Results of a high-resolution genome screen of 437 Alzheimer's disease families

Alzheimer's disease (AD) is a devastating neurodegenerative disorder of late life with complex inheritance. Mutations in three known genes lead to the rare early-onset autosomal dominant form of AD, while a common polymorphism (epsilon 4) in the gene encoding apolipoprotein E (APOE ) is a risk factor for more typical late-onset (>60 years) AD. A recent study concluded that there are up to four additional genes with an equal or greater contribution to the disease. We performed a 9 cM genome screen of 437 families with AD, the full National Institute of Mental Health (NIMH) sample, which has been carefully ascertained, evaluated and followed by our group over the last decade. Performing standard parametric and non-parametric linkage analyses, we observed a 'highly significant' linkage peak by Lander and Kruglyak criteria on chromosome 19q13, which probably represents APOE. Twelve additional locations-on 1q23, 3p26, 4q32, 5p14, 6p21, 6q27, 9q22, 10q24, 11q25, 14q22, 15q26 and 21q22-met criteria for 'suggestive' linkage [i.e. two-point lod score (TLS) >/=1.9 and/or multipoint lod score (MLS) >/=2.2] in at least one of our analyses. Although some of these will surely prove to be false positives, these linkage signals should provide a valuable framework for future studies aimed at identifying additional susceptibility genes for late-onset AD.     

Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin

Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42. In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB. Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity. Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity. In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins. In line with reduced enzymatic activity, the mutants also lacked cytotoxicity. Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB. Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location. Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target. Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate. This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Differential modulation of Ca(v)2.1 channels by calmodulin and Ca2+-binding protein 1

Ca(v)2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. Here we report that the neuronal Ca2+-binding protein 1 (CaBP1) modulates Ca(v)2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Ca(v)2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the alpha1 subunit of Ca(v)2.1 channels (alpha12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with alpha12.1 from transfected cells and brain extracts, and colocalizes with alpha12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. Our results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons.     

Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease.     

Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts

Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors.     

Protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a

Subunit intranasal vaccines offer the prospect of inducing combined systemic-mucosal immunity against mucosally transmitted infections such as human immunodeficiency virus. However, although human studies have demonstrated the induction of active immunity, secretory immunoglobulin A (sIgA) responses are variable, and no study has demonstrated protection by accepted vaccine-licensing criteria as measured by direct toxin-neutralizing activity. Using the genetically inactivated mutant diphtheria toxoid CRM(197) in a bioadhesive polycationic polysaccharide chitosan delivery system, we found that a single nasal immunization was well tolerated and boosted antitoxin neutralizing activity in healthy volunteers, which could be further boosted by a second immunization. The neutralizing activity far exceeded accepted protective levels and was equivalent to that induced by standard intramuscular vaccine and significantly greater than intranasal immunization with CRM(197) in the absence of chitosan. A striking but unexpected observation was that although unilateral intranasal immunization induced circulating antitoxin antibody-secreting cells, a nasal antitoxin sIgA response was seen only after the second immunization and only in the vaccinated nostril. If these data are reproduced in larger studies, an intranasal diphtheria vaccine based on CRM(197)-chitosan could be rapidly licensed for human use. However, a restricted sIgA response suggests that care must be taken in the priming-boosting strategy and clinical sampling techniques when evaluating such vaccines for the induction of local mucosal immunity.     

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

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