Human Umbilical Cord Blood Lymphocytes Research Articles

Evaluation of Calyculin A Effect on γH2AX/53BP1 Focus Formation and Apoptosis in Human Umbilical Cord Blood Lymphocytes.

Dephosphorylation inhibitor calyculin A (cal A) has been reported to inhibit the disappearance of radiation-induced γH2AX DNA repair foci in human lymphocytes. However, other studies reported no change in the kinetics of γH2AX focus induction and loss in irradiated cells. While apoptosis might interplay with the kinetics of focus formation, it was not followed in irradiated cells along with DNA repair foci. Thus, to validate plausible explanations for significant variability in outputs of these studies, we evaluated the effect of cal A (1 and 10 nM) on γH2AX/53BP1 DNA repair foci and apoptosis in irradiated (1, 5, 10, and 100 cGy) human umbilical cord blood lymphocytes (UCBL) using automated fluorescence microscopy and annexin V-FITC/propidium iodide assay/γH2AX pan-staining, respectively. No effect of cal A on γH2AX and colocalized γH2AX/53BP1 foci induced by low doses (≤10 cGy) of γ-rays was observed. Moreover, 10 nM cal A treatment decreased the number of all types of DNA repair foci induced by 100 cGy irradiation. 10 nM cal A treatment induced apoptosis already at 2 h of treatment, independently from the delivered dose. Apoptosis was also detected in UCBL treated with lower cal A concentration, 1 nM, at longer cell incubation, 20 and 44 h. Our data suggest that apoptosis triggered by cal A in UCBL may underlie the failure of cal A to maintain radiation-induced γH2AX foci. All DSB molecular markers used in this study responded linearly to low-dose irradiation. Therefore, their combination may represent a strong biodosimetry tool for estimation of radiation response to low doses. Assessment of colocalized γH2AX/53BP1 improved the threshold of low dose detection.

DNA damage response and apoptosis induced by hyperthermia in human umbilical cord blood lymphocytes

While hyperthermia (HT) is a promising modality for cancer treatment, the knowledge on mechanisms of its effect on cells is still limited. We have investigated DNA double-strand break (DSB) and apoptosis induced by HT. Umbilical cord blood lymphocytes (UCBL) were subjected to HT at 43 °C. We have treated cells for 1 h (1 h HT), 2 h (2 h HT) and by combined HT and ice treatment (both lasting 1 h). Enumeration of DSB by 53BP1/γH2AX DNA repair focus formation and early apoptosis by γH2AX pan-staining was conducted by automated fluorescent microscopy. Apoptotic stages and viability were assessed by the annexin/propidium iodide (PI) assay using flow cytometry 0, 18, and 42 h post-treatment. HT induced either immediate (2 h HT) or postponed (1 h HT) DNA damage. The levels of 53BP1 and γH2AX foci differed under the same treatment conditions, suggesting that the ratio of co-localized γH2AX/53BP1 foci to all γH2AX and also to all 53BP1 foci could be a valuable marker. The ratio of co-localized foci increased immediately after 2 h HT regardless the way of assessment. For the first time we show, by both annexin/PI and γH2AX pan-staining assay that apoptosis can be induced during or immediately after the 2 h HT treatment. Our results suggest that HT may induce DSB in dependence on treatment duration and post-treatment time due to inhibition of DNA repair pathways and that HT-induced apoptosis might be dependent or associated with DSB formation in human lymphocytes. Assessment of γH2AX pan-staining in lymphocytes affected by HT may represent a valuable marker of HT treatment side effects.

Probit analysis of comparative assays on toxicities of lead chloride and lead acetate to in vitro cultured human umbilical cord blood lymphocytes.

This work describes that cytotoxicity of lead chloride and lead acetate to in vitro cultured lymphocytes from human umbilical cord blood, using four monitoring methods namely, trypan blue staining, acridine orange/ethidium bromide staining, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake assays; lead genotoxicity to lymphocytes was monitored by comet assay. The MIC value in each method was invariably 300 mg/L for PbCl2. Lethal concentration25 (LC25) values were almost in an agreeable range: 691.83 to 831.76 mg/L; LC50 values in each method were almost in the range: 1174.9 to 1348.9 mg/L; LC100 values were in the range: 3000 to 3300 mg/L, for lead chloride. Similarly, The MIC value in each method were invariably 150 mg/L; LC25 values were almost in the range: 295.12 to 371.53 mg/L; LC50 values were in the range: 501.18 to 588.84 mg/L; LC100 value was 1500 mg/L in all assays, for lead acetate. The comet assay also indicated that the LC100 values were 3300 mg/L lead chloride and 1500 mg/L lead acetate. Thus, both cytotoxicity and genotoxicity were recorded at 3300 mg/L lead chloride and 1500 mg/L lead acetate with lymphocytes.

The Human Immunodeficiency Virus tat Gene Enhances Replication of Human Herpesvirus-6

Human herpesvirus-6 (HHV-6) can enhance the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) in cells doubly infected by HIV-1 and HHV-6. HHV-6 enhances transcription of the HIV-1 long terminal repeat, and several HHV-6 trans-activator genes have been identified. Since HIV-1 and HHV-6 have similar cellular tropism, and since HIV-1 trans-activates other herpesviruses, a reciprocal interaction between the two viruses is possible. Interactions between HIV-1 and HHV-6 were analyzed in human umbilical cord blood (CB) lymphocytes and in a T-cell line by transfaction and infection experiments. CB cells dually infected with HIV-1 and HHV-6 showed an increase in HHV-6 infectious titer, an increase in HHV-6-specific immediate early RNA, and an increase in HHV-6 protein synthesis. Similarly, T-lymphocyte cells transfected with the entire HIV-1 proviral genome displayed an increase in HHV-6. When T-cells were transfected with a plasmid containing the HIV-1 tat gene under control of the simian virus (SV40) promoter and infected with HHV-6, higher levels of HHV-6 proteins and infectious virus were detected. Therefore, the presence of HIV-1 gene products, such as tat, can lead to an activation of HHV-6 expression. Since HHV-6 is cytopathic, its activation by HIV-1 may accelerate the depletion of CD4+ T-cells in infected individuals.

Frequent isolation of human herpesvirus 7 from saliva

We obtained isolates of human herpesvirus 7 (HHV-7) from 6 of 8 healthy adults by culturing saliva with human umbilical cord blood lymphocytes. These isolates were identified as HHV-7 on the basis of comparisons of restriction endonuclease fragment profiles and hybridization with HHV-7 strain RK DNA. The isolates could be differentiated from HHV-7 strain RK and from each other by their restriction endonuclease fragment profiles. We confirm the finding of frequent isolation of HHV-7 from saliva of healthy adults and report the first dual isolation of human herpesvirus 6 (HHV-6) and HHV-7 from a single saliva specimen. We also describe an in situ hybridization assay that can distinguish between HHV-6 and HHV-7.

Immune Modulation Exerted by Thymic Humoral Factor (Thf-γ2), on T-Cell Subsets and Il-2 Production of Umbilical Cord Blood Lymphocytes

The effect of a synthetic thymic humoral factor, THF-gamma 2, on the immune competence of T-cells was investigated in vitro on lymphocytes from human cord blood (UCBL). It was found that preincubation of UCBL with THF-gamma 2 caused an increase in the percentage of cells expressing the CD4 or the CD8 differentiation antigens, but did not affect the percentage of CD3 cells. The effect of THF-gamma 2 on PHA-induced IL-2 secretion was also studied and found that a 3hr preincubation with THF-gamma 2, prior to suboptimal PHA stimulation caused an increase in the IL-2 activity of the treated UCBL cultures. This effect was THF-gamma 2 dose dependent with an optimum in the range of 300-600 ng/ml and was not influenced by irradiation of the UCBL. These results indicate that THF-gamma 2, a synthetic octapeptide, modulates the immune state and response of human umbilical cord blood lymphocytes.

Primary EBV Infection of Human Umbilical Cord Lymphocytes and EBV Genome-negative Lymphoblastoid Cell Lines (Bjab and Ramos)

The appearance of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and induction of EBV-induced early antigen (EA) in human umbilical cord blood lymphocytes (HUCLs) and two EBV genome-negative Burkitt's lymphoma (BL) lines (BJAB and Ramos) were studied by infection with EBVs prepared from three different cell lines: marmoset cell line (B95-8) derived from infections mononucleosis, BL-derived cell line (P3HR-1) and human epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. B95-8 virus can transform HUCLs but cannot superinfect Raji cells. P3HR-1 virus can transform HUCLs cells but cannot transform HUCLs. NPC-KT virus can transform HUCLs and can superinfect Raji cells. We have examined the time sequence of EBNA appearance and EA induction in HUCLs, BJAB cells and Ramos cells, in order to determine if three different strains of EBV differ in their abilities to infect their cells. We found that all three strains of EBV can induce EBNA in HUCLs, BJAB cells and Ramos cells. On the other hand, we found that P3HR-1 virus and NPC-KT virus can induce EA in BJAB cells and Ramos cells, but B95-8 virus cannot induce EA in their cells.

Inverse relationship between constitutive gamma interferon production and human T-cell lymphoma/leukemia virus expression in cultured T lymphocytes.

Particular interest in human T lymphocyte lymphoma/leukemia virus (HTLV) derives from the close association of HTLV with several types of human mature T lymphocyte malignancies and the strong possibility that HTLV is the causative agent of this group of leukemias and lymphomas. This is the first report to show that HTLV expression in T lymphocytes cultured in vitro is inversely proportional to constitutive gamma interferon production. Of 16 fresh T lymphocyte cultures established from patients with mature T lymphocyte neoplasias, 3 were grown continuously for over 3 years and 13 were grown for 2 to 8 months in culture. Of the 16 cultures, 9 were HTLVp19 positive and interferon negative, whereas the remaining 7 were HTLVp19 negative or weakly positive and also interferon positive (12 to 105 U/ml). The prototype HTLV-positive T-cell line (HUT102) was examined over a long-term culture and after selective cell cloning for high virus yield. Results indicate that early-passage, low-HTLV-producing HUT102 cells constitutively produced significant levels of gamma-immune interferon. In late-passage and cloned HUT102 cells, an increase in HTLV production was concordant with a decrease in constitutive interferon production and the loss of mature T lymphocyte antigens. Transformation of human umbilical cord blood lymphocytes by HTLV was possible only after cocultivation with the non-interferon, high virus-producing, cloned HUT102 T lymphocytes. The inverse relationship between interferon and HTLV production was also observed when normal human umbilical cord blood and adult T lymphocytes were transformed by HTLV and maintained in culture.

Restricted expression of human T-Cell leukemia-lymphoma virus (HTLV) in Transformed human umbilical cord blood lymphocytes

The productive infection and transformation of fresh human lymphocytes by several HTLV isolates have recently been reported. We extend these observations here with the description of multiple immortalized, non-producer, human umbilical cord blood lymphocyte cultures developed by cocultivation or fusion of fresh cells with T cells cultured from leukemia-lymphoma patients. These transformed neonatal leukocytes exhibit morphological, cytochemical, and other phenotypic characteristics similar to those of other HTLV-infected cells but, in contrast to the usual productive infection seen, these cells contain only low amounts of viral proteins and do not release virus particles. These cells contain at least one copy of HTLV proviral DNA/cell and transcribe viral RNA similar in size to virus-producing cells. Virus expression in these cultures was not enhanced by IUdR treatment. These cell cultures should be useful in studies of the regulation of viral expression in human cells and of the viral proteins and nucleic acids involved in T-cell immortalization and growth.

Rescue of transforming Epstein-Barr virus (EBV) from EBV-genome-positive epithelial hybrid cells transfected with subgenomic fragments of EBV DNA.

Transfection experiments using subgenomic fragments of the B95-8 strain of Epstein-Barr virus (EBV) DNA and EBV genome (HR-1)-positive epithelial/Burkitt hybrid cells (D98/HR-1) were carried out to determine whether an interaction between the transfecting virus fragment(s) and the endogenous HR-1 EBV genome could take place. Expression of EBV-specific antigens, including early antigen and virus capsid antigen, was examined in transfected cells by immunofluorescence. Attempts were also made to recover biologically active EBV from the D98/HR-1 cells after transfection with cloned fragments of B95-8 DNA. We found that D98/HR-1 cells transfected with the BamHI H or H, F, and X fragments were positive for early antigen 3 days after transfection. Spent media from transfected D98/HR-1 cells maintained for 20-30 days in culture were pooled, filtered, concentrated, and used as a potential source of virus to inoculate human umbilical cord blood lymphocytes. No evidence of transformation was observed with such preparations. However, if spent medium from D98/HR-1 cell cultures was first treated with iododeoxyuridine (to induce EBV DNA synthesis and replicative cycle) and then transfected with the BamHI H, F, and X fragments of B95-8 DNA and used to infect cord blood lymphocytes, transformation was obtained. A lymphoblastoid cell line derived in this manner, designated HI-HFX, is an EBV nuclear antigen-positive nonproducer cell line. Similar results were obtained with preparations from iododeoxyuridine-treated D98/HR-1 cells transfected with the EB 26-36 fragment of B95-8 DNA cloned in a Charon 4A vector. The EB 26-36 fragment contains the BamHI H, F, and X regions.

Transformation in the presence of dimethyl sulfoxide facilitates recovery of Epstein-Barr virus.

Human umbilical cord blood lymphocytes were immortalized by infection with Epstein-Barr virus (EBV) in the presence of various concentrations of dimethyl sulfoxide (DMSO). 13 continuous lymphoblastoid cell lines were established. DMSO did not affect the efficiency of transformation and establishment of these lines. After a period of 2 months in culture, the lines were tested for EBV production by lethal irradiation (6,000 rad) from a 60Co source and subsequent cocultivation with primary umbilical cord lymphocytes. Cell lines transformed in the presence of 2% DMSO yielded transforming activity in 46.6% of test cultures, compared with 9.5% for lines established without DMSO in the medium. These findings imply that a permanent change in the host/virus relationship resulted from a brief exposure (48 h) to DMSO at the time of transformation.

Immunoglobulin properties of Epstein-Barr virus transformed human umbilical cord and adult peripheral blood lymphocytes

Lymphocytes from umbilical cords and peripheral adult blood were transformed with Epstein-Barr virus. Cultures of these transformed cells were studied for cell-associated and secreted immunoglobulins (Ig) and for their stability after long-term culture. The adult cell lines produced intracellular and extracellular Ig of all five classes but there was a paucity of IgA in cord cell cultures. Cells producing cytoplasmic Ig were at least 10 times more numerous than those secreting Ig. Both cord and adult lines appeared relatively stable after months of culture indicating that both may be useful in the preparation of immunological reagents.

Enhancement of Epstein-Barr virus-induced transformation of human lymphocytes by teleocidin

Teleocidin, a naturally occurring tumor promoter, enhanced colony formation of human umbilical cord blood lymphocytes in soft agar iduced by the B95-8 strain of Epstein-Barr virus. Colonies were formed most efficiently in the presence of teleocidin at a concentration of 0.1 ng/ml. Possible mechanisms of this enhancement are discussed.

Epstein-Barr virus and Herpesvirus saimiri: sensitivity to interferons and interferon inducers.

The in vitro sensitivity of oncogenic herpesviruses, Epstein-Barr virus (EBV), and Herpesvirus saimiri (HVS) to human interferon produced by normal human leukocytes (Le), lymphoblastoid cell lines (LYI), and diploid fibroblasts (Fi) was studied. Four virus strains were used: HVS S295C, the highly oncogenic HVS S396-O, the transforming B95-8 strain of EBV, and the nontransforming P3HR1 strain of EBV. All interferons were active when applied to the cells after absorption of HVS and P3HR1-EBV, although different amounts were required to achieve 50% inhibition of HVS-induced cytopathic effect or EBV-induced early antigen (EA) expression. Transformation of human umbilical cord blood lymphocytes (HCBL) by the B95-8 strain of EBV was prevented only by Le and LYI. In these experiments, the most effective inhibitor of the oncogenic herpesviruses was Le, and the least effective was Fi. The effect of polynucleotides poly(I).poly(C) and the complex of poly(I).poly(C) with poly-L-lysine and carboxymethylcellulose on HVS and EBV was also studied. Their inhibitory action was proportionate to the ability of herpesvirus-infected cells to produce interferon. Thus owl monkey kidney cells, which produce relatively high levels of interferon, required nanogram quantities of polynucleotides to become resistant to HVS. Transformation of HCBL by B95-8-EBV was also prevented by poly(I).poly(C). In Raji cells superinfected with P3HR1-EBV, polynucleotides failed to stimulate interferon, and higher EBV-induced EA expression was observed. The percentage of P3HR1 and Raji cells spontaneously expressing EBV-associated antigens remained unchanged after exposure to either interferon or polynucleotides.

Recovery of transforming EBV from non‐producer cells after superinfection with non‐transforming P3HR‐1 EBV

Cells of the Raji and NC37 lines can be induced by chemical inducers, such as BrdUrd and IdUrd, or the tumor-promoter TPA to EA-expression only, but do not reveal any VCA synthesis. After superinfection by nontransforming P3HR-1 EBV, however, a varying percentage of the cell population shows VCA synthesis and releases infectious viral particles. The recovered virus differs biologically from P3HR-1 EBV since it transforms human umbilical cord blood lymphocytes into EBNA-positive lymphoblastoid cell lines. Cells of these established lines are susceptible to renewed infection by P3HR-1 EBV which results in EA induction and VCA synthesis. Only cells of one line, NC37-R1, spontaneously produce VCA and EBV particles, which reveal transforming properties and do not induce EA upon superinfection of Raji cells. Infection of P3HR-1 EBV-converted BJA-B cells also leads to EA and VCA induction and the release of viral particles. In contrast to particles recovered from Raji and NC37 cells, no transforming activity was detectable in these virus preparations. According to these data, we propose that viral genomes persisting within Raji and NC37 cells are defective and become complemented by the superinfecting P3HR-1 virus.

Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus

Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells.

Demonstration of a Surface Antigen on Epstein-Barr Virus Genome-Carrying Lymphoid Cells: Distinction from the Virus-Determined Membrane Antigen

A surface antigen (SA) was detected on Epstein-Barr virus (EBV)-carrying lymphoid cell lines by indirect membrane immunofluorescence with an antiserum from a rabbit immunized with Raji cells; the antiserum had been extensively absorbed with normal human blood and tonsil cells. The SA was not detected on normal human umbilical cord and adult peripheral blood lymphocytes or EBV-negative cell lines. Incidences of the SA and EBV-determined membrane antigen (MA) on certain EBV-carrying cell lines were not compatible. Antibody against SA was differentially absorbed by the SA-positive MA-negative cell lines whereas MA antibody was absorbed by MA-positive SA-negative cell lines. The results of cross-absorption tests of antiserum against Raji cells or P3HR-1 cells suggested that SA may contain more than one antigenic determinant.

Abortive growth of human lymphocytes carrying a dormant Epstein-Barr viral genome.

When P3HR-1 lymphoblastoid cells expressing Epstein-Barr virus (EBV)-related antigens at very low frequency were cocultivated with human umbilical cord blood lymphocytes and the cell-free mixed culture fluid was applied to fresh cord lymphocytes, cells morphologically distinct from normal lymphocytes became evident after one to two weeks' exposure. The abnormal cells became abundant after one month and were easily identified by B-cell markers and a variety of morphologic abnormalities. Such abnormal B-lymphocytes appeared to be negative for EBV-determined nuclear antigen (EBNA), but when the immunofluorescence-negative cells were exposed to pokeweed mitogen and 5-iododeoxyuridine, a striking EBNA induction occurred. The growth of these abnormal cells was limited and they could be maintained for no more than three months. The implications of these findings are discussed in relation to the biological activity of EBV.

Epstein-Barr Virus Infection of Cryopreserved Umbilical Cord Blood Lymphocytes

SummaryWe have described the application of cryopreservation technology to freeze storage of human primary umbilical cord blood lymphocytes and have shown that the cryopreserved cells remain as sensitive to EB virus infection and transformation as the fresh autologous lymphocytes. The methodology has been satisfactorily applied to primary peripheral blood lymphocytes from adult human sources, and has been a convenient time and resource saving advance.

Comparative Studies on the Induction of Virus-Associated Nuclear Antigen and Early Antigen by Lymphocyte-Transforming (B95–8) and Nontransforming (P3HR-1) Strains of Epstein-Barr Virus

The kinetics of the appearance of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and early antigen (EA) in different EBV-infected target cell systems was studied. In addition, attention was given to the effect of pokeweed mitogen (PWM) and phosphonoacetic acid (PAA) (an inhibitor of herpesvirus DNA replication in infected cells) on the expression of EBNA and EA in EBV genome-negative target cells. The results obtained show that both of the EBV strains used (B95–8 and P3HR-1) induce EBNA at different time periods in the EBV genome-negative BJA-B cells and that P3HR-1 EBV is unable to induce either EBNA or EA in human umbilical cord blood lymphocytes (CBL) even after prestimulating these cells with PWM. Thus, our data present additional evidence for biological differences between B95–8 and P3HR-1 strains of EBV. EBNA is indeed the very first EBV-induced intracellular antigen to appear in an EBV-infected cell, and it is possible that the blast state may play an important role in the early induction of EBNA in CBL (as compared to BJA-B cells). Our data also suggest that the resident EBV genome in Raji cells may contribute, through an unknown mechanism, to the early expression of EA following superinfection with P3HR-1 EBV. Furthermore, our results also indicate that PAA does not inhibit the expression of EBNA or EA in BJA-B cells. The importance of these findings in relation to EBV-lymphocyte interaction and particularly in relation to cell transformation by EBV is discussed.