficiency after definitive treatment of acromegaly.

OBJECTIVE: Although growth hormone (GH) replacement is prescribed for patients with hypopituitarism due to many etiologies, it is not routinely prescribed for patients with GH deficiency (GHD) after cure of acromegaly (acroGHD). This study was designed to investigate the effect of GH replacement on cardiac parameters in acroGHD. DESIGN: We prospectively evaluated for 12months 23 patients with acroGHD: 15 subjects on GH replacement and eight subjects not on GH replacement. Main outcome measures included LV mass corrected for body surface area (LVM/BSA) and measures of diastolic dysfunction (E/A ratio and deceleration time), as assessed by echocardiography. RESULTS: After 12months of follow-up, there were no differences between the GH-treated group and the untreated group in LVM/BSA (GH: 74.4±22.5g/m(2) vs untreated: 72.9±21.3g/m(2), p=0.89), E/A ratio (GH: 1.21±0.39 vs untreated: 1.08±0.39, p=0.50) or deceleration time (GH: 224.5±60.1ms vs untreated: 260±79.8ms, p=0.32). The overall degree of diastolic function was similar between the groups with 42.9% of untreated subjects and 50% of GH-treated subjects (p=0.76) classified as having normal diastolic function at follow-up. CONCLUSIONS: There were no significant differences in LVM/BSA or parameters of diastolic function in patients with a history of acromegaly treated for GHD as compared to those who were untreated. These data are reassuring with respect to cardiovascular safety with GH use after treatment for acromegaly, although further longer term study is necessary to evaluate the safety and efficacy of GH treatment in this population.

Model-based navigation of left and right ventricular leads to optimal targets for cardiac resynchronization therapy: a single-center feasibility study.

BACKGROUND: Left ventricular (LV) and right ventricular pacing site characteristics have been shown to influence response to cardiac resynchronization therapy (CRT). This study aimed to determine the clinical feasibility of image-guided lead delivery using a 3-dimensional navigational model displaying both LV and right ventricular (RV) pacing targets. Serial echocardiographic measures of clinical response and procedural metrics were evaluated. METHODS AND RESULTS: Thirty-one consecutive patients underwent preimplant cardiac MRI with the generation of a 3-dimensional navigational model depicting optimal segmental targets for LV and RV leads. Lead delivery was guided by the model in matched views to intraprocedural fluoroscopy. Blinded assessment of final lead tip location was performed from postprocedural cardiac computed tomography. Clinical and LV remodeling response criteria were assessed at baseline, 3 months, and 6 months using a 6-minute hall walk, quality of life questionnaire, and echocardiography. Mean age and LV ejection fraction was 66 ± 8 years and 26 ± 8%, respectively. LV leads were successfully delivered to a target or adjacent segment in 30 of 31 patients (97%), 68% being nonposterolateral. RV leads were delivered to a target or adjacent segment in 30 of 31 patients (97%), 26% being nonapical. Twenty-three patients (74%) met standard criteria for response (LV end-systolic volume reduction ≥ 15%), 18 patients (58%) for super-response (LV end-systolic volume reduction ≥ 30%). LV ejection fraction improved at 6 months (31 ± 8 versus 26 ± 8%, P=0.04). CONCLUSIONS: This study demonstrates clinical feasibility of dual cardiac resynchronization therapy lead delivery to optimal targets using a 3-dimensional navigational model. High procedural success, acceptable procedural times, and a low rate of early procedural complications were observed. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01640769.

The link between vitamin D metabolism and sleep medicine.

Vitamin D is a hormone that interacts with intranuclear receptors to effect transcriptional changes in many cell types including those in gut, bone, breast, prostate, brain, skeletal muscle, and the immune system. Inadequacy of vitamin D is widely prevalent, and leads to the classic diseases of bone demineralization as well as to more recently recognized problems such as nonspecific pain and noninflammatory skeletal myopathy, which may disrupt sleep and directly cause daytime impairment. Emerging lines of evidence suggest that low vitamin D levels increase the risk for autoimmune disease, chronic rhinitis, tonsillar hypertrophy, cardiovascular disease, and diabetes. These conditions are mediated by altered immunomodulation, increased propensity to infection, and increased levels of inflammatory substances, including those that regulate sleep, such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, and prostaglandin D2 (PD2). Together, the recent reports suggest a role for inadequate vitamin D in the development of symptoms of wake impairment commonly associated with sleep disorders. Persistent inadequacy of vitamin D may also increase the risk for obstructive sleep apnea via promotion of adenotonsillar hypertrophy, airway muscle myopathy, and/or chronic rhinitis. Much remains to be learned concerning the complex relationship between chronically low levels of vitamin D, normal sleep, sleep disruption, and daytime neurocognitive impairment.

Significance of electrocardiographic right bundle branch block in trained athletes.

We sought to determine the clinical and physiologic significance of electrocardiographic complete right bundle branch block (CRBBB) and incomplete right bundle branch block (IRBBB) in trained athletes. The 12-lead electrocardiographic and echocardiographic data from 510 competitive athletes were analyzed. Compared to the 51 age-, sport type-, and gender-matched athletes with normal 12-lead electrocardiographic QRS complex duration, the 44 athletes with IRBBB (9%) and 13 with CRBBB (3%) had larger right ventricular (RV) dimensions, as measured by the basal RV end-diastolic diameter (CRBBB 43 ± 3 mm, IRBBB 38 ± 6 mm, normal QRS complex 35 ± 4 mm, p <0.001) and RV end-diastolic area (CRBBB 33 ± 5, IRBBB 27 ± 7, and normal QRS complex 23 ± 3 cm(2); p <0.001). Athletes with CRBBB also had a relative reduction in the RV systolic function at rest as assessed by the RV fractional area change and peak systolic tissue velocity. Finally, QRS prolongation was associated with parallel increases in interventricular dyssynchrony (basal RV to basal lateral left ventricular peak systolic tissue velocity time difference: CRBBB 112 ± 15, IRBBB 73 ± 33, normal QRS complex 43 ± 39 ms, p <0.001). Despite these findings, no athlete with CRBBB or IRBBB was found to have pathologic structural cardiac disease. In conclusion, among trained athletes, CRBBB and IRBBB appear to be markers of a structural and physiological cardiac remodeling triad characterized by RV dilation, a relative reduction in the RV systolic function at rest, and interventricular dyssynchrony.

Growth hormone status predicts left ventricular mass in patients after cure of acromegaly.

CONTEXT: Growth hormone excess and growth hormone deficiency (GHD) are both associated with increased cardiovascular morbidity. A specific acromegaly-related cardiomyopathy has been described, characterized in part by increased left ventricular mass (LVM). Growth hormone deficiency is associated with reduced LVM. Following cure of acromegaly with surgery or radiation therapy, GHD may develop; however, its effects on cardiac morphology and function in this population are not established. OBJECTIVE: We hypothesized that the development of GHD in patients with prior acromegaly would be associated with cardiac morphologic and functional changes that differ from those in patients who are GH sufficient following cure of acromegaly. DESIGN: A cross-sectional study was conducted in a Clinical Research Center. Study participants consisted of three groups of subjects (n=34): I. Cured acromegaly with GHD (n=15), II. Cured acromegaly with GH sufficiency (n=8), and III. Active acromegaly (n=11). Main outcome measures included cardiac morphology and function, using echocardiography parameters. RESULTS: Mean age and BMI, 44.6 ± 2.3 years (SEM) and 30.7 ± 1.3 kg/m², respectively, were not different among the three groups. Mean peak GH values were: I. 2.8 ± 0.4 ng/ml; II. 30.1 ± 9.1 ng/ml (p=0.0002.) In group I, left ventricular mass, indexed to body surface area (LVMi), was within the normal range in all patients; moreover, left ventricular (LV) geometry was normal. At least 50% of patients in groups II and III had elevated LVMi, and in 50% of patients, LV geometry was abnormal, indicating pathologic hypertrophy. Ejection fraction was similar between all three groups. There were no significant differences in diastolic function. CONCLUSIONS: Patients who develop GHD following cure of acromegaly do not demonstrate elevated LV mass, in contrast to patients with a history of acromegaly but normal GH levels or to patients with active acromegaly. This suggests that GH status after treatment of acromegaly correlates with LV mass, and that, in GH sufficient patients, reversal of remodeling may be slower than previously thought. These data suggest that it will be important to determine whether GH replacement alters left ventricular morphology over time.

Comparison of procedural and in-hospital outcomes of percutaneous balloon aortic valvuloplasty in patients >80 years versus patients < or =80 years.

Percutaneous balloon aortic valvuloplasty (PBAV) is a procedure used for palliation, bridging to surgery, and as an integral step in the procedure for percutaneous aortic valve replacement. Older patients with severe aortic stenosis are thought to have greater risk for adverse perioperative events than younger patients. The aim of this study was to evaluate the outcomes of patients aged >80 years and those aged < or =80 years who underwent PBAV to identify factors associated with adverse clinical outcomes. This was a retrospective study of 111 consecutive patients with severe symptomatic aortic stenosis who underwent retrograde PBAV at Massachusetts General Hospital from December 2004 to December 2008. Forty-nine patients (44%) were men, and the mean age for the whole group was 82 +/- 8 years. Patients were divided into 2 age groups: those aged >80 years (n = 73) and those aged < or =80 years (n = 38). Procedural outcomes, complications, and in-hospital adverse events were compared. Multivariate logistic regression was used for the adjusted analysis. Nearly 90% of patients were in New York Heart Association class III or IV. Patients aged >80 years had lower baseline ejection fractions (43.5% vs 56.1%, p <0.01) and smaller aortic valve areas (0.59 vs 0.73 cm(2), p <0.01). Although the 2 age groups had a similar percentage of aortic valve area increase (55.5% vs 45.2%, p = 0.28), those aged >80 years had smaller post-PBAV aortic valve areas (0.89 vs 1.02 cm(2), p <0.05). Overall, in-hospital mortality was 8.1%, with no significant differences between the groups. Advanced age was not an independent predictor of in-hospital death, myocardial infarction, stroke, cardiac arrest, or tamponade; however, patients aged >80 years had a significantly higher incidence of intraprocedural emergent intubation and cardiopulmonary resuscitation compared to the younger group. New York Heart Association class was the only independent predictor of worse in-hospital outcomes. In conclusion, compared to younger patients, those aged >80 years had less favorable preprocedural characteristics for PBAV but similar overall in-hospital clinical outcomes. Patients aged >80 years had significantly higher incidence of emergent intubation and cardiopulmonary resuscitation during PBAV.

Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A.

BACKGROUND: Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response. METHODS: Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response. RESULTS: We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naïve and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII. CONCLUSIONS: These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products.

Engineered drug-resistant immunocompetent cells enhance tumor cell killing during a chemotherapy challenge.

Establishment of immunocompetent cell mediated anti-tumor immunity is often mitigated by the myelosuppressive effects during administration of chemotherapy. We hypothesized that protecting these immune cells from drug induced toxicities may allow for the combined administration of immunotherapy and chemotherapy. Using a SIV-based lentiviral gene transfer system we delivered the drug-resistant variant P140KMGMT into the immunocompetent cell lines NK-92 and TALL-104, and the myelogenous leukemia cell line, K562, which is a target for both NK-92 and TALL-104 cells. Genetically engineered immunocompetent cells developed significant resistance to temozolomide compared to non-modified cells, and genetic modification of these cells did not affect their ability to kill K562 cells. We then evaluated the effectiveness of drug-resistant immunocompetent cell mediated killing of tumor cells in the presence and absence of chemotherapy. During a chemotherapy challenge the cytotoxic activity of non-modified immunocompetent cells was dramatically impaired. However, when combined with chemotherapy, genetically-modified immune cells retained their cytotoxic activities and efficiently killed non-modified target cells. These results show that engineering immunocompetent cells to withstand chemotherapy challenges can enhance tumor cell killing when chemotherapy is applied in conjunction with cell-based immunotherapy.

Resolution of hypersomnia following identification and treatment of vitamin d deficiency.

A 28-year-old woman was evaluated for 4 months of excessive daytime sleepiness (EDS), after an overnight polysomnogram (PSG) revealed neither sleep disordered breathing nor a sleep related movement disorder. A full sleep evaluation revealed the presence of heavy daytime napping and pervasive fatigue. Epworth Sleepiness Scale (ESS) Score was 10/24. No features characteristic for depression or narcolepsy were present. Chronic pain in the low back and thighs, as well as chronic daily headaches were identified as potential sleep-disrupting forces. Risk factors for hypovitaminosis D included limited natural sun exposure, dark skin tone, and obesity. A 25-hydroxyvitamin D level was low, at 5.9 ng/mL. Vitamin D supplementation was initiated at a dose of 50,000 IU once weekly, and EDS improved within 2 weeks. One week later, a PSG with next-day multiple sleep latency testing (MSLT) failed to show significant pathology. At follow-up, she reported resolution of thigh pain and headaches, with a significant improvement in her low back pain syndrome. EDS had resolved, and her ESS score was 1/24. Follow-up 25-hydroxyvitamin D level was normal at 39 ng/mL. Mechanisms for her clinical improvement could include enhanced sleep quality due to resolution of hypovitaminosis D-associated noninflammatory myopathy, or a possible immunomodulatory effect of vitamin D decreasing central nervous system (CNS) homeostatic sleep pressure via its effects on tumor necrosis factor-alpha (TNF-α) and/or prostaglandin D2. More research is needed to determine if patients presenting with EDS should be more broadly screened for vitamin D deficiency.

Gender effects on cardiac valvular function in hyperprolactinaemic patients receiving cabergoline: a retrospective study.

BACKGROUND: Ergot-derived dopamine agonists are associated with increased risk of valvular dysfunction in Parkinson's disease. The risk of valvular disease associated with lower doses of cabergoline used to treat prolactinomas remains controversial. OBJECTIVE: To determine whether there is an association of cabergoline and valvular function in patients with hyperprolactinaemia according to gender. DESIGN: Case-record retrospective study. SETTING: Outpatient neuroendocrine clinical centre at a tertiary care hospital. STUDY PARTICIPANTS: One hundred patients (48 men and 52 women) with hyperprolactinaemia who had an echocardiogram while receiving cabergoline for at least 6 months. CONTROLS: One hundred controls (48 men and 52 women) selected from Massachusetts general hospital (MGH) database of echocardiograms without clinically significant findings, matched to patients for age, gender, body mass index (BMI) and hypertension. MAIN OUTCOME MEASURE: Echocardiogram. RESULTS: There were no significant differences in valvular function in patients compared with controls. However, women patients had a higher prevalence of mild tricuspid regurgitation (TR) than female controls (15.4%vs. 1.9%, P = 0.03). Among men only, patients had more trace TR than controls (68.8%vs. 45.8%, P = 0.02). The mild valvular regurgitation in patients was not clinically significant and did not correlate with dose, duration or cumulative dose. CONCLUSIONS: Overall cabergoline was not associated with valvulopathy. However, subdivided by gender, hyperprolactinaemic men and women had higher prevalence of trace or mild TR, respectively, compared with gender matched controls. There may be gender differences in valvular dysfunction associated with cabergoline. Longer term, larger studies are necessary to evaluate definitively an effect of cabergoline on valvular function in hyperprolactinaemic patients.

Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Comparison of factor VIII transgenes bioengineered for improved expression in gene therapy of hemophilia A.

Successful gene therapy of hemophilia A depends on the sustained expression of therapeutic levels of factor VIII (fVIII). Because of mRNA instability, interactions with resident endoplasmic reticulum (ER) chaperones, and the requirement for carbohydrate-facilitated transport from the ER to the Golgi apparatus, fVIII is expressed at much lower levels from mammalian cells than other proteins of similar size and complexity. A number of bioengineered forms of B domain-deleted (BDD) human fVIII have been generated and shown to have enhanced expression. Previously, we demonstrated that recombinant BDD porcine fVIII exhibits high-level expression due to specific sequence elements that increase biosynthesis via enhanced posttranslational transit through the secretory pathway. In the current study, high-expression recombinant fVIII constructs were compared directly in order to determine the relative expression of the various bioengineered fVIII transgenes. The data demonstrate that BDD porcine fVIII expression is superior to that of any of the human fVIII variant constructs tested. Mean fVIII expression of 18 units/10(6) cells/24 hr was observed from HEK-293 cells expressing a single copy of the porcine fVIII transgene, which was 36- to 225-fold greater than that of any human fVIII transgene tested. Furthermore, greater than 10-fold higher expression was observed in human cells transduced with BDD porcine fVIII versus BDD human fVIII-encoding lentiviral vectors, even at low proviral copy numbers, supporting its use over other human fVIII variants in future hemophilia A gene therapy clinical trials.

Percutaneous coronary intervention and 1 year survival in patients treated with fibrinolytic therapy for acute ST-elevation myocardial infarction.

AIMS: To assess the predictors of 1 year mortality in patients treated with fibrinolytic therapy for ST-segment elevation myocardial infarction (STEMI) and to determine whether a strategy of early percutaneous coronary intervention (PCI) improves outcome. METHODS AND RESULTS: Consecutive patients (n = 474) admitted to our unit (1998-2001) with STEMI were treated with fibrinolytic therapy. For each patient, age, gender, admission via mobile coronary care unit (MCCU), infarct location, initial systolic blood pressure and Killip class, prior history of ischaemic heart disease, hypertension, diabetes mellitus, smoking status, family history, hyperlipidaemia, and in-hospital PCI (n = 154) were recorded. Mortality at 1 year was obtained from medical records (n = 473). Binary logistic regression analysis was performed to determine independent predictors of 1 year mortality. Mortality in the non-PCI group was 21 vs. 7% in the PCI group. Independent predictors of 1 year mortality were age (risk ratio 1.12, 95% CI 1.08-1.15, P < 0.0001), initial SBP < or = 80 mmHg (risk ratio 4.34, 95% CI 1.68-11.2, P = 0.002), initial Killip class > or = 3 (risk ratio 2.97, 95% CI 1.42-6.2, P = 0.004), and lack of in-hospital PCI (risk ratio 0.39, 95% CI 0.19-0.81, P = 0.012). Although the PCI group were younger (P = 0.007), more likely to be admitted via the MCCU (P = 0.008), with a shorter pain to needle time (P = 0.04), multivariable analysis adjusted for these differences. CONCLUSION: In-hospital PCI in patients treated with fibrinolytic therapy for STEMI is associated with a substantial reduction in 1 year mortality.

Spontaneous coronary artery dissection presenting as cardiac tamponade.

Spontaneous dissection of the left main coronary artery is the least common of all dissections involving the coronary arteries. It usually occurs in young women, especially in the peripartum or early postpartum period. We describe the case of a 59-year-old man with no previous history of atherosclerotic heart disease who presented in cardiac tamponade and was found to have a spontaneous left main stem coronary artery dissection at cardiac catheterization. Emergency revascularization was carried out with the patient remaining symptom-free 4 months after surgery.