Prevalence of weak D phenotypes in the general population of Québec, Canada: A focus on weak D type 42.

BACKGROUND AND OBJECTIVES: Weak D type 42 accounts for an unusually high proportion of weak D phenotypes in Québec (Canada), which contrasts with other predominantly White populations. However, its prevalence in the general population is unknown. We estimated the prevalence of weak D type 42 and other common weak D phenotypes in Québec. MATERIALS AND METHODS: We screened for RHD*01W.42 alleles among 1000 individuals of CARTaGENE-a cohort representative of Québec's population. The prevalence of weak D type 42 was calculated based on the allele frequency of RHD*01W.42 and d (i.e., all recessive alleles that confer a D- phenotype), assuming a Hardy-Weinberg equilibrium. This prevalence was then leveraged to calculate that of other common weak D phenotypes, using published prevalence estimates among weak D phenotypes. RESULTS: Two individuals harboured the RHD*01W.42/RHD*01 heterozygous genotype. Assuming an allele frequency of 38.19% for d, the overall prevalence of weak D type 42 was 0.08%. The following prevalence estimates were also obtained: 0.44% for all weak D phenotypes and 0.07%, 0.01% and 0.04% for weak D types 1, 2 and 3, respectively. CONCLUSION: Québec has the highest documented prevalence of weak D type 42, which was estimated at 0.08%.

Seroprevalence of SARS-CoV-2 antibodies among blood donors in Québec: an update from a serial cross-sectional study.

OBJECTIVES: We previously estimated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies following the first pandemic wave at 2.23% in Québec, Canada. Following the much bigger second wave in fall 2020 and early 2021, we estimated the seroprevalence of anti-SARS-CoV-2 in Québec during the first months of 2021. METHODS: Blood samples from regular, asymptomatic (for ≥ 14 days) donors were collected between January 25, 2021 and March 11, 2021. Anti-SARS-CoV-2 seropositivity was assessed using an enzyme-linked immunosorbent assay that captures antibodies directed against the receptor binding domain of the SARS-CoV-2 spike (and hence cannot discriminate between infection- and vaccine-induced seropositivity). Seroprevalence estimates were adjusted for regional distribution, age, and sex. RESULTS: Samples from 7924 eligible donors were analyzed, including 620 (7.8%) vaccinated donors and 7046 (88.9%) unvaccinated donors (vaccination status unknown for 258 (3.3%) donors). Overall, median age was 51 years; 46.4% of donors were female. The adjusted seroprevalence was 10.5% (95% CI = 9.7-11.3) in the unvaccinated population and 14.7% (95% CI = 13.8-15.6) in the overall population. Seroprevalence gradually decreased with age and was higher among donors who self-identified as having a racial/ethnic background other than white, both in the overall and in the unvaccinated populations. CONCLUSION: The seroprevalence of SARS-CoV-2 antibodies significantly increased in Québec since spring 2020, with younger persons and ethnic minorities being disproportionately affected. When compared with the cumulative incidence rate reported by public health authorities (i.e., 3.3% as of March 11, 2021), these results suggest that a substantial proportion of infections remain undetected despite improvements in access to COVID-19 testing.

RéSUMé: OBJECTIFS: Lors d'une première étude, nous avons estimé la séroprévalence des anticorps contre le syndrome respiratoire aigu sévère coronavirus 2 (SRAS-CoV-2) après la première vague pandémique à 2,23 % au Québec, Canada. Cette seconde étude estime la séroprévalence de l'anti-SRAS-CoV-2 au Québec lors de la deuxième vague pandémique. MéTHODES: Des échantillons de donneurs de sang asymptomatiques (≥ 14 jours) ont été prélevés entre le 25 janvier et le 11 mars 2021. La séropositivité a été évaluée à l'aide d'un dosage immuno-enzymatique qui capture les anticorps dirigés contre la protéine Spike du récepteur de domaine de liaison du SARS-CoV-2 (et ne peut donc distinguer l'immunité induite par l'infection et la vaccination). La séroprévalence a été ajustée en fonction de l'âge et du sexe par région. RéSULTATS: Des échantillons de 7 924 donneurs ont été analysés, dont 620 (7,8 %) étaient vaccinés et 7 046 (88,9 %) étaient non vaccinés (statut vaccinal inconnu pour 258 (3,3 %) donneurs). Dans l'ensemble, l'âge médian était de 51 ans et 46,4 % des donneurs étaient des femmes. La séroprévalence ajustée était de 10,5 % (IC 95 % = 9,7 à 11,3) dans la population non vaccinée et de 14,7 % (IC 95 % = 13,8 à 15,6) dans la population globale. La séroprévalence diminuait progressivement avec l'âge et était plus élevée chez les donneurs d'origine ethnique autre que blanche. CONCLUSION: La séroprévalence anti-SRAS-CoV-2 a considérablement augmenté au Québec depuis le printemps 2020, les personnes plus jeunes et les minorités ethniques étant plus touchées. Comparés au taux d'incidence cumulatif signalé par la santé publique (c.-à-d. 3,3 % au 11 mars 2021), ces résultats suggèrent qu'une proportion importante d'infections reste non détectée.

SARS-CoV-2 seroprevalence among blood donors in Québec, and analysis of symptoms associated with seropositivity: a nested case-control study.

OBJECTIVES: A substantial proportion of individuals infected with SARS-CoV-2 do not experience noticeable symptoms typical of COVID-19. Our objectives were to evaluate the impact of the first wave of the pandemic in Québec by measuring SARS-CoV-2 antibody seroprevalence in a convenience sample of healthy blood donors and to study the association between seropositivity and the occurrence of COVID-19 symptoms. METHODS: The study design was a cross-sectional serological survey with a nested case-control study. Residual blood samples from donations collected between May 25 and July 9, 2020 (well before vaccination rollout) in the province of Québec were tested for anti-Spike RBD antibodies by ELISA. Seropositive donors and a control group of seronegative donors were questioned about prior COVID-19 symptoms. All qualified blood donors were eligible for participation. RESULTS: A total of 7691 blood donors were included in the study. After adjustments, the seroprevalence rate was 2.2% (95% CI 1.9-2.6). Seropositive donors reported one or more symptoms in a proportion of 52.2% (95% CI 44.2-60.1); this proportion was 19.1% (95% CI 13.4-26.1) among seronegative donors, suggesting that approximately 50-66% of all infections were asymptomatic. Univariate analysis of associations between symptoms and seropositivity revealed that except for rhinorrhea, all symptoms were significantly associated with seropositivity. CONCLUSION: Assuming that blood donors are fairly representative of the general adult population, this study shows that less than 3% of 18-69-year-olds have been infected during the first wave of the pandemic in the province of Québec. Our data also confirm that many infections escaped detection, including a substantial proportion that were asymptomatic.

RéSUMé: OBJECTIFS: Une proportion substantielle de personnes infectées par le SRAS-CoV-2 ne présentent pas de symptômes visibles typiques de la COVID-19. Nos objectifs étaient d'évaluer l'impact de la première vague de la pandémie au Québec en mesurant la séroprévalence des anticorps anti-SRAS-CoV-2 chez les donneurs de sang en bonne santé, et d'étudier l'association entre la séropositivité et la survenue des symptômes de la COVID-19. MéTHODES: Le design de l'étude était une enquête de sérologie transversale avec une étude cas-témoins nichée dans la cohorte. Des échantillons de sang provenant de dons recueillis entre le 25 mai et le 9 juillet 2020 (bien avant le déploiement de la vaccination) dans la province de Québec ont été testés pour les anticorps anti-spicule RBD (Receptor Binding Domain) par ELISA. Les donneurs séropositifs et un groupe témoin de donneurs séronégatifs ont été interrogés sur les symptômes spécifiques à la COVID-19. Tous les donneurs qualifiés pour le don de sang étaient éligibles à participer à l'étude. RéSULTATS: Au total, 7 691 donneurs de sang ont été inclus dans l'étude. Après ajustements, le taux de séroprévalence était de 2,2 % (IC à 95% 1,9­2,6). Les donneurs séropositifs ont signalé un ou plusieurs symptômes dans une proportion de 52,2 % (IC à 95% 44,2­60,1); cette proportion était de 19,1 % (IC à 95% 13,4­26,1) parmi les donneurs séronégatifs, ce qui suggère qu'entre 50 % et 66 % de toutes les infections étaient asymptomatiques. Une analyse univariée des associations entre les symptômes et la séropositivité a révélé qu'à l'exception de la rhinorrhée, tous les symptômes étaient significativement associés à la séropositivité. CONCLUSION: En supposant que les donneurs de sang sont assez représentatifs de la population adulte générale, cette étude montre que moins de 3 % des 18­69 ans ont été infectés lors de la première vague de la pandémie dans la province du Québec. Nos données confirment également que de nombreuses infections n'ont pas fait l'objet d'un test moléculaire de dépistage, y compris une proportion importante qui était asymptomatique.

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than two million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. STUDY DESIGN AND METHODS: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike-specific antibodies in the plasma of convalescent donors. RESULTS AND CONCLUSION: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

Marker-free coselection for CRISPR-driven genome editing in human cells.

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

MiR-146a potentially promotes IVIg-mediated inhibition of TLR4 signaling in LPS-activated human monocytes.

IVIg is used as an immunomodulatory agent in inflammatory disorders such as sepsis. IVIg also affects monocyte differentiation and functions, two processes in which microRNAs play a crucial role. Monocytes detect microorganisms through pathogen recognition receptors (PRRs) such as TLR4. MiR-146a has been shown to supress NF-κB and IRF3 activity, two key components of TLR4 signaling. To evaluate whether miR-146a is involved in the anti-inflammatory effects of IVIg, monocytes were treated with LPS or IVIg alone or, alternately, first activated with LPS followed by washing and addition of IVIg. MiR-146a, IRF3, TNF-α, IL-1ß, IL-6, IL-10, IFN-ß, TGF-ß1 and IL-1Ra expression was analyzed by qPCR, while IRAK1, TRAF6 and IκBα expression was measured by Western blotting. We found that addition of IVIg to LPS-activated monocytes significantly upregulated the expression of miR-146a, which was associated with a significant reduction in the expression of its targets IRF3 and its regulated gene IFN-ß. Furthermore, expression of IRAK1, TRAF6, and consequently NF-κB activation, was also reduced in LPS-activated monocytes following addition of IVIg, whereas TGF-ß1, IL-10 and IL-1Ra were increased. Our results thus suggest that miR-146a is a mediator of IVIg effects in inflammatory disorders, point to an important role for miR-146a in the control of inflammation during sepsis and highlight a new mechanism by which IVIg exerts its anti-inflammatory effects in sepsis.

Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.

Biodegradation of nitroglycerin from propellant residues on military training ranges.

Nitroglycerin (NG) is often present in soils and sometimes in pore water at antitank firing positions due to incomplete combustion of propellants. Various degradation processes can contribute to the natural attenuation of NG in soils and pore water, thus reducing the risks of groundwater contamination. However, until now these processes have been sparsely documented. This study aimed at evaluating the ability of microorganisms from a legacy firing position to degrade dissolved NG, as well as NG trapped within propellant particles. Results from the shake-flask experiments showed that the isolated culture is capable of degrading dissolved NG but not the nitrocellulose matrix of propellant particles, so that the deeply embedded NG molecules cannot be degraded. Furthermore, the results from column experiments showed that in a nutrient-poor sand, degradation of dissolved NG may not be sufficiently rapid to prevent groundwater contamination. Therefore, the results from this study indicate that, under favorable soil conditions, biodegradation can be an important natural attenuation process for NG dissolving out of fresh propellant residues. In contrast, biodegradation does not contribute to the long-term attenuation of NG within old, weathered propellant residues. Although NG in these old residues no longer poses a threat to groundwater quality, if soil clean-up of a legacy site is required, active remediation approaches should be sought.

Suppression of protein phosphatase 2A activity enhances Ad5/F35 adenovirus transduction efficiency in normal human B lymphocytes and in Raji cells.

Investigation of the molecular processes which control the development and function of lymphocytes is essential for our understanding of humoral immunity, as well as lymphocyte associated pathogenesis. Adenovirus-mediated gene transfer provided a powerful tool to investigate these processes. We have previously demonstrated that adenoviral vector Ad5/F35 transduces plasma cell lines at a higher efficiency than primary B cells, owing to differences in intracellular trafficking. Given that phosphatases are effectors of intracellular trafficking, here we have analyzed the effects of a panel of phosphatase inhibitors on Ad5/F35 transduction efficiency in B lymphocytes in the present study. FACS analysis was conducted to determine Ad5/F35-EYFP transduction efficiency in lymphoid cells, including human primary B cells, following serine/threonine phosphatase (PSP) inhibitor treatment. We further used confocal microscopy to analyze intracellular trafficking and fate of CY3 labeled Ad5/F35 vectors, in PSP treated lymphoid cell. Finally, we analyzed the MAPK pathway by Western blot in PSP treated cells. Adenoviral transduction efficiency was unresponsive to inhibition of PP1 whereas inhibition of PP2A by cantharidic acid, or PP1 and PP2A by okadaic acid, substantially increased transduction efficiency. Importantly, confocal microscopy analyses revealed that inhibition of PP2A shut down adenovirus recycling. Moreover, inhibition of PP2A resulted in increased phosphorylation of AKT, ERK1/2 and MEK1/2. Taken together, these results suggest that Ad5/F35 is more efficiently transduced in cells following PP2A inhibition. Our results are in agreement with reports indicating that PP2A is involved in the formation of recycling vesicles and might be of interest for gene therapy applications.

Overexpression of PAX5 induces apoptosis in multiple myeloma cells.

PAX5 is an essential transcription factor for the commitment of lymphoid progenitors to the B-lymphocyte lineage. PAX5 suppression results in retrodifferentiation of B lymphocytes to an uncommitted progenitor cell stage, whereas PAX5 suppression in mature B lymphocytes leads to further development into plasma cells. Here, we have analyzed the fate of plasma cell lines following PAX5 reexpression. Human B cell lines were infected with Ad5/F35 adenoviruses encoding either EYFP or PAX5. Expression analysis of specific plasma cell transcription factors (IRF4, Blimp-1 and XBP-1) suggests that PAX5 reexpression does not induce retrodifferentiation of plasma cells into B lymphocytes. Interestingly, the viability of RPMI-8226 and U266 multiple myeloma cell lines markedly declined at 4-7 days post-transduction, whereas other plasma cell lines maintained their viability. Apoptosis analysis through Annexin V measurement also revealed a higher level of apoptosis in PAX5-expressing myeloma cell lines. Finally, Western blot analysis of pro- and anti-apoptotic proteins revealed that the anti-apoptotic protein MCL-1 was down-modulated in PAX5-transduced multiple myeloma cell lines. In conclusion, our results show that the expression of PAX5 in plasma cell lines induces apoptosis exclusively in multiple myelomas. This might represent a potential therapeutic avenue in the treatment of multiple myeloma.

2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

Adenovirus 5 and chimeric adenovirus 5/F35 employ distinct B-lymphocyte intracellular trafficking routes that are independent of their cognate cell surface receptor.

Gene transfer applications with adenovirus (Ad) type 5 are limited by its native tropism, hampering their use in several cell types. To address this limitation, several Ad vectors bearing chimeric fiber have been produced to take advantage of the different cellular receptors used by other subgroups of Ads. In this study, we have compared the transduction efficiency of Ad5 and the chimeric Ad5/F35 in primary human B lymphocytes and B-cell lines as a function of the developmental stage. We found that transduction efficiencies of the two Ads differ independently of their targeted cellular receptor but are related to the intracellular localization of the virus. In efficiently transduced cells, Ads were localized in early endosomes or cytosol, whereas in poorly transduced cells they were localized within late endosomes/lysosomes. Finally, we demonstrate that treatment of cells with phosphatase inhibitors known to redirect endocytosis towards caveolae, increased Ad5/F35 transduction efficiency.

Comparison of promoter activities for efficient expression into human B cells and haematopoietic progenitors with adenovirus Ad5/F35.

Adenoviral gene transfer into human B lymphocytes and haematopoietic progenitors would allow the characterization of their function on cellular growth, differentiation and survival. Efficient gene expression is however strongly dependent on the promoter used. In this study, we investigated the relative strength of various promoters by following and measuring the expression of the reporter gene EYFP in human peripheral B lymphocytes, cord blood CD34(+) cells and the megakaryocytic cell line M-07e. The murine PGK promoter provided the best level of transgene expression in CD34(+) cells among the four promoters tested, followed closely by the CMV promoter, and to a lesser extend by a CMV promoter with a beta-globin/IgG chimeric intron, whereas the human CD40 promoter provided the lowest levels of expression. In contrast, the strongest promoters in B lymphocytes were the two CMV promoters. Surprisingly, even the best promoters were unable to induce transgene expression in more than 75-80% of the primary B and CD34(+) cells, even though 100% of the cells were infected. Finally and in contrast to retroviruses, only a minority of B lymphocytes and CD34(+) cells were able to induce the transcription of IRES-containing bicistronic expression cassettes from adenovirus.

Efficient gene transfer into normal human B lymphocytes with the chimeric adenoviral vector Ad5/F35.

The failure to efficiently introduce genes into normal cells such as human B lymphocytes limits the characterization of their function on cellular growth, differentiation and survival. Recent studies have shown that a new adenoviral vector Ad5/F35 can efficiently transduce human haematopoietic CD34+ progenitor cells. In this study, we compared the gene transfer efficiencies of the Ad5/F35 vector to that of the parental vector Ad5 in human B lymphocytes. Peripheral blood B cells obtained from healthy individuals were cultured in vitro using CD40-CD154 system. Normal B lymphocytes were infected with replication-defectives Ad5 and Ad5/F35, both containing the GFP reporter gene, and transduction efficiencies were monitored by flow cytometry. Ad5 was highly ineffective, infecting only about 5% of human B lymphocytes. In contrast, Ad5/F35 transduced up to 60% of human B lymphocytes and GFP expression could be detected for up to 5 days post infection. Importantly, physiology of B lymphocytes such as proliferation, viability and antibodies secretion were unaffected following Ad5/F35 transduction. Finally, we observed that memory B lymphocytes were more susceptible to Ad5/F35 infection than naïve B lymphocytes. Thus, our results demonstrate that the adenoviral vector Ad5/F35 is an efficient tool for the functional characterization of genes in B lymphopoiesis.

Maintenance of Epstein-Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2.

Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein-Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.

Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures.

B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.