André Darveau

Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

ABSTRACT A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

Simultaneous detection and identification of hepatitis A virus and rotavirus by multiplex nucleic acid sequence-based amplification (NASBA) and microtiter plate hybridization system

Human rotavirus and hepatitis A virus (HAV) are two of the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for their detection in foodstuffs. In this study, a multiplex nucleic acid sequence-based amplification (NASBA) system was developed to detect specifically and simultaneously human rotavirus and HAV. Two sets of primers selected from published nucleic acid sequences were used in the NASBA mixture to amplify viral RNA from both viruses. Denaturing gel electrophoresis revealed two distinct RNA products with 268 and 474 nucleotides amplified from rotavirus and HAV, respectively. The specificity of the multiplex NASBA was confirmed by a microtiter plate hybridization and detection system and by Northern blot analysis using specific oligonucleotide probes. The presence of non-homologous nucleic acid and non-target microorganisms did not have any effect on the specificity of the multiplex NASBA. Using the optimized NASBA and microtiter plate hybridization conditions, as little as 400 PFU ml x (-1) of HAV and 40 PFU ml x (-1) of rotavirus were detected. The multiplex NASBA system offers advantages over monoplex virus detection systems in terms of turnaround time and cost-effectiveness.

CD40/CD40 Homodimers Are Required for CD40-induced Phosphatidylinositol 3-Kinase-dependent Expression of B7.2 by Human B Lymphocytes

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels. Oligomerization of CD40 leads to a rapid and significant increase in the disulfide-linked CD40/CD40 homodimer formation, a response that could be prevented using a thiol-alkylating agent. Formation of CD40/CD40 homodimers was found to be absolutely required for CD40-mediated activation of phosphatidylinositol 3-kinase, which, in turn regulated B7.2 expression. In contrast, CD40 monomers provided the minimal signal emerging from CD40, activating p38 MAP kinase and inducing homotypic B cell adhesion. CD40/CD40 homodimer formation was totally independent of TRAF1/2/3/5 associations with the threonine at position 254 in the cytoplasmic tail of the CD40 molecules. This finding may be vital to better understanding the molecular mechanisms that govern cell signaling triggered by CD40/CD154 interactions.

All Patients with Persistent Polyclonal B Cell Lymphocytosis Present Bcl-2/Ig Gene Rearrangements

The bcl-2 gene belongs to a class of oncogenes involved in the inhibition of apoptosis. Most follicular lymphomas are associated with the t(14;18) translocation that juxtaposes the bcl-2 gene located on chromosome 18 to the immunoglobulin gene locus located on chromosome 14. Consequently, the bcl-2 gene is overly expressed and leads to an accumulation of mature clonal B cells. Prolonged survival of the B cell clone appears to be the early event in tumorigenesis, creating an increased risk of cumulative mutations. Interestingly, bcl-2/Ig gene rearrangements may be identified in nearly 50% of normal individuals but the outcome of normal individuals carrying high levels of t(14;18) is not well defined. Persistent polyclonal B cell lymphocytosis (PPBL) is a unique polyclonal lymphoproliferative disorder mostly restricted to women. We have recently demonstrated that PPBL is also associated with multiple bcl-2/Ig gene rearrangements. In this report, we have extended our analysis to additional patients and demonstrated that all patients presented multiple detectable t(14;18) translocated clones. In addition, Bcl-2 protein expression was increased. Our findings, along with the clinical features of PPBL, make this disorder an exceptional model for the study of B-cell homeostasis.

Persistent polyclonal B-cell lymphocytosis : further evidence for a genetic disorder associated with B-cell abnormalities

Persistent polyclonal B‐cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B‐cell population is also evidenced by the presence of multiple bcl‐2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first‐degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B‐cell proliferation was shown in two additional siblings. Multiple bcl‐2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first‐degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co‐factors.

Requirement of Oxidation-dependent CD40 Homodimers for CD154/CD40 Bidirectional Signaling

It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys238 of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling.

Multiple bcl‐2/Ig gene rearrangements in persistent polyclonal B‐cell lymphocytosis

Persistent polyclonal B‐cell lymphocytosis is a benign lymphoproliferative disorder of unknown aetiology occurring exclusively in women, characterized by typical binucleated lymphocytes, polyclonal expansion of B cells and elevated serum IgM. Owing to the role of Bcl‐2 oncogene in inhibition of apoptosis, we have investigated the presence of the bcl‐2/Ig gene rearrangement. Bcl‐2/Ig gene rearrangement was determined by polymerase chain reaction targeting the usual breakpoint regions of the t(14;18). Bcl‐2/Ig gene rearrangement was identified in all six patients and, more importantly, multiple rearrangements were present in five patients. The frequency of the bcl‐2/Ig gene rearrangement is estimated to be of one translocation in 1 × 102 to 1 × 103 peripheral blood mononuclear cells. We conclude that persistent polyclonal B‐cell lymphocytosis is associated with bcl‐2/Ig gene rearrangement. These findings are of clinical importance because these patients may be misdiagnosed as having a leukaemic expression of non‐Hodgkins lymphoma.

[6] PCR-mediated synthesis of chimeric molecules

Publisher Summary The introduction of the polymerase chain reaction (PCR) has revolutionized molecular biology. Not only does it allow the amplification and detection of an infinitesimal amount of DNA, it also facilitates and accelerates most of the steps involved in common techniques used in every laboratory dealing with molecular biology. In addition, it has opened the way to the development of new technologies. Antibodies are naturally made by joining together components with different functions at a molecular level; the variable region at the amino terminus of the molecule is involved in the recognition of a given antigen by the antibody, while the constant region at the carboxy-terminal end supports the effector functions of the antibody. However, it has become more prevalent to assist nature by manipulating in vitro immunoglobulin genes to create new reagents. PCR is used to facilitate the creation of chimeric molecules derived from antibodies. The chapter presents three examples of increasing complexity pertaining to immunoglobulin genes. The approach that is described in the chapter can be used for any proteins for which DNA is available.

Tuning of CD40–CD154 Interactions in Human B-Lymphocyte Activation: A Broad Array of In Vitro Models for a Complex In Vivo Situation

Naive and memory B-lymphocyte populations can be activated through the binding of CD154 to CD40, a receptor that is constitutively expressed on the surface of these cells. Models based on the in vitro stimulation of human B lymphocytes through CD40 have greatly contributed to our understanding of the human immune response in healthy individuals and patients suffering from immune disorders. The nature of the engineered CD40 ligands is as diverse as the in vitro models used in studies of CD40-activated B lymphocytes. Monoclonal anti-CD40 antibodies, recombinant CD154 proteins, soluble CD154+ membranes as well as CD154+ cell lines have turned out to be very useful tools, and are still in use today. As for any receptor–ligand interaction, parameters such as duration and strength of contact, timing, affinity, and receptor density are major determinants of CD40 binding by CD154 or anti-CD40. Furthermore, variation in the intensity of CD40 stimulation has been shown to influence proliferation, differentiation and immunoglobulin secretion of human hybridomas, B-cell lines, tonsil and blood B lymphocytes. The objective of this review is to present an overview of the great diversity of CD40 agonists used in in vitro models of B-lymphocyte activation, with a particular emphasis on variations in the resulting strength of CD40 signaling generated by these models. A better understanding of these models could open up new avenues for the rational use of human B lymphocytes as antigen-presenting cells in cellular therapies.

PRODUCTIVE INFECTION OF NORMAL CD40-ACTIVATED HUMAN B LYMPHOCYTES BY HIV-1

ObjectiveAntigen-driven B-cell proliferation and maturation occur in germinal centers present in lymphoid tissues. This process is highly dependent on functional interactions between B and T lymphocytes. In vitro activation of CD40 present on B cells mimics B cell-T cell interactions and allows the proliferation of normal Epstein-Barr virus (EBV)-negative B lymphocytes. In HIV-1-seropositive individuals, B cells become exposed to free viral particles and to infected T lymphocytes while migrating through germinal centers. The effect of HIV-1 viral exposure on CD40-activated B lymphocytes was therefore examined. MethodsFreshly isolated B lymphocytes were cultured in vitro through activation of CD40. B-cell proliferation, HIV-1 infectivity and viral production were monitored following B-lymphocyte exposure to HIV-1. In addition, HIV-mediated fusion between infected B cells and uninfected CD4+ T lymphocytes was assessed in a co culture assay. ResultsEBV-negative, CD40-activated human B lymphocytes were directly infected by HIV-1. The infection significantly reduced their proliferation rate. Viral production was detected in B-cell culture supernatant. Numerous fusion events indicated that HIV-1 infection of B lymphocytes could spread to T lymphocytes following HIV-1-mediated fusion of these two cell types. ConclusionIn view of the importance of B cell-T cell interactions in the maintenance of a functional immune system, disruption of B-lymphocyte development could have direct implications on the course of AIDS progression.