Search

In the Ninth Workshop, the DR4 sera revealed some heterogeneity, and it appeared that some of the patterns correlated with B alleles and/or DW splits (1). There was also some difficulty distinguishing well characterized lymphoblastoid cell lines (LCLs) which would have been very useful, but few were available at the time. Furthermore, many laboratories experienced difficulty with DR and DQ typing on LCLs largely because of extra reactions.

The classical technique of producing hybridomas involves the fusion of B lymphocytes, albeit at a low efficiency (approximately 1 hybrid per 105–106 cells), with an appropriate fusion partner, typically a myeloma or lymphoblastoid cell line, via the cumbersome and somewhat unpredictable polyethylene glycol (PEG) procedure.1 The fusion partner simply acts as a vector, supplying immortalization functions to an immunoglobulin-secreting lymphocyte. Recently, however, other approaches have become available that have the potential to generate immortalized, immunoglobulin-secreting lymphocytes with a higher efficiency. The aim of this chapter is to provide an introduction to an alternative method of generating immortalized lymphocytes—that of a form of DNA-mediated gene transfer known as electroporation.

A human lymphoblastoid cell line with ability to perform oxidative metabolism of various chemicals is mutated by the direct addition of an intact particulate soot. This experiment demonstrates that materials associated with combustion-generated particulates are biologically available and able to cause genetic changes in metabolically competent human cells.

Natural isolates of Epstein-Barr virus (EBV) were analyzed from individuals from California who were healthy or suffering from infectious mononucleosis (IM) and from individuals from Southern China who were healthy or suffering from nasopharyngeal carcinoma (NPC) or other carcinomas. The B95-8 Bam Hl I probe distinguished two strains of EBV. Type C strains of EBV are prevalent in Southern China and are missing a Bam Hl site in the Bam Hl I region of the genome. The type D strains, on the other hand, retain this restriction site and prevail in California.

The study of signals involved in the generation and maintenance of cytotoxic T lymphocytes (CTLs) has been facilitated by the availability of various techniques based on the mixed leukocyte culture (MLC) test (Bain et al. 1964; Bach and Hirschhorn 1964). In addition to the proliferative response in an MLC, CTLs are generated (Hayry and Defendi 1970; Hodes and Svedmyr 1970; Solliday and Bach 1970).

The occurrence of a malignant tumor that is seen relatively frequently in African children was first highlighted as a clinicopathological entity by Burkitt (1958, 1962a) about two decades ago. Children with such tumors were familiar to some medical workers in tropical Africa and have been documented in hospital records since the early decades of this century (Burkitt, 1962a). Case reports of the peculiar jaw manifestation of the disease were even published by a few doctors before the epoch-making report of Burkitt (1958). The tumor was characterized as a malignant lymphoma by O’Conor and Davies (1960) following which the eponymous title of Burkitt’s lymphoma was generally adopted (Berard et al 1969).

Theoretically, potent oncogenic viruses may persist in their natural host species but must reach a symbiotic equilibrium that maintains infection without causing severe pathological conditions during or before the reproductive age. Tumor development may then be a biological accident, occurring when control mechanisms, which usually ensure a relatively harmless virus-host relationship, break down or fail to function properly.

Mutants of lymphoblastoid cell lines have helped to identify, dissect, and locate genes of defined HLA products particularly within the HLA-D region [1], They can be used to characterize still unknown human MHC antigens and genes.

The effect of deoxyadenosine (dAdo) with deoxycoformycin on the induction of 2',5'-oligoadenylate synthetase by interferon was investigated. After semi-purification through poly(I):poly(C) gel, the activity was similar in control and dAdo-treated cells. However, the activity in the crude extract decreased with rising concentrations of dAdo. On the other hand, the level of 2'-phosphodiesterase, which is also induced by interferon and degrades 2',5'-oligoadenylate, showed no significant change after dAdo treatment. Thus, the crude extract was speculated to contain an inhibitor of 2',5'-oligoadenylate synthetase. Further characterization of the inhibitor revealed that inhibition was not due to dATP accumulation in cells.

Human lymphoblastoid cell line WI-L2-729-HFZ was fused with human lymph-node lymphocytes in one fusion and with human spleen cells in another fusion to generate human-human hybridomas. In both, increasing doses of insulin were added to the HAT medium immediately after the PEG-mediated cell fusion (10(-1)-10(-5) IU/ml) and the number of clones formed was determined 3 weeks later. 10(-3) IU/ml of insulin resulted in a 2- to 5-fold increase in the number of clones generated compared to the control plates. In view of the known difficulties in generating high-yield human-human hybridoma fusions, it is suggested that the use of insulin-supplemented HAT medium may provide a more efficient way in obtaining such clones.

The thioether nucleoside, 5′-methylthioadenosine (MTA) (Figure 1) is a product of transpropylamine reactions which lead to the synthesis of spermidine and spermine (Figure 2)(1). These polyamines are ubiquitous in mammalian cells (2). Their synthesis, and concomitantly the production of MTA, increases during periods of rapid growth (3). MTA does not accumulate in mammalian cells. Rather, the nucleoside is cleaved by MTA Phosphorylase (5′-methylthiadenosine: orthophosphate methylthioribosyltransferase), to yield adenine and 5-methylthioribose 1-phosphate (Figure 2)(4).

Epstein-Barr virus (EBV)- and fetal calf serum (FCS)-specific cytotoxic human T cells can be generated in vitro , and have been shown to be HLA-antigen restricted. In the present work, peripheral blood mononuclear cells were stimulated with the γ-irradiated autologous lymphoblastoid cell line (LCL) grown previously in FCS, human serum, or without serum. The induction and generation of cytotoxic T cells was carried out exclusively in culture medium containing autologous serum. With EBV-seronegative responders, FCS-grown stimulator LCL generated a FCS-specific cytotoxic T cell response. AB serum-grown LCL generated only a weak response, except at a high stimulatory dose, where the response tended to be essentially nonspecific. EBV-seropositive responders, in contrast, gave a typical secondary EBV-specific response regardless of the serum in which the LCL had been grown previously; no FCS response was detected. Dose-response and cold target inhibition studies confirmed these results. EBV immunity obviously plays a major role in the T cell response to the autologous LCL, which can no longer be viewed simply as a form of autologous mixed leucocyte reaction.

Previously, two B (B1 and B2)- and four T (T1, T2, T3, T4)-lymphocyte subpopulations have been identified in human blood smears by bacterial adherence. Here, to study the functional differences between these subpopulations the T1T2 cells were separated from T3T4 cells by selective adherence to Escherichia coli-2 4 monolayers. The adherent cells (T1T2 cells) responded well to concanavalin A in 3-day cultures and in mixed lymphocyte culture (MLC) in 6-day cultures and developed into cells specifically cytotoxic for allogeneic lymphocytes. The nonadherent cells (T3T4 cells) cultured for the same length of time were poorly responsive to concanavalin A, variably responsive in MLC, and poorly active in specific cytotoxicity. The T3T4 cells were naturally cytotoxic for allogeneic lymphocytes and for a normal lymphoblastoid cell line. We concluded that the T cells that bind E. coli-2 (T1T2 cells) are functionally different from those that do not bind (T3T4 cells).

Significantly decreased activities of the ectoenzyme 5′-nucleotidase (EC 3.1.3.5) in peripheral lymphocytes of patients with primary hypogammaglobulinemia have been described. There is conflicting evidence in the literature whether this concerns the X-linked type of the disease or rather the socalled “adult onset” hypogammaglobulinemia1,2,3 . Nevertheless, it seemed interesting to study the properties of human 5′-nucleotidase (5′-N) more closely. For reasons of availability of starting material the following approach was chosen: First the placental 5′-N was purified and characterized followed by an extensive comparison of the properties of this enzyme with 5′-N from human lymphocytes and lymphoblastoid cells

A new lymphoblastoid cell line, DG-75, was investigated for its ability to produce interferon. DG-75 cells, previously shown to be free of Epstein-Barr virus genome and receptors, could be grown in submerged culture and could produce interferon in titers comparable to interferon produced by Namalva cells. The interferon produced was similar in size to the Namalva interferon as determined by gel filtration in Ultrogel AcA54. The DG-75 cells present a new source of large quantities of interferon which may be safer for human use than the Namalva interferon.

Human Ia molecules have been isolated from the DR5 homozygous cell line Swei by alloantisera, and analyzed by 2-D gel electrophoresis, tryptic peptide mapping, and lectin affinity chromatography. Two distinct α chains and three distinct β chains have been identified. These chains associate to form molecules composed of α1β2, α1β3 and α2β1. The α1β2 + α1β3 molecules bear some oligosaccharide structures not found on the α2β1 molecule.

THE persistence in a latent state of the herpes simplex virus in neuronal cells1–3 and of the Epstein–Barr virus (EBV) in lymphoblastoid cells4,5 has been documented. Cytomegaloviruses (CMVs) have been isolated in the past from leukocytes of congenitally infected babies6 and found in peripheral leukocytes by in situ RNA–DNA cytohybridisation (C. H. Huang, P. Neiman and J. S. Pagano, unpublished data). EBV-positive lymphoblastoid cell lines have been established from three of 12 patients with CMV mononucleosis7,8 but the possible persistence of CMVs in these lines was not documented. We have now found an unexpressed CMV genome in an EBV-positive lymphoblastoid cell line.

The results of animal studies have long suggested that human interferons should be studied as antiviral and antitumour agents in medicine (see reviews by Finter 1973; Oxman 1973), but clinical trials have been hindered by the shortage of suitable material. Primary human leukocytes obtained from transfusion blood and processed as described by Strander and Cantell (1966,1967) have until recently provided nearly all the interferon used in humans. In 1976, Finnish workers made 1011 IU crude interferon from this source (Cantell and Hirvonen 1977), and have since increased their output severalfold (K. Cantell, personal communication). Such preparations have played a key role in the development of clinical studies. Nevertheless, it is obvious that human blood could not provide enough cells to make interferon in really large amounts. Furthermore, only limited quality control procedures can be applied to individual white blood cell lots, and for example there are few countries where the incidence of hepatitis in blood donors is as low as in Finland.

Of eight lymphoblastoid cell lines studied five were insensitive to both the anticellular and antiviral activities of human leukocyte interferon, and two were sensitive to both activities. One line could not be fully evaluated since it was not possible to study its sensitivity to the antiviral activity.

The promoting effects on cell proliferation of the 105K glycoprotein (105Kgp), purified from sera of chickens to which a Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C (MSB1-41C), had been transplanted, were examined using culture cells from various sources. The MSB1-41C line as well as the other chicken lymphoblastoid cell lines examined were sensitive to the 105Kgp. The growth-promoting effects of 105Kgp showed a biphasic pattern depending upon the amount of 105Kgp added into the culture medium. These findings indicate that the 105Kgp may be a promoting factor for chicken growing cells, especially lymphoblastoid cell lines.

Ataxia—telangiectasia (AT, McKusick 20890) is an autosomal recessive disorder characterized by progressive cerebellar ataxia starting in early infancy and leading to complete disability by the age of 10 years, and oculocutaneous telangiectasias, appearing at 3–5 years of age. The disease is clinically and genetically heterogeneous (Balbi et al., 1972). The basic molecular defect is unknown. AT patients show low levels or complete absence of IgA, are highly prone to lympho-reticular proliferative disorders and show hypersensitivity to ionizing radiations (Swift et al., 1976; Paterson et al., 1979; Painter et al., 1980; Gatti, 1984). Life expectancy can be shortened because patients may succumb to pulmonary infections or malignancies.

The use of a B-lymphoblastoid cell line (B-LCL) as a stimulator cell in the mixed leukocyte reaction (MLR) was investigated in patients with immunodeficiency disorders. The kinetics of the MLR stimulated by B-LCL are similar to those stimulated by normal allogeneic leukocytes, however, B-LCL stimulate a greater quantitative response. Comparison of LCL-stimulated and normal allogeneic lymphocyte-stimulated MLR in 32 patients and normal controls demonstrated that variation in the MLR was reduced when B-LCL were used as stimulator cells. The decrease in variability allowed for more sensitivity in the determination of abnormal responses; 9 of 32 patients had abnormal B-LCL-stimulated MLC responses, compared with 5 of 32 patients with abnormal responses to normal allogeneic leukocytes. Dose-response studies showed that vigorous responses could be obtained with low doses of B-LCL stimulator cells which served to better define deficient patient responses. Several patients demonstrated dissociation between the B-LCL-stimulated MLR and mitogen responses. The use of a B-LCL as a stimulator cell in the MLR is a valuable tool for the assessment of the immune status of patients with a variety of immunodeficiency disorders.

Publisher Summary The cell substrates that are used for human lymphoblastoid interferon (HLyIF) production are derived from human lymphoblastoid cell lines, most of which are established from Burkitt's lymphoma cases. Such cells are grown in submerged culture, and after suitable viral induction, some lines produced HLyIF in reasonable amounts. Human lymphoblastoid cells are first screened for interferon production. Large amounts are produced by several lymphoblastoid cell lines, the best of which is found to be the Namalva lymphoblastoid cell line. Most production of HLyIF is now being carried out with the Namalva cells. For separation of cells, the fequipment that may be used are batch and continuous-flow centrifuges, bottle laboratory centrifuge, industrialscale tubular continuous-flow centrifuge, and basket centrifuges. There are three main steps in the production of HLyIF: Cell propagation, HLyIF induction, and HLyIF purification. Namalva cells are then available for IF induction. Using this method, several parallel lines of scaled-up cultures should be maintained continuously.

A 2.5-year-old boy with a ring chromosome 10 was described. Clinical features included developmental retardation, short stature, mild mental retardation and cryptorchidism. He had a history of photohypersensitivity. By using PHA stimulated peripheral blood lymphocytes, Epstein-Barr virus transformed lymphoblastoid cell line and cultured skin fibroblasts, his karyotype was identified to be mos45,XY,−10/46,XY/46,XY,r(10) (p15.3q26.3).

Tunicamycin inhibited the synthesis of glycosylated mu-chains and kappa-chains in the human lymphoblastoid cell line, Daudi. Nonglycosylated IgM could not be detected on the surface of tunicamycin-treated cells by cell surface iodination techniques even under conditions where membrane IgM was re-expressed in control cultures following the enzymatic stripping of the existing membrane IgM. Biosynthetic labeling and subsequent immunochemical analysis indicated that the nonglycosylated mu and kappa-chains failed to efficiently assemble into monomeric IgM units. In a previous study (Dulis et al., J. biol. Chem., 1982), we have shown that the nonglycosylated mu- and kappa-chains are rapidly catabolized. The lack of expression of nonglycosylated IgM could be due to the rapid catabolism of the nonglycosylated polypeptide chains and/or to the inability to form functional monomeric IgM molecules. Thus glycosylation may be required to protect the newly synthesized polypeptide chains from intracellular catabolic events and to maintain proper conformational foldings of the polypeptide chains to allow for the assembly of subunits into functional units and their ultimate expression.

A human leukemia-associated differentiation antigen has been identified by a monoclonal antibody (A12) raised to the lymphoblastoid cell line NALM-1. The A12 antigen was expressed on the surface of leukemic cells from patients with common acute lymphoblastic leukemia (c-ALL) as well as on cells of the hematopoietic cell lines NALM-1, Reh-6, Raji, Daudi, CEM, and 8402 as determined by an antibody-binding radioimmunoassay, as well as by indirect immunofluorescence and FACS analysis. This antigen was not detected on normal blood lymphocytes, normal bone-marrow cells or leukemic cells from patients with acute myeloid leukemia (AML). The A12 antigen had an apparent molecular weight of 100 kD as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and appeared to be related to if not identical with the acute lymphoblastic leukemia antigen CALLA described by others. Cross-blocking experiments indicated that preincubation of NALM-1 cells with antibody A12 or J5 (anti-CALLA) could block subsequent binding of 125I-labeled A12 and J5 antibody. These results suggest that the two monoclonal antibodies recognize identical or closely located antigenic sites. The surface membrane expression of A12 antigen in NALM-1 cells was modulated when the cells were cultured in the presence of A12 antibody. Under these conditions, the expression of Ia antigens was unaffected. Re-expression of A12 antigen occurred within 24 h after transfer of the modulated cells into medium devoid of monoclonal antibody.

A lymphoblastoid cell line derived from the spleen of a chicken infected with an Australian strain of reticuloendotheliosis virus (REV) and designated RE-LB was established in suspension culture. The presence of REV antigen in the cells was demonstrated by the indirect fluorescent antibody test, while ultrathin sections of the RE-LB line cells revealed C-type particles. Infection of day-old-chickens with the cellular and cell-free culture fluid of the line produced 100 per cent and 50 per cent mortality, repectively. The line is probably transformed because the cells were agglutinated by concanavalin A and grew in soft agar.

Cultured skin fibroblast-like (FL) cells from patients with Werner syndrome grow poorly: reduced growth potential has been reported by more than 11 laboratories for cultures from more than 26 patients, using many different tissue culture media. This characteristic is of importance to research in Werner syndrome for several reasons. It remains to be determined whether poor growth is a primary or a secondary manifestation of the basic molecular defect, and the relationship is not yet clear between reduced growth of cultured skin fibroblasts and the other manifestations of Werner syndrome in vitro and in vivo. This characteristic also makes it difficult to obtain sufficient growth for clonal studies or for biochemical studies that require a large number of cells.

Homologous DNA from a human lymphoblastoid cell line is taken up in macromolecular form by human skin fibroblasts in culture. Experiments utilizing the heavy buoyant density and ultraviolet-light sensitivity of DNA containing 5-bromouracil reveal that the homologous DNA is retained in the recipient cells in relatively undegraded form for over 20 h. Similar experiments with heterologous DNA from bacteriophage T7 show that this DNA undergoes extensive degradation after entry into the fibroblasts and that the products of degradation are readily incorporated into DNA synthesized de novo .

A human lymphoblastoid cell line derived from a normal donor has been synchronized with a double thymidine block. The affinity of the cell membrane for ruthenium red was examined during different phases of the cell cycle. The thickness of the cell coat increased from G1 to G2. The significance of this finding is discussed.

Background RT-qPCR is a sensitive and increasingly used method for gene expression quantification. To normalize RT-qPCR measurements between samples, most laboratories use endogenous reference genes as internal controls. There is increasing evidence, however, that the expression of commonly used reference genes can vary significantly in certain contexts. Results Using the Genevestigator database of normalized and well-annotated microarray experiments, we describe the expression stability characteristics of the transciptomes of several organisms. The results show that a) no genes are universally stable, b) most commonly used reference genes yield very high transcript abundances as compared to the entire transcriptome, and c) for each biological context a subset of stable genes exists that has smaller variance than commonly used reference genes or genes that were selected for their stability across all conditions. Conclusion We therefore propose the normalization of RT-qPCR data using reference genes that are specifically chosen for the conditions under study. RefGenes is a community tool developed for that purpose. Validation RT-qPCR experiments across several organisms showed that the candidates proposed by RefGenes generally outperformed commonly used reference genes. RefGenes is available within Genevestigator at http://www.genevestigator.com., BMC Genomics, 12, ISSN:1471-2164

Normal immunosuppressive protein, prepared from human plasma by DEAE-cellulose chromatography, inhibits DNA synthesis in human cell lines of lymphocytes of both T and B origin. It also inhibits [3H]thymidine incorporation in mouse cell lines. Normal immunosuppressive protein was able to inhibit the proliferation of these cells, although they were already transformed and had a high rate of DNA synthesis. On the other hand, it does not inhibit myeloid cells or fibroblasts.

SUMMARY Continous passage of MDCC-RP1, a highly tumorigenic Marek's disease (MD) lymphoblastoid cell line, in cell culture resulted in a gradual loss in ability of the cell line to cause progressive tumors in susceptible day-old chicks. Inoculation of day-old chicks with high-cell-culture-passaged (187th to 417th) nontumorigenic MDCC-RP1 cells gave excellent protection against challenge at 8 days with low-passaged tumorigenic MDCC-RP1 cells but failed to protect against primary tumors caused by inoculation with MD virus. Vaccination with the herpesvirus of turkeys, on the other hand, protected the chickens well against primary tumors caused by MD virus and against transplantable tumors caused by tumorigenic MDCC-RP1 cells, but it did not protect as well against another MD lymphoblastoid cell line, MDCC-RP4. It is unlikely, therefore, that vaccines prepared from passaged MDCC-RP1 cell lines will have value for protecting chickens against MD in the field. RESUMEN

Background Comparative gene expression profiling analysis is useful in discovering differentially expressed genes associated with various diseases, including mental disorders. Autism spectrum disorders (ASD) are a group of complex childhood-onset neurodevelopmental and genetic disorders characterized by deficits in language development and verbal communication, impaired reciprocal social interaction, and the presence of repetitive behaviors or restricted interests. The study aimed to identify novel genes associated with the pathogenesis of ASD. Methods We conducted comparative total gene expression profiling analysis of lymphoblastoid cell lines (LCL) between 16 male patients with ASD and 16 male control subjects to screen differentially expressed genes associated with ASD. We verified one of the differentially expressed genes, FOXP1, using real-time quantitative PCR (RT-qPCR) in a sample of 83 male patients and 83 male controls that included the initial 16 male patients and male controls, respectively. Results A total of 252 differentially expressed probe sets representing 202 genes were detected between the two groups, including 89 up- and 113 downregulated genes in the ASD group. RT-qPCR verified significant elevation of the FOXP1 gene transcript of LCL in a sample of 83 male patients (10.46 ± 11.34) compared with 83 male controls (5.17 ± 8.20, P = 0.001). Conclusions Comparative gene expression profiling analysis of LCL is useful in discovering novel genetic markers associated with ASD. Elevated gene expression of FOXP1 might contribute to the pathogenesis of ASD. Clinical trial registration Identifier: NCT00494754