Raymond G. Watts

Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial

BACKGROUND In an open-label phase 1 trial, gene delivery of the trophic factor neurturin via an adeno-associated type-2 vector (AAV2) was well tolerated and seemed to improve motor function in patients with advanced Parkinsons disease. We aimed to assess the safety and efficacy of AAV2-neurturin in a double-blind, phase 2 randomised trial. METHODS We did a multicentre, double-blind, sham-surgery controlled trial in patients with advanced Parkinsons disease. Patients were randomly assigned (2:1) by a central, computer generated, randomisation code to receive either AAV2-neurturin (5·4 × 10¹¹ vector genomes) injected bilaterally into the putamen or sham surgery. All patients and study personnel with the exception of the neurosurgical team were masked to treatment assignment. The primary endpoint was change from baseline to 12 months in the motor subscore of the unified Parkinsons disease rating scale in the practically-defined off state. All randomly assigned patients who had at least one assessment after baseline were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT00400634. RESULTS Between December, 2006, and November, 2008, 58 patients from nine sites in the USA participated in the trial. There was no significant difference in the primary endpoint in patients treated with AAV2-neurturin compared with control individuals (difference -0·31 [SE 2·63], 95% CI -5·58 to 4·97; p=0·91). Serious adverse events occurred in 13 of 38 patients treated with AAV2-neurturin and four of 20 control individuals. Three patients in the AAV2-neurturin group and two in the sham surgery group developed tumours. INTERPRETATION Intraputaminal AAV2-neurturin is not superior to sham surgery when assessed using the UPDRS motor score at 12 months. However, the possibility of a benefit with additional targeting of the substantia nigra and longer term follow-up should be investigated in further studies. FUNDING Ceregene and Michael J Fox Foundation for Parkinsons Research.

Idiopathic Thrombocytopenic Purpura: A 10-Year Natural History Study at the Childrens Hospital of Alabama

Childhood idiopathic thrombocytopenic purpura (ITP) is a common disorder. However, single-institution, long-term, natural history data are limited. The objective of this paper is to review presenting features, response to therapy, and natural history of ITP treated at a single pediatric academic medical center. A retrospective chart review was made for all children (ages birth-18 years) diagnosed with ITP (ICD 287.3) and treated at the Childrens Hospital of Alabama/University of Alabama at Birmingham between 1993 and 2003. Four hundred nine patients were identified (49% male, 51% female; mean age: 5.85 years; range: 1 month-17 years). There was no seasonal variation of presentation. The mean platelet count was 19k (0-120k). Bone marrow aspiration (BMA) was performed in 72% but altered the diagnosis or therapy in no patient. Treatment consisted of corticosteroids in 256 (92% response), intravenous immunoglobulin (IVIG) in 125 (87% response), Win-Rho D in 58 (91% response), and no therapy in 71(100% response). Response was defined as increase in platelet count to > 50k. There was no difference in response to any therapy. No patients died. One patient presented with a CNS hemorrhage at presentation, responded to therapy, and survived. Twenty-three of 409 patients (6%) experienced clinical bleeding requiring hospitalization or blood transfusion. Chronic ITP (persistence > 6 months) was noted in 99 patients (24%). Chronic patients presented at an older age (7.8 vs 5.2 years for acute only, p<0.001), and with higher platelet counts (27k vs 17k, p<0.001). The risk of chronic ITP was partially predicted by presenting platelet count > 50k and age > 10 years, or both; 50% of patients presenting with these features developed chronic ITP vs 24% overall rate. Splenectomy was curative in 30/31 (97%) patients. There was no postsplenectomy sepsis. Of 99 patients with chronic ITP, 25 responded to splenectomy, 37 resolved at a mean of 20.3 months after diagnosis (7-96 months), 36 had persistent mild thrombocytopenia (50k-125k), and 1 failed to respond to any treatment including splenectomy. Overall, 91% of cases resolved with therapy or observation. ITP is a common pediatric disease presenting at any age with low morbidity and mortality. Most cases can be managed by pediatricians without hematology referral. Several equally successful therapeutic options exist. Chronic cases present at an older age with higher platelet counts. Up to 50% of cases of chronic ITP will resolve with ongoing follow-up. The overall prognosis in childhood ITP is excellent.

Valproic acid-induced cytopenias: Evidence for a dose-related suppression of hematopoiesis

Valproic acid is an important agent in the management of complex seizure disorders. The drug is being used increasingly in higher doses as a single antieonvulsant agent that produces a good response and has an acceptable level of toxicity. 1 The hematologic side effect primarily reported has been thrombocytopenia, which is believed to be mainly of idiosyncratic or immunologic origin. 2 Neutropenia and erythrocyte aplasia have also been reported, but the cause is unknown. We report a patient with severe erythrocyte aplasia and mild neutropenia associated with valproate monotherapy. The cytopenias were temporally related to escalation of VPA dose. In vitro studies of normal adult bone marrow indicated that VPA levels in the therapeutic range for high-dose valproate monotherapy directly suppressed the growth of hematopoietic progenitors.

Venous thromboembolism in pediatric patients: epidemiologic data from a pediatric tertiary care center in Alabama.

Venous thrombosis is an infrequent but serious cause of hospitalization in children. The epidemiology and natural history remains incompletely defined, especially in geographically distinct regions of the United States. We thus evaluated thrombosis in a single childrens hospital over a 3-year period. Of 41,906 hospitalizations, 92 children were identified for review. The incidence of thrombosis was 21.9 per 10,000 admissions (0.22%). Venous thrombosis was of equal incidence in African-American and white patients. Locations of thrombosis included deep venous (51%), pulmonary (21%), renal vein (8%), intrahepatic (8%), and intracranial (12%). Risk factors for thrombosis included central catheter (32%), malignancy (18%), systemic infection (21%), neurologic disability (9%), cardiac (4%), nephrotic syndrome (3%), and autoimmune (6%). Six of 92 patients (7%) had thrombophilia. Positive family history of venous thromboembolism (VTE) or thrombophilic disorder predicted an abnormal test. Treatment included low-molecular-weight heparin (n=53), coumadin (n=12), heparin (n=10), tissue plasminogen activator (n=6), argatroban (n=1), thrombectomy (n=2), inferior vena cava filter (n=2), and no treatment (n=23). Seventy-seven percent demonstrated resolution of the VTE, 14% had persistent or recurrent VTE, and 9% died. Causes of death were malignancy, prematurity, septicemia, and congenital heart disease. Venous thrombosis is a serious comorbidity in hospitalized children. In our population, African-Americans had an equal incidence of VTE as whites. Positive family history predicted thrombophilia.

Pretreatment and routine echocardiogram monitoring during chemotherapy for anthracycline‐induced cardiotoxicity rarely identifies significant cardiac dysfunction or alters treatment decisions

The widespread use of anthracycline chemotherapy has contributed to improved outcomes in children with cancer. The most feared complication of the anthracyclines is cardiotoxicity. Routine echocardiographic monitoring typically is used before, during, and after treatment to minimize cardiotoxicity. The ideal use of screening before and during chemotherapy remains uncertain.

Tailored chemotherapy for malignant lymphoma arising in the setting of posttransplant lymphoproliferative disorder after solid organ transplantation.

Posttransplant lymphoproliferative disorder is a clinical challenge. Traditional chemotherapy results in tumor response, but toxicity and transplant rejection limit survival. The authors treated seven patients with malignant lymphoma after organ transplant with chemotherapy tailored to individual patient response. Chemotherapy consisted of vincristine, cyclophosphamide, and prednisone, with or without doxorubicin (Adriamycin; Pharmacia & Upjohn, Peapack, NJ, U.S.A.), or the ProMACE-CytaBOM regimen. Six of seven patients (86%) showed a complete response to treatment, with five of seven (71%) alive disease-free at 9 to 72 months (mean 38.2) after treatment. The results show that chemotherapy tailored to individual patient response is a safe and effective therapy for malignant lymphoma arising in patients with posttransplant lymphoproliferative disorder.

Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed “inhibitors”. A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2–5 µg/ml (∼1–33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects’ immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40–80% of FVIII-specific antibodies in most samples were directed against this domain.

Operative management and outcomes in children with congenital bleeding disorders: a retrospective review at a single haemophilia treatment centre

Summary.  Establishing haemostasis for surgical procedures in children with inherited bleeding disorders is challenging. Providers are often hesitant to undertake surgeries in children with bleeding disorders out of fear of bleeding complications. To review the preoperative management and haemorrhagic complications of children with inherited bleeding disorders at our institution, we conducted a retrospective electronic medical record review from 1999 to 2010. Primary focus was review of bleeding complications and factor replacement strategies. A total of 168 procedures were performed in 66 children. Fifteen procedures (8%) in four children were performed in the presence of high‐titre factor inhibitors. Procedures included central venous catheter (CVL) placement or revision (41%), otolaryngology procedures (23%), dental (11%), non‐synovectomy orthopaedic procedures (8%), synovectomy (5%), circumcision (5%) and miscellaneous (7%). All patients received preoperative factor replacement (100% in haemophilia patients) followed by various factor replacement regimens postoperatively. No deaths or life‐threatening bleeding occurred with any procedure. Twelve of 168 procedures (7%) were complicated by bleeding. Tonsillectomy was the most common procedure complicated by haemorrhage 4 of 15 (26%) followed by nasal surgery (3/7 bleeds = 43%). The CVL surgeries were remarkably free of complications with only 1/69 (1.4%) with bleeding. Surgical procedures are safe in children with bleeding disorders with adequate planning and factor replacement. Bleeding remains a problem in a subset of patients and requires ongoing haematological involvement and oversight. Delayed bleeding following T&A was especially common and suggests a need for close follow‐up and ongoing factor coverage for this group of patients.

Combination chemotherapy with ifosfamide and etoposide is effective in the treatment of central nervous system metastasis of childhood neuroblastoma

Childhood neuroblastoma is a neural crest‐derived tumor that presents most commonly during this period of life. In disseminated form, it is resistant to cure by chemotherapy. The tumor tends to recur in diverse locations after an initial clinical response. The parenchyma of the central nervous system (CNS) is a rare location for meta‐static disease and typically represents terminal disease spread. Therefore, effective therapy for CNS metastasis of neuroblastoma has not been reported. The authors describe the case of a child who had a large parenchymal CNS metastasis at the time of initial recurrence of Stage IV neuroblastoma. Chemotherapy with ifosfamide and etoposide resulted in complete resolution of this lesion.

A clinically applicable technique to study cytoskeletal dynamics in normal and abnormal polymorphonuclear leukocytes isolated from small volume blood samples.

Shape change and motility of polymorphonuclear leukocytes (PMNs) are essential for host defense and require dynamic reorganizations of microfilamentous cytoskeleton by reversible polymerization of G-actin into filaments (F-actin). Although clinical disorders of actin polymerization are rare, recently described simple methodologies for assaying actin dynamics in PMNs make the technique readily applicable to clinical studies. To develop a clinically useful F-actin assay, the authors investigated the optimal preparation conditions for PMN isolation that resulted in the least in vitro cytoskeletal activation and evaluated the variability in actin dynamics in acutely and chronically infected patients. Basal and chemotactic factor-activated PMN F-actin content was measured by a previously described flow cytometric technique in fixed, permeabilized, NBD phallacidin-stained PMNs isolated by centrifugation in Percoll or Ficoll-Hypaque density gradients or by counter-current elutriation. F-actin content is expressed as mean fluorescent channel or relative fluorescence intensity. Basal F-actin in PMNs prepared from countercurrent elutriation (mean fluorescent channel = 79.0 ± 4.5, n = 6) or by Ficoll Hypaque (82.0 ± 3.5, n = 4) was significantly higher than endotoxin free, Percoll purified PMNs, whether purified in bulk (56.1 ± 7.9, n = 8) or by the small volume modification applicable to clinical studies (53.3 ± 8.7, n = 15). Basal Ficoll Hypaque purified PMNs have evidence of shape change, whereas endotoxin free, Percoll purified PMNs are smooth and round and represent the most basal cell equivalent in F-actin content to a circulating PMN. In contrast, maximal F-actin in PMNs observed 45 seconds after N-formyl-L-methionyl-L-leucyl-L-phenylalanine activation (10–9 M) was highest in Ficoll Hypaque (relative fluorescence intensity = 2.21 ± 0.30, n = 4), then countercurrent elutriation (1.77 ± 0.25, n = 6) and finally, large (1.60 ± 0.21, n = 8) or mini volume (1.64 ± 0.28, n = 12) endotoxin free, Percoll purified PMNs, suggesting that preparative regimens can activate the cytoskeleton of PMNs and may affect clinical evaluation of actin dynamics in clinical or basic science studies. With the small volume (5.0 mL) modification of the endotoxin free, Percoll technique, the authors compared actin dynamics in patients with acute and chronic infections with those of healthy volunteers. This novel technique offers unique opportunities for study of the cytoskeleton in PMNs from healthy and unhealthy patients and defines optimal conditions for clinical investigations of cytoskeletal dynamics.