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At least 109 records · Page 6Linked to original sources
PubMed1991

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta.

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.

CD4 Antigens↗
PubMed2001

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones.

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62. 2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

Animals↗
PubMed2002

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos.

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.

Animals↗
PubMed1985

Inhibition of herpes simplex virus replication in vitro by human cytotoxic T cell clones and natural killer cell clones.

The abilities of human cytotoxic T cell (CTL) clones and natural killer (NK) cell clones to inhibit the replication of herpes simplex virus (HSV) in vitro were shown. The specificities of clones inhibiting HSV replication were the same as those of cytotoxicity in HSV type specificity and HLA restriction, i.e. HSV type 1 (HSV-1) and HSV type 2 (HSV-2) common CTL clones inhibited the replication of both HSV-1 and HSV-2 in autologous cells, but not in allogeneic cells. HSV-1-specific CTL clones inhibited the replication of HSV-1 in autologous cells; however, the replication of HSV-2 was not inhibited. NK cell clones inhibited the replication of HSV-1 and HSV-2 in autologous and allogeneic cells. This is the first demonstration that human virus-specific CTL clones and NK cell clones directly limit virus replication in vitro.

Cell Line↗
PubMed1997

Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus.

An improved version of the previously obtained cloning vector pCass was constructed by partially duplicating the 35S promoter used to drive the transient transcription of cloned viral cDNAs. Full-length cDNAs of the three genomic RNAs of tomato aspermy cucumovirus (TAV) cloned in this improved pCass (designated pCass2) gave a 3-fold higher infectivity in two plant species tested than the same cDNAs cloned in pCass1 with only a single 35S promoter. Host range, symptoms, morphology of viral particles and viral progeny RNAs induced by these sets of infectious cDNA clones analysed were identical to those induced by the wild-type virus. A mutant of genomic TAV RNA 3 containing a 163 nt deletion in the 3' untranslated region was stably maintained in the progeny RNAs, indicating that these cDNA clones may facilitate a study of virus function. This is the first report of infectious cDNA clones of TAV as well as of infectious cDNA clones with a duplicated 35S promoter of CaMV.

Cloning, Molecular↗
PubMed1988

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.

Amino Acid Sequence↗
PubMed2003

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

Amino Acid Sequence↗
PubMed1990

CD8+ suppressor T cell clone capable of inhibiting the antigen- and anti-T cell receptor-induced proliferation of Th clones without cytolytic activity.

A CD8+ Ts clone 13G2 was established from lymph node cells of bovine alpha s1-casein-primed C57BL/6 mice by in vitro antigenic stimulation followed by maintenance with IL-2-containing medium. The clone suppressed the Ag-induced proliferative responses of CD4+ Th cell clones without detectable cytotoxicity for both APC and responding T cells. The clone was able to suppress the in vitro proliferative response and antibody formation of Ag-primed lymph node cells. The suppression was Ag-nonspecific and not restricted to the MHC. The clone was able to suppress the proliferation of Th clones induced by an immobilized anti-TCR antibody in which APC was absent. The clone was, however, unable to suppress the proliferation of Th clones induced by anti-CD3 or IL-2. Thus, the mechanism of suppression by 13G2 was found to be due to a direct action on Th by inhibiting a consequence of signal transduction initiated through the TCR.

Animals↗
PubMed1990

Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones.

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.

Animals↗
PubMed1988

Establishment of antigen-specific and MHC-restricted B cell hybridoma clones: a model for studying antigen presentation to T cell lines and clones.

Presentation of GAT by GAT-specific hybridoma B cell clones to GAT-specific T cell lines and clones was investigated. B cell clones expressed surface IgG2b, Ia and Fc receptors, and did not secrete immunoglobulins. The clones formed rosettes with GAT-SRBC which could be inhibited by monoclonal antibodies against GAT idiotype. Presentation of GAT by the clones was MHC-restricted and highly efficient, i.e., a few hundred B cells or very low concentration of GAT stimulated proliferation of GAT-specific T cell lines. The fusion partner, M12.4.5.2, clone presented GAT much less efficiently. Although GAT-specific B cell clones presented also heterologous antigen beef insulin to insulin-reactive T cell lines, they were not able to present insulin in the absence of soluble antigen. Presentation of GAT was inhibited by pretreatment of the clones with monoclonal antibodies against I-E, mouse Ig and GAT-idiotype, as well as related antigens GA and GT. The rate of antigen uptake was much shorter compared to resting B cells for cells pulsed with GAT for 4 hours presented the antigen well. The studies suggest that a membrane-associated Il-1 may be required in the presence of antigen presentation by B cells. The results also revealed that antigen-specific hybridomas can present specific antigen in part with association of surface immunoglobulins which may be involved in antigen uptake and focusing to T cells.

Animals↗
PubMed1982

Analysis of cloned T cell function. I. Dissection of cloned T cell proliferative responses using cyclosporin A.

CsA interferes in a specific manner with the expansion of T cell clones in that it inhibits the antigen-driven component of the proliferative responses made by cloned helper T cells, cloned conventional cytolytic T cells, and cloned helper-independent cytolytic T cells. Cloned helper T cells and helper-independent cytolytic T cells, which share the ability to proliferate when cultured with specific alloantigen, fail to proliferate when cultured with specific alloantigen, fail to proliferate in response to this stimulus in the presence of CsA (10 to 100 ng/ml). In contrast, the proliferation observed when these cells are cultured with exogenous growth factors (but not alloantigen) is little influenced by as much as 1000 ng/ml CsA. When cloned helper T cells or helper-independent cytotoxic T cells are cultured with alloantigen plus exogenous growth factor, additive or synergistic proliferation occurs. However, CsA (10 to 1000 ng/ml) blocks only the component of proliferation induced by alloantigen, and leaves the lymphokine-driven component intact. CsA has similar effects on the proliferation of cloned conventional cytolytic T cells. Thus, CsA separates cloned T cell proliferation into two components: one driven by contact with alloantigens, the other driven by contact with mitogenic lymphokines.

Animals↗
PubMed1981

Cloning of functional human T lymphocytes by limiting dilution: impact of filler cells and interleukin 2 sources on cloning efficiencies.

Conditions for the cloning of alloactivated human lymphocytes by limiting dilution in the presence of interleukin 2 (IL 2) and filler cells have been investigated. Cloning efficiencies were extremely high (at least 40%) when IL 2 derived from pooled peripheral blood mononuclear cells (PBM), stimulated with phytohaemagglutinin in the presence of irradiated Epstein-Barr virus-transformed lymphoid cell lines (LCL), was used in combination with irradiated pooled PBM as filler cells. Cloning efficiencies were reduced almost twofold using autologous filler cells and were further reduced dramatically using autologous IL2, although some clones could still be obtained. In addition, cloning efficiencies were acceptable (over 30%) when IL 2 produced spontaneously from the leukaemic cell Jurkat (M-N) was used. In contrast, sheep erythrocytes and LCL as filler cells failed to allow successful cloning, although LCL were beneficial in the expansion of cloned populations. Other factors being equal, the use of pooled PBM as filler cells and highly active preparations of IL 2 will be the method of choice for cloning functional human T lymphocytes.

Cell Division↗
PubMed1994

Characterization of human serum-resistant and serum-sensitive clones from a single Trypanosoma brucei gambiense parental clone.

A protocol was developed to select clones of Trypanosoma brucei gambiense having different levels of resistance to normal human serum. Human serum-resistant clones were selected from a single parental clone by continuous serum treatment of infected immunosuppressed mice. Human serum-sensitive revertant clones were also obtained by continuous passage of resistant clones in immunosuppressed mice but without human serum pressure. It has been demonstrated that our trypanosome clones express distinct but stable levels of resistance. The variant antigenic type of each clone was characterized serologically and by 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. After selective pressure with human serum, variant antigen-type differences always occurred among clones in which different human serum susceptibilities were found. The work reported here demonstrates that in our T. brucei gambiense immunosuppressed mouse model there is a predictable association between variant antigen type and human serum resistance.

Animals↗
PubMed1975

Radioactive antigen suicide of an anti-DNP (2,4-dinitrophenyl) clone. II. Follow-up of clones relatively resistant to radioactive antigen suicide when initially selected.

After suicide of the anti-dinitrophenyl (DNP) clone E21 in a previous experiment (Eur. J. Immunol. 1975. 5:58) several new clones (S clones) appeared promptly in the recipients. There was compelling evidence that they were then resistant to suicide, relative to E21. We now report further studies on these S clones and their antibodies. The antibodies of half of the S clones had affinities ten to one hundred times higher than E21 (3 x 10(-7) M); none was lower. The four tested were also able to bind DNP on the particular conjugate used for the suicide procedure. Three of the S clones were serially propagated, they showed a very great capacity for proliferation, transferring into 510, 85 and 110 recipients each. When their cells were tested at subsequent passages, they recovered early after inhibition by radioactive antigen. Two S clones may still have been somewhat refractory to suicide but, probably in all three, rapid proliferation was largely responsible for the recovery of the few cells escaping suicide, and helped them to appear suicide resistant.

Age Factors↗
PubMed1997

Leishmania major: a clone with low virulence for BALB/c mice elicits a Th1 type response and protects against infection with a highly virulent clone.

BALB/c mice are highly susceptible to infection with Leishmania major and generally develop a severe, nonhealing form of disease following parasite inoculation. As opposed to protective Th1 type immune responses which develop in resistant strains of mice, BALB/c mice develop predominant Th2 type responses characterized by the production of high levels of IL-4, but only low levels of IFN-gamma. However, BALB/c mice will develop resistance and Th1 type responses following the inoculation of very low numbers of L. major promastigotes. In this study, we have examined the effects of parasite virulence on the immune response and disease phenotype in susceptible BALB/c mice. Two clones of L. major were isolated which differed with respect to their in vitro growth rates as promastigotes and their virulence for mice. One rapidly growing clone, L.m.F1, was highly virulent in BALB/c mice and produced nonhealing infections characterized by predominant Th2 type responses. In contrast, a slow-growing clone, L.m.S2, was less virulent in BALB/c mice and produced self-healing infections at parasite doses equivalent to those which produced progressive disease with the more virulent clone. Mice which healed infections with the L.m.S2 clone developed responses characterized by elevated production of IFN-gamma and were resistant to a challenge infection with the virulent L.m.F1 clone. These results suggest that the virulence of individual parasite clones may influence both the course of disease and the phenotype of the immune response which develops during infection.

Animals↗
PubMed1992

Murine cytolytic CD8+ T cell clones generated in a high cloning efficiency, accessory cell-free culture system express a restricted lymphokine profile.

To determine whether cytolytic T lymphocyte activity is associated with a particular lymphokine profile, lymphokine synthesis was analyzed in a large panel of primary clones derived from single murine CD8+ lymph node T cells. The clones were generated at high efficiency (60-70%), under conditions that had been optimized for the induction of specific cytolytic activity, by culture with phorbol 12-myristate 13-acetate, ionomycin, IL-2, and IFN-gamma for 6 days and then with IL-2 and IL-6 for a further 2 days. When the clones were restimulated for 24 hr with IL-2 and immobilized antibodies to CD3, CD8, and LFA-1, most secreted IL-3 and IFN-gamma and about a third secreted TNF. Although levels of production of IL-3 and IFN-gamma were positively correlated with each other (r = 0.85) and weakly correlated with cytolytic activity (r = 0.68 and 0.55, respectively). TNF titers were unrelated to any other function measured. None of the clones tested secreted detectable IL-4 or IL-6. Similarly, analysis of several clones for lymphokine mRNA expression following cDNA amplification by the polymerase chain reaction revealed the presence of IL-2, IL-3, IFN-gamma, TNF-alpha, and GM-CSF but not IL-4 or IL-6 transcripts. Cytolytic CD8+ T cell clones generated in this high efficiency, accessory cell-independent cloning system therefore expressed a restricted lymphokine profile characterized by the synthesis of IL-3 and IFN-gamma, with variable production of TNF.

Animals↗
PubMed1986

Cloned helper T cell for IgE: characterization of T cells cloned from an atopic donor with a high serum IgE.

T cells cloned from nonatopic and atopic donors were examined for the ability to help IgE production in vitro. Experiments with clones from nonatopic donors were unsuccessful in spite of isolation of four clones that promoted IgM and IgG when these were mixed with B cells from nonatopic donors and pokeweed mitogen. We reasoned that T cells regulating IgE might be found with greater frequency in atopic donors with very high serum IgE. Two T cell clones from an atopic donor with a serum IgE of 3000 IU/ml were found to drive atopic B cells to produce up to 10,000 pg/ml of IgE in vitro. This promotion of IgE secretion occurred in the absence of mitogen stimulus and appeared to decrease with higher ratios of T cells:B cells. One of the clones (A-H4) also promoted the production of IgM and IgG in addition to the IgE; however, when this clone was carried in culture for several months, this activity for IgM and IgG fell markedly, whereas help for IgE decreased less. We have speculated that these two T cell clones, with unusual activation properties, may be representative of a subset of atopic T cells responsible for maintaining the circulating B cells that spontaneously secrete IgE in vitro in patients with high serum IgE.

B-Lymphocytes↗
PubMed1988

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

Animals↗