ABSTRACT
The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.
Subject(s)
AIDS Vaccines/therapeutic use , Clinical Trials as Topic/standards , Cryopreservation/standards , HIV Infections/therapy , Immunologic Tests/standards , Laboratories/standards , Laboratory Proficiency Testing/standards , Leukocytes, Mononuclear/immunology , Monitoring, Immunologic/standards , Specimen Handling/standards , Africa , Cell Survival , Consensus , Cooperative Behavior , Guideline Adherence/standards , HIV Infections/diagnosis , HIV Infections/immunology , HIV Infections/virology , Humans , International Cooperation , Leukocytes, Mononuclear/virology , Longitudinal Studies , Observer Variation , Practice Guidelines as Topic/standards , Predictive Value of Tests , Program Development , Program Evaluation , Quality Control , Reproducibility of Results , Time Factors , Treatment Outcome , United States , WorkflowABSTRACT
Mutations in the gene encoding the cell adhesion molecule L1 or its close homologue, CHL1 (close homologue of L1), cause brain dysfunction in both humans and mice. Here we report that prepulse inhibition (PPI) of the acoustic startle response is impaired in mice deficient in either L1 or CHL1. This newly identified feature may provide a basis for using these mice as models for sensorimotor gating impairment found in some neuropsychiatric disorders such as schizophrenia.
Subject(s)
Ion Channel Gating/physiology , Neural Cell Adhesion Molecule L1/physiology , Neural Inhibition/physiology , Reflex, Startle/physiology , Acoustic Stimulation , Animals , Cell Adhesion Molecules , Female , Ion Channel Gating/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Models, Animal , Neural Cell Adhesion Molecule L1/deficiency , Neural Inhibition/genetics , Proteins/physiology , Reflex, Startle/geneticsABSTRACT
Individuals expressing an APOE4 genotype demonstrate increased Alzheimer's disease (AD) neuropathology and a decreased onset age. The APOE4 gene may act by modulating the CNS immune response. Using human monocyte-derived macrophages (MDM), we show a significantly greater increase in NO production during immune activation in MDM from APOE4 AD patients compared to normal, age-matched individuals or to AD patients with an APOE 3/3 genotype. Microglia and peritoneal macrophages from APOE4 targeted replacement mice demonstrate a similar increase in NO compared to the APOE3 targeted replacement mice. The enhanced macrophage responsiveness and the increased production of NO in APOE4 AD patients may predispose the CNS to an increased potential for nitration and nitrosation, consistent with the redox imbalance and neuroinflammatory state seen in AD.
Subject(s)
Apolipoproteins E/genetics , Genotype , Macrophages/metabolism , Nitric Oxide/metabolism , Aged , Alzheimer Disease/blood , Alzheimer Disease/complications , Alzheimer Disease/genetics , Animals , Apolipoprotein E3 , Apolipoprotein E4 , Apolipoproteins E/deficiency , Case-Control Studies , Cells, Cultured , Dementia/blood , Dementia/complications , Dementia/genetics , Female , Humans , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Male , Mice , Mice, Knockout , Microglia/enzymology , Middle Aged , Monocytes/enzymology , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Species SpecificityABSTRACT
The mechanism linking the APOE4 gene with increased susceptibility for Alzheimer's disease (AD) and poorer outcomes following closed head injury and stroke is unknown. One potential link is activation of the innate immune system in the CNS. Our previously published data demonstrated that apolipoprotein E regulates production of nitric oxide, a critical cytoactive factor released by immune active macrophages. To determine if immune regulation is different in the presence of apolipoprotein E4 compared to apolipoprotein E3, we have measured NO production by peritoneal and CNS macrophages (microglia) cultured from transgenic mice that only express the human apoE4 or apoE3 protein isoform. Significantly more NO was produced in APOE4 mice compared to APOE3 transgenic mice that only express human apoE3 protein. Similarly, monocyte derived macrophages from humans carrying APOE4 gene alleles also produce significantly greater NO than those individuals with APOE3. The mechanism for this isoform-specific difference in NO production is not known and multiple sites in the NO production pathway may be affected. Expression of inducible nitric oxide synthase (iNOS) mRNA and protein are not significantly different between the APOE3 and APOE4 mice, suggesting that induction of iNOS is not a primary cause of the increased NO production in APOE4 animals. One alternative regulatory mechanism that demonstrates isoform specificity is arginine transport, which is greater in microglia from APOE4 transgenic mice compared to microglia from APOE3 mice. Increased transport is consistent with an increased production of NO and may reflect a direct or indirect effect of the APOE genotype on microglial arginine uptake and microglial activation in general. Overall, greater NO production in APOE4 carriers where characteristically high levels of oxidative/nitrosative stress are found in diseases such as AD provides a mechanism that potentially explains the genetic association between APOE4 and human diseases.
