Hepatitis A virus attachment to agri-food surfaces using immunological, virological and thermodynamic assays.

AIMS: This study was designed to investigate the ability of hepatitis A virus (HAV) to attach to various food contact surfaces. METHODS AND RESULTS: HAV attachment was demonstrated after elution of attached viruses from solid surfaces by an immunofluorescent method using anti-HAV-specific antibodies and confocal microscopy. Attachment and survival of HAV on stainless steel, copper, polythene and polyvinyl chloride (PVC) at 20 and 4 degrees C after 2 and 4 h were quantified by plaque assay. HAV was shown to attach almost instantaneously to all four surfaces tested. Attachment of HAV depended on initial viral concentration and was slightly greater at 4 degrees C. The total surface energy (gammaTOT), nonpolar Lifshitz-Van der Waals (gammaLW) and polar short range (gammaSR) hydrogen-bonding components for HAV and each surface as well as total free energy of the system were determined by contact angle measurements using an extended Young equation [Young (1805) Philosophical Transactions of The Royal Society (London) 95, 65-87). The calculation of these parameters predicted the favourable conditions for attachment of HAV to all four surfaces tested. CONCLUSION: HAV particles attach to stainless steel, copper, polythene and PVC at 20 and 4 degrees C and the total free energy of the interaction is optimal for this attachment. SIGNIFICANCE AND IMPACT OF THE STUDY: Comprehension of viral attachment to the solid surfaces will permit to successfully disinfect these surfaces and to establish a better surveillance programme for control of viral food-borne illnesses.

Effectiveness of commercial disinfectants for inactivating hepatitis A virus on agri-food surfaces.

Six commercial disinfectants were tested for their efficacy in inactivating hepatitis A virus in solution or attached to agri-food surfaces. Disinfectant I contains 10% quaternary ammonium plus 5% glutaraldehyde; disinfectant II contains 12% sodium hypochlorite; disinfectant III contains 2.9% dodecylbenzene sulfonic acid plus 16% phosphoric acid; disinfectant IV contains 10% quaternary ammonium; disinfectant V contains 2% iodide; and disinfectant VI contains 2% stabilized chlorine dioxide. Among these, disinfectants I and II were shown to be the most effective in inactivating hepatitis A virus in solution. The efficacy of these disinfectants was further tested against hepatitis A virus attached to common agri-food surfaces, including polyvinyl chlorine, high-density polyethylene, aluminum, stainless steel, and copper. Disinfectant II was shown to be the most effective, with a maximum inactivation level of about 3 log10. The inactivation efficacy was shown to be affected by the concentration of the active ingredient, the contact time between the disinfectant and the contaminated surfaces, and the incubation temperature. In general, hepatitis A virus was shown to be highly resistant to most disinfectants tested, and high concentrations of active ingredient were needed to achieve acceptable inactivation levels.

Detection of hepatitis A virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription-PCR.

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 x 10(2) PFU/ml) was obtained with NASBA, compared to 50 PFU (1 x 10(4) PFU/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.

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.

Lack of CD40-dependent B-cell proliferation in B lymphocytes isolated from patients with persistent polyclonal B-cell lymphocytosis.

Persistent B-cell lymphocytosis (PPBL) is a haematological disorder diagnosed primarily in adult female smokers that is characterized by a polyclonal increase in peripheral blood B lymphocytes and a moderate elevation of serum IgM. B lymphocyte-associated cellular abnormalities, such as the occurrence of multi-lobed nuclei, increased bcl2/Ig gene rearrangements and the identification of an extra long-arm chromosome (i3)(q10) in the B-cell population, indicate that PPBL could be part of a multi-step process leading to the emergence of a malignant B lymphoproliferation. However, the resulting impact on cellular functional properties remains to be elucidated. Our goal was to address that aspect via the study of B-cell activity following stimulation through CD40, a key molecule of the tumour necrosis factor receptor superfamily involved in B lymphocyte development. In contrast to normal B cells, PPBL B lymphocytes were unable to respond to the proliferative signal delivered in vitro by CD40, indicating a defect in the CD40 activation pathway. Polymerase chain reaction amplification and sequencing of the receptor as well as FACScan analysis of patient B lymphocytes dismissed the possibility of a defect in either CD40 structure or expression. Moreover, Western blot analysis of tyrosine phosphorylation, an early event in the CD40-signalling cascade, was similar in patients and controls, leading to the conclusion that the defect affecting B lymphocytes in PPBL patients is probably located downstream of that signalling cascade.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Production of a diagnostic monoclonal antibody in perennial alfalfa plants.

The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs.

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.

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 x 10(2) to 1 x 10(3) 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-Hodgkin's lymphoma.

Interference of HIV-1 Nef in the sphingomyelin transduction pathway activated by tumour necrosis factor-alpha in human glial cells.

OBJECTIVE: The HIV-1 nef gene product, thought to interact with mediators of cell signalling, is overexpressed during the restricted HIV-1 infection of human astrocytes. This infection can be reactivated following exposure to tumour necrosis factor (TNF)-alpha. We examined the possibility that Nef alters the TNF-alpha-induced cell signalling in astroglioma cells through the sphingomyelin pathway. METHODS: Sphingomyelinase activation by TNF-alpha was analysed in U251MG glial cells constitutively expressing Nef and compared with U251MG cells stably transfected with the expression vector alone. The consequent effect on the cellular proliferative response and induction of nuclear factor NF-kappa B and AP-1 binding activities were examined. RESULTS: A marked enhancement in the levels of ceramide, a product of the sphingomyelin hydrolysis, was observed in U251MG-Nef upon stimulation with TNF-alpha. In contrast, ceramide levels in control cells were barely increased under similar conditions. A concomitant reduction of sphingomyelin level occurred in U251MG-Nef cells. In addition, the reduced survival rate of U251MG cells resulting from TNF-alpha activation was prevented in the presence of Nef. Furthermore, electrophoretic mobility shift assays indicated that nef expression inhibits AP-1 activation without altering the induction of NF-kappa B. CONCLUSION: These results strongly suggest that nef expression in U251MG cells modulates the sphingomyelinase signalling pathway triggered by TNF-alpha, thus leading to important modifications in the activation and proliferation of glial cells. They also provide new insights to explain the widespread reactive astrogliosis observed in AIDS-associated neuropathological disorders.

Detection of a homologue of bcl-2 in plant cells.

An emerging family of bcl-2-like genes has been identified from nematode to humans. These genes play a role in the maintenance of homeostasis. Its members have highly conserved domains that are important for their dimerization. Since nothing is known about the importance of these genes in plant cells, we have investigated their presence in an alga as well as in three higher plants both by Western analysis and by immunocytochemistry. Immunoblots revealed the presence of a protein immunoreacting with the anti-bcl-2 polyclonal antibody in leaves of tobacco plants. Furthermore, immunocytochemical localization has shown that this protein is mainly associated with mitochondria, plastids, and nuclei of plant cells. Taken together, our results suggest that bcl-2 is a protein highly conserved throughout evolution.

HIV-1 LTR activity in human CD40-activated B lymphocytes is dependent on NF-kappaB.

CD40-stimulated human B lymphocytes are highly permissive to a productive infection by the human immunodeficiency virus type 1. In these cells, nuclear factors involved in activation of the HIV-1 LTR, which contains the transcriptional control elements of the virus, are unknown. Transient expression assays with plasmids containing deleted parts of the LTR region linked to a reporter gene showed that the NF-kappaB binding site was essential for HIV-1 LTR activity in CD40-stimulated B lymphocytes. In addition, electrophoretic mobility shift and supershift assays revealed that important NF-kappaB binding activity composed of at least p50, p65, and c-Rel NF-kappaB subunits was present in nuclei of CD40-stimulated B cells. These results confirm at a molecular level the ability of HIV-1 to replicate in B cells and that this activity is strongly associated with NF-kappaB.

Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes.

In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40-dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12 myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40-dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor kappa B (NF- kappa B). In contrast, the cAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-kappa B binding sequence from the kappa light chain as a probe, revealed that both p65 (RelA) and c-Rel were present in CD40-stimulated B cells. While PKC depletion did not alter the NF-kappa B level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-kappa B level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes.

Induction of LFA-1 independent human B cell proliferation and differentiation by binding of CD40 with its ligand.

Engagement of CD40 on resting B cells in the presence of IL-4 triggers B cell proliferation, differentiation and homotypic adhesion. This study was designed to investigate the role of LFA-1/ICAM-1 interactions in homotypic adhesion and proliferation of CD40-activated human B lymphocytes. Freshly isolated B cells were cultured in vitro in the presence of IL-4 and of L cells expressing CD40L, the CD40 ligand. The addition to the culture medium of LFA-1 and ICAM-1 antibodies inhibited homotypic B lymphocyte adhesion. However, these antibodies failed to affect B lymphocyte proliferation and antibody production. These results indicate that aggregation and proliferation are independent events although both induced by CD40 activation.

Productive infection of normal CD40-activated human B lymphocytes by HIV-1.

OBJECTIVE: Antigen-driven B-cell proliferation and maturation occur in germinal centres 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 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 centres. The effect of HIV-1 viral exposure on CD40-activated B lymphocytes was therefore examined. METHODS: Freshly 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 coculture assay. RESULTS: EBV-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. CONCLUSION: In 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.

Inhibition of protein synthesis by the human immunodeficiency virus type 1 nef gene product.

During productive infection of human T lymphocytes in cell culture, the expression of human immunodeficiency virus type 1 is temporally regulated by virus-encoded regulatory proteins. Among these Nef, whose function has not been clearly elucidated, is thought to alter CD4+ T cells. We examined the possibility that the nef gene interferes with the translation process in a cell-free system. The results demonstrate that the nef gene product mediates an inhibitory effect on protein synthesis. Conversely, the use of antisense nef mRNA did not affect translation. Further observations suggest that this inhibitory effect is an inherent property of the nef gene product itself and not of its mRNA. The data show that the translational repression directed by Nef is a general phenomenon, acting on its own and on other messengers used as reporter mRNAs. We propose that, as a consequence, Nef can play an important role in the pathogenesis of AIDS.

The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties.

The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the cellular defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growth-suppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) and a resultant block to protein synthesis initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetratricopeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably transfected NIH 3T3 cells with a eukaryotic expression plasmid containing p58 cDNA under control of the cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, growing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2 alpha phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one mechanism by which the protein induces malignant transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis.

Nuclear localization of the protein encoded by the Wilms' tumor gene WT1 in embryonic and adult tissues.

The human Wilms' tumor gene WT1 encodes a putative transcription factor implicated in tumorigenesis and in specifying normal urogenital development. We have studied the distribution of WT1 protein and mRNA using immunohistochemistry and in situ hybridization. Monoclonal antibodies were raised against a peptide specific to the first alternative splice site of WT1. Two antibodies specifically reacted on Western blot to this WT1 isoform. Immunofluorescence localized WT1 protein to podocytes during mesonephric and metanephric development. In situ hybridization revealed a similar pattern of expression except that WT1 mRNA was also present in metanephric blastema and renal vesicles. Messenger RNA expression was most pronounced in the kidneys during early fetal development and declined thereafter. In contrast, WT1 protein was readily detectable in glomerular podocytes throughout adulthood. WT1 protein in Wilms' tumor was present in blastema and glomeruloid structures. Expression in the female gonad was linked to the different stages of granulosa cell development. In the male gonad, expression was restricted to Sertoli cells and their precursors, the embryonic tunica albuginea and the rete testis. The intracellular distribution of the WT1 protein was investigated by confocal laser microscopy and was demonstrated to be exclusively nuclear. The nuclear distribution and the selective pattern of expression support the proposed role of WT1 as a transcription factor active during urogenital development. The persistence of WT1 expression in the adult kidney suggests a role in homeostasis of the podocyte.

Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system.

The use of peripheral B lymphocytes in the successful preparation of human monoclonal antibodies by hybridoma technology is highly dependent on lymphocyte activation procedures. We studied the ability of peripheral human B lymphocytes cultured in vitro and activated through their CD40 antigen (CD40 system) (Banchereau et al., 1991) to form antibody-secreting heterohybridomas after fusion with murine X63Ag8.653 myeloma cells. The frequency of antibody-secreting heterohybridomas formation was greatly increased (15 times) by culture of B cells in the CD40 system. The CD40 system offers many advantages over other procedures of B lymphocyte activation representing a significant technological advance in the preparation of human monoclonal antibodies by standard hybridoma technology.

Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.

We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.