Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Symptomatic spinal cord malperfusion after stent-graft coverage of the entire descending aorta

The study aims to identify risk constellations for symptomatic spinal cord malperfusion in patients undergoing extensive stent-graft coverage of the thoracic aorta.

Tuberculosis in HIV programmes in lower-income countries: practices and risk factors

BACKGROUND: Tuberculosis (TB) is a common diagnosis in human immunodeficiency virus (HIV) infected patients on antiretroviral treatment (ART). OBJECTIVE: To describe TB-related practices in ART programmes in lower-income countries and identify risk factors for TB in the first year of ART. METHODS: Programme characteristics were assessed using standardised electronic questionnaire. Patient data from 2003 to 2008 were analysed and incidence rate ratios (IRRs) calculated using Poisson regression models. RESULTS: Fifteen ART programmes in 12 countries in Africa, South America and Asia were included. Chest X-ray, sputum microscopy and culture were available free of charge in respectively 13 (86.7%), 14 (93.3%) and eight (53.3%) programmes. Eight sites (53.3%) used directly observed treatment and five (33.3%) routinely administered isoniazid preventive treatment (IPT). A total of 19 413 patients aged ≥16 years contributed 13 227 person-years of follow-up; 1081 new TB events were diagnosed. Risk factors included CD4 cell count (>350 cells/μl vs. <25 cells/μl, adjusted IRR 0.46, 95%CI 0.33–0.64, P < 0.0001), sex (women vs. men, adjusted IRR 0.77, 95%CI 0.68–0.88, P = 0.0001) and use of IPT (IRR 0.24, 95%CI 0.19–0.31, P < 0.0001). CONCLUSIONS: Diagnostic capacity and practices vary widely across ART programmes. IPT prevented TB, but was used in few programmes. More efforts are needed to reduce the burden of TB in HIV co-infected patients in lower income countries.

Limb apraxia in multiple sclerosis: prevalence and impact on manual dexterity and activities of daily living

To evaluate the prevalence and impact of limb apraxia on manual dexterity and activities of daily living (ADLs) in patients with multiple sclerosis (MS).

Bilateral central crystalline corneal deposits four years after intacs for myopia

PURPOSE: To report a case of bilateral central crystalline keratopathy in the anterior stroma occurring 4 years after Intacs implantation. METHODS: A 45-year-old woman underwent bilateral uncomplicated Intacs implantation for myopia. The postoperative course was uneventful. However, between 3 and 4 years after surgery, the patient developed central opacifications of the anterior stroma in both eyes, reducing best spectacle-corrected visual acuity. RESULTS: Intacs were explanted. Confocal microscopy, electron microscopy of the explanted ring segments, and microbiology studies were performed. Opacities were still detectable at the slit-lamp microscope up to 8 months after explantation. CONCLUSIONS: This is the first report on central corneal opacifications after Intacs implantation for myopia. The opacities could be the result of chronic metabolic stress or the beginning of lipid-like changes in another more central corneal localization.

Design, synthesis, and biological evaluation of somatostatin-based radiopeptides

The prototypes for tumor targeting with radiolabeled peptides are derivatives of somatostatin. Usually, they primarily have high affinity for somatostatin receptor subtype 2 (sst2), and they have moderate affinity for sst5. We aimed at developing analogs that recognize different somatostatin receptor subtypes for internal radiotherapy in order to extend the present range of accessible tumors. We synthesized DOTA-octapeptides based on octreotide by replacing Phe3 mainly with unnatural amino acids. The affinity profile was determined by using cell lines transfected with sst1-5. Internalization was determined by using AR42J, HEK-sst3, and HEK-sst5 cell lines, and biodistribution was studied in rat tumor models. Two of the derivatives thus obtained showed an improved binding affinity profile, enhanced internalization into cells expressing sst2 and sst3, respectively, and better tumor:kidney ratios in animals.

Internal iliac artery embolization before endovascular repair of aortoiliac aneurysms with a nitinol vascular occlusion plug

PURPOSE: To evaluate the acute and midterm effectiveness of a novel vascular occlusion device for embolization of the internal iliac artery (IIA) before endovascular repair of aortoiliac aneurysms. MATERIALS AND METHODS: Between March 2005 and April 2006, nine men (mean age, 75 years +/- 5; range, 66-83 y) with aortoiliac aneurysms underwent bifurcated endovascular stent-graft procedures. All these patients were referred specifically for embolization. Pre- and perioperatively, eight patients underwent unilateral embolization and one underwent bilateral embolization of the IIA to prevent type II endoleak. Via a contralateral femoral approach with a 6-F or 8-F sheath, the embolization procedure was performed with an Amplatzer Vascular Plug, a self-expandable cylindrical device consisting of a nitinol-based wire mesh. Technical success, clinical outcome, and complications were evaluated. Follow-up at 3, 6, and 12 months was performed with clinical and radiologic examinations. RESULTS: IIA embolization was technically successful in all cases and no procedure-related complications occurred. Imaging at discharge and at 3-, 6-, or 12-month follow-up was accomplished in all nine patients. Control computed tomography and magnetic resonance angiography did not reveal retrograde perfusion of the aneurysmal sac, ie, type II endoleak. Three of nine patients (33.3%) reported symptoms of buttock claudication that did not resolve completely. Clinical symptoms such as bowel ischemia or sexual dysfunction were not observed. CONCLUSIONS: The midterm results of this study suggest that preoperative IIA embolization with a nitinol vascular occlusion plug during endovascular treatment of aortoiliac aneurysms is safe and effective.

Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing

PURPOSE: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. METHODS: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. RESULTS: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. CONCLUSIONS: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.

Addition of dextran sulfate to blood cardioplegia attenuates reperfusion injury in a porcine model of cardiopulmonary bypass

OBJECTIVE: Contact of blood with artificial surfaces and air as well as ischemia/reperfusion injury to the heart and lungs mediate systemic and local inflammation during cardiopulmonary bypass (CPB). Activation of complement and coagulation cascades leads to and accompanies endothelial cell damage. Therefore, endothelial-targeted cytoprotection with the complement inhibitor and endothelial protectant dextran sulfate (DXS, MW 5000) may attenuate CBP-associated myocardial and pulmonary injury. METHODS: Eighteen pigs (DXS, n=10; phosphate buffered saline [PBS], n=8) underwent standard cardiopulmonary bypass. After aortic cross-clamping, cardiac arrest was initiated with modified Buckberg blood cardioplegia (BCP), repeated after 30 and 60 min with BCP containing either DXS (300 mg/10 ml, equivalent to 5mg/kg) or 10 ml of PBS. Following 30 min reperfusion, pigs were weaned from CPB. During 2h of observation, cardiac function was monitored by echocardiography and invasive pressure measurements. Inflammatory and coagulation markers were assessed regularly. Animals were then sacrificed and heart and lungs analyzed. RESULTS: DXS significantly reduced CK-MB levels (43.4+/-14.8 ng/ml PBS, 35.9+/-11.1 ng/ml DXS, p=0.042) and significantly diminished cytokine release: TNFalpha (1507.6+/-269.2 pg/ml PBS, 222.1+/-125.6 pg/ml DXS, p=0.0071), IL1beta (1081.8+/-203.0 pg/ml PBS, 110.7+/-79.4 pg/ml DXS, p=0.0071), IL-6 (173.0+/-91.5 pg/ml PBS, 40.8+/-19.4 pg/ml DXS, p=0.002) and IL-8 (304.6+/-81.3 pg/ml PBS, 25.4+/-14.2 pg/ml DXS, p=0.0071). Tissue endothelin-1 levels were significantly reduced (6.29+/-1.90 pg/100mg PBS, 3.55+/-1.15 pg/100mg DXS p=0.030) as well as thrombin-anti-thrombin formation (20.7+/-1.0 microg/ml PBS, 12.8+/-4.1 microg/ml DXS, p=0.043). Also DXS reduced cardiac and pulmonary complement deposition, neutrophil infiltration, hemorrhage and pulmonary edema (measured as lung water content, 81+/-3% vs 78+/-3%, p=0.047), indicative of attenuated myocardial and pulmonary CPB-injury. Diastolic left ventricular function (measured as dp/dt(min)), pulmonary artery pressure (21+/-3 mmHg PBS, 19+/-3 mmHg DXS, p=0.002) and right ventricular pressure (21+/-1 mmHg PBS, 19+/-3 mmHg DXS p=0.021) were significantly improved with the use of DXS. CONCLUSIONS: Addition of DXS to the BCP solution ameliorates post-CPB injury and to a certain extent improves cardiopulmonary function. Endothelial protection in addition to myocyte protection may improve post-CPB outcome and recovery.

P450 oxidoreductase deficiency: a new disorder of steroidogenesis affecting all microsomal P450 enzymes

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase (POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients' phenotypes, but that assays of POR-supported 17alpha-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism.

5-year results comparing mineral trioxide aggregate and adhesive resin composite for root-end sealing in apical surgery

INTRODUCTION Recent meta-analyses of the outcome of apical surgery using modern techniques including microsurgical principles and high-power magnification have yielded higher rates of healing. However, the information is mainly based on 1- to 2-year follow-up data. The present prospective study was designed to re-examine a large sample of teeth treated with apical surgery after 5 years. METHODS Patients were recalled 5 years after apical surgery, and treated teeth were classified as healed or not healed based on clinical and radiographic examination. (The latter was performed independently by 3 observers). Two different methods of root-end preparation and filling (primary study parameters) were to be compared (mineral trioxide aggregate [MTA] vs adhesive resin composite [COMP]) without randomization. RESULTS A total of 271 patients and teeth from a 1-year follow-up sample of 339 could be re-examined after 5 years (dropout rate = 20.1%). The overall rate of healed cases was 84.5% with a significant difference (P = .0003) when comparing MTA (92.5%) and COMP (76.6%). The evaluation of secondary study parameters yielded no significant difference for healing outcome when comparing subcategories (ie, sex, age, type of tooth treated, post/screw, type of surgery). CONCLUSIONS The results from this prospective nonrandomized clinical study with a 5-year follow-up of 271 teeth indicate that MTA exhibited a higher healing rate than COMP in the longitudinal prognosis of root-end sealing.