Reduced cell turnover in bovine leukemia virus-infected, persistently lymphocytotic cattle

Although nucleotide analogs like bromodeoxyuridine have been extensively used to estimate cell proliferation in vivo, precise dynamic parameters are scarce essentially because of the lack of adequate mathematical models. Besides recent developments on T cell dynamics, the turnover rates of B lymphocytes are largely unknown particularly in the context of a virally induced pathological disorder. Here, we aim to resolve this issue by determining the rates of cell proliferation and death during the chronic stage of the bovine leukemia virus (BLV) infection, called bovine persistent lymphocytosis (PL). Our methodology is based on direct intravenous injection of bromodeoxyuridine in association with subsequent flow cytometry. By this in vivo approach, we show that the death rate of PL B lymphocytes is significantly reduced (average death rate, 0.057 day(-1) versus 0.156 day(-1) in the asymptomatic controls). Concomitantly, proliferation of the PL cells is also significantly restricted compared to the controls (average proliferation rate, 0.0046 day(-1) versus 0.0085 day(-1)). We conclude that bovine PL is characterized by a decreased cell turnover resulting both from a reduction of cell death and an overall impairment of proliferation. The cell dynamic parameters differ from those measured in sheep, an experimental model for BLV infection. Finally, cells expressing p24 major capsid protein ex vivo were not BrdU positive, suggesting an immune selection against proliferating virus-positive lymphocytes. Based on a comparative leukemia approach, these observations might help to understand cell dynamics during other lymphoproliferative disease such as chronic lymphocytic leukemia or human T-cell lymphotropic virus-induced adult T-cell leukemia in humans.     

Viral expression directs the fate of B cells in bovine leukemia virus-infected sheep

The host immune response is believed to tightly control viral replication of deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). However, this assumption has not been definitely proven in vivo. In order to further evaluate the importance of the immune response in the BLV model, we studied the fate of cells in which viral expression was transiently induced. Using a dual fluorochrome labeling approach, we showed that ex vivo induction of viral expression induces higher death rates of B cells in vivo. Furthermore, cyclosporine treatment of these animals indicated that an efficient immune response is required to control virus-expressing cells.     

Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep

Lymphocyte homeostasis is determined by a critical balance between cell proliferation and death, an equilibrium which is deregulated in bovine leukemia virus (BLV)-infected sheep. We have previously shown that an excess of proliferation occurs in lymphoid tissues and that the peripheral blood population is prone to increased cell death. To further understand the mechanisms involved, we evaluated the physiological role of the spleen in this accelerated turnover. To this end, B lymphocytes were labeled in vivo using a fluorescent marker (carboxyfluorescein diacetate succinimidyl ester), and the cell kinetic parameters (proliferation and death rates) of animals before and after splenectomy were compared. We show that the enhanced cell death observed in BLV-infected sheep is abrogated after splenectomy, revealing a key role of the spleen in B-lymphocyte dynamics.     

Enhanced apoptosis of peripheral CD5-negative B lymphocytes from chronically hepatitis C virus-infected patients: reversal after antiviral treatment

Whereas enhanced peripheral T-cell apoptosis and its association with autoimmunity have recently been reported, the apoptotic status of peripheral B cells in chronic hepatitis C virus (HCV) infection remains ambiguous. We therefore sought to investigate the sensitivity of peripheral B cells to apoptosis and to assess the possible benefits of antiviral treatment in mitigating these effects. Spontaneous apoptosis, the extent of apoptosis rescue, and NF-kappaB expression in peripheral B cells were studied in patients with chronic HCV infections (group 1), in sustained responders after antiviral treatment (group 2), and in healthy controls (group 3). For group 1, spontaneous B-cell apoptosis was increased (26% +/- 4.6%) and apoptosis rescue was altered (39%) compared to group 3 (18% +/- 5% and 50%, respectively; P = 0.001). In contrast, apoptosis and apoptosis rescue were similar for groups 2 and 3. Enhanced B-cell apoptosis was associated with decreased NF-kappaB expression and was found only in CD5-negative (CD5(neg)) B cells, whereas CD5(pos) cells were apoptosis resistant. Chronic HCV infection is associated with enhanced peripheral B-cell apoptosis and decreased apoptosis rescue. Successful antiviral treatment reverses these abnormalities to the levels seen in healthy individuals. The relative resistance of the CD5(pos) B-cell subpopulation to apoptosis may play a role in HCV-related autoimmunity and lymphoproliferation.     

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.     

The CD40-inducible Bcl-2 family member A1 protects B cells from antigen receptor-mediated apoptosis

CD40 activation is necessary for thymus-dependent humoral immune responses and rescuing both phenotypically immature WEHI-231 B lymphoma cells from B cell antigen receptor-induced cell death and germinal center B cells from spontaneous apoptosis. As some effects of CD40 are probably mediated by differences in gene expression, cDNA expression arrays and RNase protection assays were used to identify the anti-apoptotic Bcl-2 homolog A1 as a CD40-inducible gene in B cell lines and purified germinal center B cells. Sustained CD40-induced A1 upregulation correlated with CD40-mediated rescue of WEHI-231 cells from anti-IgM-induced apoptosis. Moreover, overexpression of A1 specifically protected WEHI-231 cells from anti-IgM-induced apoptosis but not cell death triggered by certain other stimuli.     

Differential susceptibility of peripheral blood CD5 and CD5 B cells to apoptosis in chronic hepatitis C patients

A body of evidence has suggested a close link between chronic hepatitis C virus (HCV) infection and B cell abnormalities, including mixed cryoglobulinemia, rheumatoid factor (RF) production, and lymphoproliferative disorders that may develop into non-Hodgkin’s lymphoma. Recent studies have demonstrated the expansion of CD5⁺ B cells in the peripheral blood of chronic hepatitis C patients (CHC). As CD5⁺ B cells, which are capable of producing autoantibodies and RF, are apparently crucial for the development of HCV-associated pathogenesis, the fate of both the CD5⁺ and CD5⁻ B cell subsets upon chronic HCV infection is of interest. In this study, the degree to which chronic HCV infection induces apoptosis in each B cell subset was investigated. Our results demonstrated that peripheral CD5⁻ B cells were more susceptible to apoptosis than CD5⁺ B cells in CHC. Furthermore, plasma levels of IL-4, IL-10, and IL-12 were significantly elevated in CHC, thus suggesting that these interleukins protect CD5⁺ B cells from apoptosis. The rationale for the differential susceptibility of distinct B cell subsets in CHC is also discussed with regard to extrahepatic manifestations associated with chronic HCV infection.     

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.     

Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle

In the present study, we monitored Foxp3⁺ T cells in bovine leukemia virus (BLV)‐infected cattle. By flow cytometric analysis, the proportion of Foxp3⁺CD4⁺ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3⁺CD4⁺ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN‐γ mRNA expression, suggesting that Foxp3⁺CD4⁺ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.     

Induction of transformed phenotypes in sheep fibroblasts by culture fluids from cells persistently infected with bovine leukemia virus

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf-form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology, after treatment with 5'-iodo-2'-deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2-deoxy-D-glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration of 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.     

Presence of 5''-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells.

The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp.     

CXC chemokine ligand 13 and CC chemokine ligand 19 cooperatively render resistance to apoptosis in B cell lineage acute and chronic lymphocytic leukemia CD23+CD5+ B cells

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23+CD5+ and CD23+CD5- B cells from cord blood (CB) and patients with B cell lineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23+CD5+ B cells at high frequency, but not on CD23+CD5- B cells. Although no significant chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-alpha-mediated apoptosis in B-ALL and B-CLL CD23+CD5+ B cells, but not in the cells from CB. B-ALL and B-CLL CD23+CD5+ B cells express elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23+CD5+ B cells, and further rescue the cells from TNF-alpha-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells, use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

Cell surface expression of the bovine leukemia virus-binding receptor on B and T lymphocytes is induced by receptor engagement

Bovine leukemia virus (BLV), one of the most common infectious viruses of cattle, is endemic in many herds. Approximately 30-40% of adult cows in the United States are infected by this oncogenic C-type retrovirus and 1-5% of animals will eventually develop a malignant lymphoma. BLV, like the human and simian T cell leukemia viruses, is a deltaretrovirus but, in contrast with the latter, the BLV receptor remains unidentified. In this study, we demonstrate that the amino-terminal 182 residues of the BLV envelope glycoprotein surface unit encompasses the receptor-binding domain. A bona fide interaction of this receptor-binding domain with the BLV receptor was demonstrated by specific interference with BLV, but not human T cell leukemia virus, envelope glycoprotein-mediated binding. We generated a rabbit Ig Fc-tagged BLV receptor-binding domain construct and ascertained that the ligand binds the BLV receptor on target cells from multiple species. Using this tool, we determined that the BLV-binding receptor is expressed on differentiating pro/pre-B cells in mouse bone marrow. However, the receptor was not detected on mature/quiescent B cells but was induced upon B cell activation. Activation of human B and T lymphocytes also induced surface BLV-binding receptor expression and required de novo protein synthesis. Receptor levels were down-regulated as activated lymphocytes returned to quiescence. In the human thymus, BLV-binding receptor expression was specifically detected on thymocytes responding to the IL-7 cytokine. Thus, expression of the BLV-binding receptor is a marker of enhanced metabolic activity in B cells, T cells, and thymocytes.     

Adherence of group B streptococci isolated from man and cattle to human and bovine epithelial cells

Proof of adherence of group B streptococci (GBS) to human and bovine vaginal epithelial cells and to bovine cells of milk cisternae of the mammary gland was employed as a criterion determining the possibility of colonization of these organs with GBS, or as another method of testing the transfer of GBS between man and cattle GBS of both human and animal origin adhered to human epithelial cells in a similar way On the other hand, a significantly stronger adherence of bovine GBS to vaginal epithelial cells and cells of milk cisternae of cattle was found than of human GBS Thus the direction of colonization man—animal is more prob able than the opposite way Neither in animal nor in human strains a correlation between the equipment of strains with type antigens and intensity of adherence could be found     

CD2 is expressed by a subpopulation of normal B cells and is frequently present in mature B-cell neoplasms

BACKGROUND: CD2 is expressed by T and natural killer (NK) cells and has been reported in T/NK cell lineage neoplasms as well as in immature B-lymphoblastic and myeloid leukemias. Although CD2+ B-cells have been identified in normal fetal and postnatal thymus, they have not been reported in adults. METHODS: We retrospectively reviewed flow cytometric immunophenotypic data on consecutive low-grade B-cell leukemias and lymphomas to investigate the frequency of CD2 expression. We also reviewed samples from normal healthy donors to determine whether there is a normal CD2+ B-cell population. RESULTS: CD2 expression (partial or complete) was observed in 13 of 83 (16%) chronic lymphocytic leukemias (CLL), 16 of 29 (55%) follicle center lymphomas (FCL), 3 of 12 (25%) hairy cell leukemias (HCL), 0 of 6 mantle cell lymphomas (MCL), 8 of 28 (29%) large cell lymphomas (LCL), and in 0 of 5 marginal zone/mucosa-associated lymphoid tissue lymphomas (MZL/MALT). We determined that 5.74 +/- 2.46% (mean +/- SD) of normal peripheral blood B cells and 6.48 +/- 1.62 % (mean +/- SD) of normal bone marrow B cells coexpress CD2. CONCLUSIONS: CD2 expression in B-cell neoplasia is a more prevalent phenomenon than previously appreciated. Normal CD2+ B-cell populations are observed in adults and may represent the nonmalignant counterpart of CD2+ B-cell neoplasms.