Evaluation of an automated hematologyanalyzer (CELL-DYN 4000) for counting CD4+ T helper cells at low concentrations

In human immunodeficiency virus (HIV) infection, CD4 cell counts are useful in defining the disease state, monitoring antiviral treatment, and identifying patients at risk for opportunistic infections. Counting CD4 cells typically relies on traditional immuno-flow cytometric analyzers that require opening the tube for manipulation of the blood sample. In addition to automated blood cell counting, the CELL-DYN 4000 hematology analyzer performs a completely enclosed and automated analysis of the T lymphocyte subsets. We studied the performance characteristics of this method in blood samples containing low levels of CD4+ T cells. In one set of experiments, we emulated low level CD4 counts by use of a CD4 Positive Isolation Kit to deplete the CD4+ cells from blood samples. We used the FACScan analyzer for reference counts. Measurements were made exactly 12 hr after venepuncture in samples that were stored at room temperature. In normal samples and those with low CD4+ cell counts, there was excellent correlation between the results of the CELL-DYN and FACScan methods. Using the CELL-DYN 4000 analyzer, the precision of CD4+ and CD8+ T-cell counts was high (CV = 2 to 8%). The CD4+ T-cell count was linear over a wide range (35 to 1640 cells/microl). This study shows that CD4 and CD8 T cell counts using the CELL-DYN 4000 analyzer is suitable for normal samples and also for those with low CD4+ T cell counts. The method is rapid and automated, and blood specimens remain enclosed, minimizing the biohazard of exposure to blood of HIV patients.     

Further characterization of memory T cells existing in a case of CD8 deficiency

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.     

Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Summary Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.     

Loss of Hrs in the Central Nervous System Causes Accumulation of Ubiquitinated Proteins and Neurodegeneration

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs^flox/flox;SynI-cre mice. Notably, the hrs^flox/flox;SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs^flox/flox;SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.     

Somatic mosaicism of CAG repeat in dentatorubral-pallidoluysian atrophy (DRPLA)

An unstable expansion of CAG repeat in the coding region ofthe DRPLA gene on chromosome 12p is the mutation specific forhereditary dentatorubralpallidoluysian atrophy (DRPLA). We studiedthe CAG expansion in brain and other tissues from six unre latedDRPLA patients. The CAG repeat lengths showed distinct difterencesbetween tissues. The sizes of the CAG expansion in various regionsof the brain except the cerebellum were generally larger byseveral repeats than in other peripheral tissues. Brain samplesshowed greater variation of the expansion compared with othertissues, but neither the size of the CAG expansion nor the degreeof CAG repeat variation parallels the detailed findings of neuropathologicalinvolvement. We conclude that somatic instabilities of the CAGrepeat cause tissue variability of the CAG repeat size in DRPLAbut other region or cell type-specific factors would be involvedto explain the selectivity of cell damage in DRPLA.     

Chemiluminescence of whole blood. II. Application to clinical examination of phagocytic functions of whole blood from various types of disease

Measurements of whole blood chemiluminescence (CL) were carried out by using 0.1 ml of blood to evaluate phagocytic functions from various kinds of pediatric patients. Patients with definite bacterial infections showed an elevated background CL and high peak CL per 1000 granulocytes after stimulation with zymosan. These results suggest that granulocytes of these patients had been preactivated in vivo, their phagocytic and/or metabolic activity being enhanced. Contrary to these results, blood from the patients with viral infections showed almost no abnormalities. Patients with chronic granulomatous disease showed no significant CL by this method similar to conventional ones. This method was also shown to be a useful technique for monitoring the phagocytic functions of blood after granulocyte transfusion.     

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.