High-throughput purification of viral RNA based on novel aqueous chemistry for nucleic acid isolation

BACKGROUND: Extraction protocols using magnetic solid phases offer a high potential for automation. However, commercially available magnetic-bead-based assays either lack the sensitivity required for viral diagnostics or are disproportionately expensive. METHODS: We developed an aqueous chemistry for extraction of viral nucleic acids from plasma samples by use of common magnetic silica beads. Nucleic acids were bound to the beads at acidic conditions in the presence of a kosmotropic salt and were eluted at a slightly alkaline pH. The method was implemented on a standard pipetting workstation for fully automated extraction of up to 48 samples of 240 muL plasma in 1 batch. RESULTS: The detection limit of the method was comparable to the spin-column-based QIAamp Viral RNA Mini Kit, which relies on chaotropic salts and binding to a silica membrane, as the comparison method. The 95% detection limit was 23.1 IU per PCR for HIV-1 and 10.7 IU per PCR for hepatitis C virus (HCV). Suitability for clinical routine testing was confirmed in a total of 178 HIV-1- or HCV-positive plasma samples. The method linearity (R(2)) was >0.99 for the viruses evaluated. CONCLUSIONS: Use of reagents without organic solvents allows simple and cost-effective automation of this method on common pipetting robots with low risk of contamination. Performance characteristics of the novel extraction method make it suitable for use in diagnosis of infectious diseases and viral load determinations.     

Prekallikrein-Kallikrein conversion rate as a predictor of contrast material catastrophies

An in vitro is described that attempts to detect patients with a potential for adverse systemic reactions to contrast material. This test involves measuring the rate of conversion of prekallikrein to kallikrein under certain standard conditions. In a preliminary retrospective study, the test could be used to identify such patients with a sensitivity of 88%, a specificity of 82%, and a predictive value of 79%.     

Local activation of dendritic cells leads to insulitis and development of insulin-dependent diabetes in transgenic mice expressing CD154 on the pancreatic beta-cells

The initial events leading to activation of the immune system in type 1 diabetes are still largely unknown. In vivo, dendritic cells (DCs) are thought to be the only antigen-presenting cells (APCs) capable of activating na?ve T-cells and are therefore important for the initiation of the autoimmune response. To test the effect of activating islet-associated APCs in situ, we generated transgenic mice expressing CD154 (CD40 ligand) under control of the rat insulin promoter (RIP). RIP-CD154 mice developed both insulitis and diabetes, although with different incidence in independent lines. We show that activated DCs could be detected both in the pancreas and in the draining pancreatic lymph nodes. Furthermore, diabetes development was dependent on the presence of T- and B-cells since recombination-activating gene (RAG)-deficient RIP-CD154 mice did not develop diabetes. Finally, we show that the activation of immune cells was confined to the pancreas because transplantation of nontransgenic islets to diabetic recipients restored normoglycemia. Together, these data suggest that expression of CD154 on the beta-cells can lead to activation of islet-associated APCs that will travel to the lymph nodes and activate the immune system, leading to insulitis and diabetes.     

Diskography outcomes in patients following lumbar diskectomy

The results of lumbar diskography at post-diskectomy and nonoperative disk levels in postoperative patients and patients without prior back surgery were reviewed over 3 years. Other possible predictive factors, including disk degeneration (per the Adam's classification), end-point resistance, gender, and age, also were reviewed. The results revealed that no statistically significant association was noted between the presence of a prior diskectomy and the outcome of diskography. However, disk degeneration classified as ruptured and fissured correlated statistically with positive diskography. Additionally, age between 30 and 39 years and male gender were statistically associated with a positive diskogram. Disk levels displaying a poor end point during diskography injection (not amendable to pressurization) were statistically related to ruptured or fissured disk levels and thus positive diskography. Based on these results, the assumption that disabling low-back pain presenting after lumbar procedure is due to diskogenic disease arising from the surgical level is not supported.