Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Predictive value of early HCV RNA quantitation for sustained response in nonresponders receiving daily interferon and ribavirin therapy

The prognostic value of early hepatitis C virus (HCV)-RNA load was evaluated among nonresponder patients to previous interferon (IFN) therapy treated with daily IFN and ribavirin. One hundred-six nonresponders (83 men), mean age 44.8 +/- 11 years, were treated with IFN-alpha 2b 3 MU/day for 24 weeks, followed by 3 MU x 3/week for 24 weeks plus ribavirin 1-1.2 g/day for 48 weeks. HCV RNA was quantified by Versant HCV RNA 3.0 assay (Bayer). The predictive values of the baseline and the change in viral load at week 1, 4, and 12 for sustained virological responses were analyzed using receiver operating characteristic (ROC) curves, as well as predictive values of >2 log(10) drop from baseline by weeks 1, 4, and 12 in combination with undetectable HCV RNA for sustained virological response. Thirty-two patients (30.2%) were sustained virological responders. The highest area under the curve was obtained at week 4. The unquantifiable HCV RNA level, in combination with at least a 2 log(10) drop in viral load by week 4 and week 12, had a negative predictive value of 96% and 97%, respectively. Nonresponse can be predicted as early as week 4 or week 12 in nonresponders treated with daily IFN and ribavirin.     

Presence of autoantibodies to apolipoprotein A-1 in patients with acute coronary syndrome further links autoimmunity to cardiovascular disease

Anti-apolipoprotein A-1 (Apo A-1) autoantibodies were described in autoimmune disorders such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and might be involved in the genesis of arterial and venous thrombotic events. To investigate the presence of these autoantibodies in patients with acute coronary syndrome (ACS) without other features of autoimmunity, we set up an enzyme-linked immunosorbent assay (ELISA) for anti-Apo A-1 antibodies. We used it to investigate their prevalence in ACS as compared to SLE and APS and correlated them to plasma Apo A-1 and serum amyloid A protein (SAA) concentrations. The prevalence of anti-Apo A-1 autoantibodies in the healthy control group was 1% (1/92), but was significantly higher in other groups: 21% (11/53) in ACS group (P=0.001), 13% (12/92) in SLE and/or APS group (P=0.005). Multiple linear regression revealed a significant correlation between plasma Apo A-1 (r=-0.72, P=0.013), plasma SAA concentration (r=0.76, P=0.0066) and anti-Apo A-1 IgG titre in ACS patients. The presence of anti-Apo A-1 autoantibodies in patients with ACS highlights an additional link between autoimmunity, inflammation and atherosclerosis.     

Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.     

Effect of electrical coupling on ionic current and synaptic potential measurements

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.     

HIV testing within at-risk populations in the United States and the reasons for seeking or avoiding HIV testing

OBJECTIVES: We determined proportions of high-risk persons tested for HIV, the reasons for testing and not testing, and attitudes and perceptions regarding HIV testing, information that is critical for planning prevention programs. METHODS: Cross-sectional interview study of persons at high risk for HIV infection (men who have sex with men [MSM]; injection drug users [IDUs]; and heterosexual persons recruited from gay bars, street outreach, and sexually transmitted disease clinics) among six states participating in the HIV Testing Survey (HITS) in 1995 to 1996 (HITS-I) and 1998 to 1999 (HITS-II). RESULTS: Overall testing rates were lower in the HITS-I (1226/1599 [77%]) than in the HITS-II (1375/1711 [80%]) (p =.01). Persons <25 years old tested less frequently than those >or=25 years old (HITS-I: 71% vs. 78%, respectively, p=.007; HITS-II: 63% vs. 85%, respectively, p<.001). The main reasons for testing and not testing were the same in both surveys, but the proportions of reasons for not testing differed (e.g., "unlikely exposed to HIV" [HITS-I (17%) vs. HITS-II (30%), p<.0001], "afraid of finding out HIV-positive" [HITS-I (27%) vs. HITS-II (18%), p<.0001]). Attitudes regarding HIV testing differed among tested and untested respondents, especially among MSM. CONCLUSIONS: HIV testing rates were higher in the HITS-II, but testing rates decreased among the youngest respondents. Denial of HIV risk factors and fear of being HIV-positive were the principal reasons for not being tested. Availability of new HIV therapies may have contributed to decreased fear of finding out that one is HIV infected as a reason to avoid testing. The increased proportion of persons at risk who did not test because they believed they were unlikely to have been exposed highlights the need for prevention efforts to address risk perceptions.     

Exclusion of Treacher Collins Franceschetti syndrome in a subject with tetralogy of Fallot and cryptorchidism

Genotyping with flanking DNA markers was used to ascertain Treacher Collins Franceschetti syndrome (TCOF1) in a subject affected by tetralogy of Fallot and cryptorchidism. The proband's family consisted of a father and sister who were affected by the disease, and a healthy mother. Since cardiac malformation and cryptorchidism have been associated with the TCOF1 syndrome, the proband was suspected to be a carrier of the mutated gene. Microsatellite markers D5S527, SPARC and D5S519, which previously mapped the TCOF1 gene within a 2.1-cM interval on chromosome 5 (5q32–33.1), were used to follow the transmission of the TCOf 1 mutated locus. Flanking markers D5S519 and D5S527 were informative and enabled us to exclude inheritance of a TCOF1 mutation to the proband, while showing that cardiac malformation and cryptorchidism were unrelated in mis patient.     

Congenital stationary night blindness: report of an autosomal recessive family and linkage analysis

Congenital stationary night blindness (CSNB) is a group of rare, non-progressive conditions of the retina characterized by abnormal rod function causing impaired night vision. Among them, the Schubert-Bornschein subgroup, itself divided into a complete and an incomplete form, is characterized by a specific electrophysiological pattern. Complete, Schubert-Bornschein CSNB is usually transmitted as a monogenic trait, and most familial cases result from mutations of the NYX gene located on the X chromosome. We report a very rare family with consanguineous, first-cousin parents, where a son and a daughter are affected with this condition, indicating autosomal recessive inheritance. As the family was too small for genome-wide linkage, we considered several candidate loci, including the sidekick SDK1 and SDK2 genes. The latter determine lamina-specific connectivity in the retina, a histological substrate of the ON pathway implicated in complete, Schubert-Bornschein CSNB. Although linkage was excluded in our family, observations like the present one may lead to the identification of a new molecular cause for this condition.     

Inflammation occurs early during the Abeta deposition process in TgCRND8 mice

Alzheimer's disease (AD) is characterized by a progressive cognitive decline leading to dementia and involves the deposition of amyloid-beta (Abeta) peptides into senile plaques. Other neuropathological features that accompany progression of the disease include a decrease in synaptic density, neurofibrillary tangles, dystrophic neurites, inflammation, and neuronal cell loss. In this study, we report the early kinetics of brain amyloid deposition and its associated inflammation in an early onset transgenic mouse model of AD (TgCRND8) harboring the human amyloid precursor protein gene with the Indiana and Swedish mutations. Both diffuse and compact plaques were detected as early as 9-10 weeks of age. Abeta-immunoreactive (Abeta-IR) plaques (4G8-positive) appeared first in the neocortex and amygdala, then in the hippocampal formation, and lastly in the thalamus. Compact plaques (ThioS-positive) with an amyloid core were observed as early as diffuse plaques were detected, but in lower numbers. Amyloid deposition increased progressively with age. The formation of plaques was concurrent with the appearance of activated microglial cells and shortly followed by the clustering of activated astrocytes around plaques at 13-14 weeks of age. This TgCRND8 mouse model allows for a rapid, time-dependent study of the relationship between the fibrillogenic process and the inflammatory response during the brain amyloidogenic process.