Site Readiness Framework to Improve Health System Preparedness for a Potential New Alzheimer's Disease Treatment Paradigm.

New therapies that address the underlying pathophysiology of Alzheimer's Disease (AD), coupled with the growth of the AD population, will transform the AD care pathway and present significant challenges to health systems. We explored real-world challenges health systems may face in delivering potential new AD therapies with diverse stakeholders. Key challenges in care included integrating primary care providers into assessment and management, availability of memory care specialists, understanding payment and coverage issues and training mid-level providers to help coordinate care and serve as a shared resource across the system. This input informed a novel Site Readiness Framework for AD, comprising self-assessment exercises to identify health system capabilities and gaps and a framework of core strategies and responsive tools to help prepare to integrate new AD therapies. These resources may help health systems improve readiness to modify care pathways to integrate new therapies for AD.

Identification of alternative splice products encoded by the human coxsackie-adenovirus receptor gene.

The human cellular receptor for group B coxsackieviruses and adenoviruses (HCAR) is a transmembrane glycoprotein which belongs to the immunoglobulin superfamily. We describe alternative splicing of the HCAR-gene and the existence of three exon-skipping splice variants in addition to the originally identified seven exon-encompassing mRNA transcript. Expression of the splice variants theoretically results in truncated proteins, possibly leading to impaired viral binding and/or the occurrence of soluble viral receptors due to the absence of the transmembranous region. Consequently, this could markedly influence the efficacy of an adenovirus subgroup C-mediated gene therapy.

Molecular analysis of the prototype coxsackievirus B5 genome.

To facilitate studies of the phylogenetic relationship between enteroviruses, in particular the prototype strain (Faulkner) of coxsackievirus B5 (CVB5F) and other CVB5 isolates and to facilitate studies of the interactions between CVB5F and the target cell, the complete nucleotide sequence of the prototype has been determined. The complete sequence was collected from three overlapping reverse transcription polymerase chain reaction (RT-PCR) generated amplicons. Molecular analysis of the CVB5F genome verified that this strain is more related to other CVB5 isolates and to swine vesicular disease virus strains than to other enteroviruses. In addition, comparison of the amino acid sequence derived from the structural genes indicated a division of group B coxsackievirus into subgroups, where CVB1, CVB3 and CVB5 constitute one group, CVB2 and CVB4 from a second group and CVB6 prototype forms a branch of its own. This observation, supported by reports describing the interactions between CVB and the cell surface, may reflect that these subgroups of group B coxsackieviruses have evolved to use slightly different approaches to carry out the complete infectious cycle within the cell.

Genomic and phylogenetic characterization of coxsackievirus B2 prototype strain Ohio-1.

The human picornavirus coxsackievirus B2 (CVB2) is often linked to several infections, from mild respiratory diseases to more severe illnesses such as myocarditis. In this study, we report the complete genome sequence of CVB2 prototype strain Ohio-1. The genome sequence was determined from reverse transcribed viral RNA, amplified with long distance PCR and used for non-radioactive sequencing. The full length PCR amplicons were used for in vitro transcription and the obtained cRNA was lipofected onto green monkey kidney cells, in order to confirm that the PCR generated sequence reflects a viable virus RNA. The CVB2 genome sequence shows a typical enterovirus genome organization with a total length of 7411 nucleotides. Phylogenetic analysis, using the CVB2 polyprotein in comparison with other enterovirus polyproteins, clearly shows that CVB2 clusters with the coxsackievirus B-like enteroviruses and is more related to coxsackievirus B4 (CVB4) than any other published CVB serotype. The grouping of CVB2 and CVB4 as one subgroup has earlier been reported in connection with receptor usage and ability to replicate in different cell lines. The exposed viral capsid proteins of CVB2 (VP1-VP3) show high similarity to other CVB proteins, except in regions that are likely to be surface epitopes.

Amplification and cloning of complete enterovirus genomes by long distance PCR.

A method for amplification and cloning of complete enterovirus cDNA genomes is described. Viral RNA was reverse transcribed using an optimized protocol and a reverse transcriptase with reduced RNase H activity. Amplicons corresponding to complete genomes of 14 prototype strains of group B coxsackieviruses and echoviruses were amplified using oligonucleotide primers derived from the Coxsackievirus B3 genomic sequence of the 5' and 3' ends and a mixture of thermostable DNA polymerases. Coxsackievirus B2 amplicon was then cloned and the terminal sequences of the insert were determined. Lipofection of individual clones resulted in productive. Coxsackievirus B2 infection. The method described makes it possible to obtain large amounts of complete enterovirus cDNAs and simplifies the construction of infectious full-length cDNA clones. Successful amplification of all enterovirus prototype strains tested emphasizes the general use of the method described, which provides a rapid and efficient alternative to traditional cloning strategies.

A competitive RT-PCR method for the quantitative analysis of cytokine mRNAs in mouse tissues.

The authors describe the design and validation of a competitive RT-PCR method for the efficient and reproducible quantitation of mRNA molecules of IFN-gamma, TNF-alpha, IL-4 and IL-10 in mouse spleen RNA extracts. Before being subjected to RT-PCR, the RNA extracts were supplemented with internal control RNAs (IC-RNAs), which were constructed by inserting DNA fragments in the cDNA of the respective cytokines. The efficiency of amplification of the target and the IC-RNA was shown to remain equal over a wide range of cycle numbers. Reproducibility was such that differences in mRNA contents that were greater than 17% could be detected between two RNA samples run in parallel. Normal mouse spleen tissue was found to contain 10(7)-10(8) molecules of TNF-alpha, IFN-gamma, IL-4 and IL-10 mRNA per micrograms total RNA extracted. Injection of animals with anti-CD3 antibody, a well-known cytokine inducer, resulted in a moderate increase in TNF-alpha and IL-10 mRNA levels (14- and 24-fold, respectively), and in a substantially greater increase in the levels of mRNA for IL-4 and IFN-gamma (199- and 851-fold, respectively). These results demonstrate an accurate and reliable quantitation of cytokine mRNA levels in animal tissues.