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At least 73 records · Page 4Linked to original sources
PubMed1995

Dengue virus-specific, HLA-B35-restricted, human CD8+ cytotoxic T lymphocyte (CTL) clones. Recognition of NS3 amino acids 500 to 508 by CTL clones of two different serotype specificities.

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas of the world. We analyzed dengue virus-specific CD8+ CD4- CTL at the clonal level to further understand the role of CD8+ CTL in dengue virus infections. Dengue virus-specific CD8+ CTL clones were established from lymphocytes of a dengue 4-immune adult. Three patterns of dengue serotype specificities were identified: 1) specific for dengue 4, 2) cross-reactive for dengue 2 and dengue 4 (subcomplex-specific); and 3) cross-reactive for all four dengue virus serotypes. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone were further analyzed. All four of the clones were HLA-B35 restricted and recognized NS3. The epitopes were mapped to amino acids (aa) 483 to 618 of NS3. The epitope was then defined by using synthetic peptides. Three dengue 4-specific clones and one dengue 2/dengue 4 cross-reactive clone recognized the same peptide (TPEGIIPTL) encompassing aa 500 to 508 of dengue 4 NS3. The peptide encompassing aa 500-508 of dengue 2 NS3 was recognized by a dengue 2/dengue 4 cross-reactive clone but was not recognized by the dengue 4-specific clones. Dengue 4-specific and dengue 2/dengue 4 cross-reactive clones used different TCR. These results indicate that CD8+ CTL clones that use different TCR and demonstrate two distinct serotype specificities recognize the same 9-mer peptide in the context of HLA-B35.

Adult↗
PubMed1987

Phenotypic and functional analysis of human CD3+ and CD3- clones with "lymphokine-activated killer" (LAK) activity. Frequent occurrence of CD3+ LAK clones which produce interleukin-2.

Clones capable of lysing fresh, uncultured tumor cells ("lymphokine-activated killer": "LAK" activity) were selected from microcultures derived from either E-rosette-positive or E-rosette-negative cell populations. All the selected clones displayed a strong cytolytic activity against the NK-sensitive K562 cell line. Two major phenotypic groups of clones could be identified: a first group expressed the CD3 differentiation antigen, present exclusively on mature T lymphocytes; however, in contrast to typical cytolytic T lymphocytes, the majority of these clones expressed the unusual CD4- CD8- phenotype, whereas the remainder were CD4- CD8+. A second group was represented by CD3- clones which, in most instances, expressed the T-cell-lineage-specific CD2 antigen. Following stimulation with phytohemagglutinin (PHA), most of the CD3+ LAK clones produced Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) whereas those expressing the CD3- phenotype did not. Since previous studies indicated that PHA may be inefficient in inducing lymphokine production by T-cell variants lacking the CD3/T cell receptor complex (TCR), CD3- clones were further stimulated with the calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA). Only 2/11 CD3- LAK clones produced small amounts of IL-2, whereas the majority released IFN-gamma. Given the peculiar phenotypic and functional properties of many CD3 + LAK clones, we suggest that they may belong to a T-cell subset distinct from typical CTLs.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Phenotypic and functional analysis of lymphokine-activated killer (LAK) cell clones. Ability of CD3+, LAK cell clones to produce interferon-gamma and tumor necrosis factor upon stimulation with tumor targets.

Lymphokine-activated killer (LAK) cells are generated by the culture of peripheral blood lymphocytes with interleukin-2 (IL-2). A variety of cells, including T-lymphocytes and natural killer (NK) cells, can be activated by IL-2 to exhibit the ability to kill multiple tumor and "modified-self" targets. Recent reports indicate that culture conditions can determine the phenotype of cells expressing LAK activity. Using limiting dilution techniques, we first generated cloned LAK cells with three culture conditions: autologous human serum (AHS) + IL-2; AHS + IL-2 + 0.1 micrograms/ml phytohemagglutinin and fetal bovine serum and IL-2. We determined that all but one of the 47 LAK cell clones generated with the three culture conditions were CD3+ and T-cell like; one NK-like clone was observed. Clones that were cytotoxic for one target could generally kill multiple targets, and the absence of phytohemagglutinin did not significantly affect the ability of the LAK cell clones to kill multiple targets. The presence of phytohemagglutinin was, however, necessary for the long-term maintenance of proliferation and cytotoxic activity of the LAK cell clones. The mechanism by which LAK cells kill tumor targets is not known. We here demonstrate that LAK cells and LAK cell clones can produce interferon-gamma and tumor necrosis factor (TNF) when stimulated with an erythroleukemia cell, K562. Five of the six CD3+, LAK cell clones tested could be stimulated by K562 cells to produce both interferon-gamma and TNF. However, the ability of the cloned LAK cells to kill K562 cells, as measured in a 4-h 51Cr-release assay, did not correlate with their ability to produce these cytokines. Furthermore, specific antibodies that neutralize the cytotoxic activity of interferon-gamma and TNF did not inhibit killing of K562 cells by LAK cells as measured with a 4-h cytotoxic assay. The cytostatic and cytotoxic activities of interferon-gamma and TNF for tumor cells are well documented, but these cytolytic activities are slower acting and exhibit their maximum effect after 48-96 h. We here propose that LAK cells kill tumor targets by a combination of cell-to-cell-mediated killing and by the release of slower acting cytostatic/cytotoxic cytokines that can inhibit the growth of tumors some distance from the effector cells.

Antibodies, Monoclonal↗
PubMed1985

Molecular cloning of the Mason-Pfizer monkey virus genome: characterization and cloning of subgenomic fragments.

The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described. An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species. The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length. A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus. Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length. These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated. The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses. Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs. Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp. Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA. Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region. No clones containing the pol-coding region have been isolated. In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies. Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected. Sequences more distantly related to the env region were also found in Old World monkeys. Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M-PMV genome, but were only weakly detectable at low stringency.

Animals↗
PubMed1983

Human allospecific T cell proliferative clones: different T cell clones expressing an apparently identical HLA class II specificity are heterogeneous in regard to their proliferative inhibition by a panel of monoclonal antibodies against human Ia-like antigens.

Four independent PLT clones displaying an apparently identical class II specificity (i.e., Dw/DR3) were found to give a heterogeneous pattern of inhibition in relation to 15 anti-class II mAb and an anti-beta 2m mAb used as a control. Some clones were inhibited by all anti-class II mAbs, irrespective of the cluster of molecular subsets with which they reacted. Such clones were also inhibited by the control anti-beta 2m mAb. Other clones were inhibited by only a few of the mAb tested. Within this group of T clones following the addition of a limiting amount of conditioned medium the inhibitory data of all independent clones with an identical specificity were inhibited by the same mAb; under these conditions, it was possible to relate the mAb inhibition patterns with the specificity of the T cell clones and these T cell specificities with the epitopic cluster/molecular subsets defined by these mAbs. A new level of T cell subset heterogeneity within T cell clones with apparent identical proliferative specificities is demonstrated, in relation to the T cell clone "susceptibility" or "resistance" to the effects of anti-class II mAbs directed towards their own Ia-like antigens.

Antibodies, Monoclonal↗
PubMed2003

Rodent malaria parasites suffer from the presence of conspecific clones in three-clone Plasmodium chabaudi infections.

We studied infection dynamics of Plasmodium chabaudi in mice infected with 3 genetically distinct clones--1 less virulent than the other 2--either on their own or in mixtures. During the acute phase of infection, total numbers of asexual parasites in mixed-clone infections were equal to those produced by the 3 clones alone, suggesting strong in-host competition among clones. During the chronic phase of the infection, mixed-clone infections produced more asexual parasites than single-clone infections, suggesting lower levels of competition than during the acute phase, and indicating that a genetically diverse infection is harder to control by the host immune system. Transmission potential over the whole course of infection was lower from mixed-clone infections than from the average of the 3 single-clone infections. These results suggest that in-host competition reduces both growth rate and probability of transmission for individual parasite clones.

Animals↗
PubMed1992

A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.

The bacteriophage P1 cloning system can package and propagate DNA inserts that are up to 95 kilobases. Clones are maintained in Escherichia coli by a low-copy replicon in the P1 cloning vector and can be amplified by inducing a second replicon in the vector with isopropyl beta-D-thiogalactopyranoside. To overcome the necessity of screening clones for DNA inserts, we have developed a P1 vector with a positive selection system that is based on the properties of the sacB gene from Bacillus amyloliquefaciens. Expression of that gene kills E. coli cells that are grown in the presence of sucrose. In the new P1 vector (pAd10sacBII) sacB expression is regulated by a synthetic E. coli promoter that also contains a P1 C1 repressor binding site. A unique BamHI cloning site is located between the promoter and the sacB structural gene. Cloning DNA fragments into the BamHI site interrupts sacB expression and permits growth of plasmid-containing cells in the presence of sucrose. We have also bordered the BamHI site with unique rare-cutting restriction sites Not I, Sal I, and Sfi I and with T7 and Sp6 promoter sequences to facilitate characterization and analysis of P1 clones. We describe here the use of Not I digestion to size the cloned DNA fragments and RNA probes to identify the ends of those fragments. The positive selection P1 vector provides a 65- to 75-fold discrimination of P1 clones that contain inserts from those that do not. It therefore permits generation of genomic libraries that are much easier to use for gene isolation and genome mapping than are our previous libraries. Also, the new vector makes it feasible to generate P1 libraries from small amounts of genomic insert DNA, such as from sorted chromosomes.

Animals↗
PubMed1999

Coexistence of widespread clones and large radial clones in early embryonic ferret cortex.

Cell lineage analysis in rodents has shown that the cerebral cortex is formed from both widespread and large radial clustered clones representing partly distinct lineages and producing differing cell types. Since previous cell lineage analysis of the ferret cortex using retroviral libraries showed that most neurons labeled at E33-E35 formed widespread clones, we determined whether clones labeled earlier in neurogenesis showed a greater tendency to form coherent radial clones. Clones labeled at E27-E29 occasionally consisted of widespread multineuron clones (13% of PCR-defined clones), but commonly consisted of small clusters of two to four neurons (65%). Moreover, 6/21 hemispheres contained a single, much larger (6-150 cells) radial cluster. Although large clusters were observed in 28% of experiments, they contained many neurons, accounting for 38% of retrovirally labeled cells. The large clusters showed at most few widely scattered sibling cells, either by histological analysis or by PCR analysis, suggesting that radial and widespread clones coexist but are lineally separate at early stages of corticogenesis. Coexistence of large radial and widespread neuronal clones appears to be an evolutionarily conserved mechanism for cortical neurogenesis.

Alkaline Phosphatase↗
PubMed2002

Construction of expression-ready cDNA clones for KIAA genes: manual curation of 330 KIAA cDNA clones.

We have accumulated information on protein-coding sequences of uncharacterized human genes, which are known as KIAA genes, through cDNA sequencing. For comprehensive functional analysis of the KIAA genes, it is necessary to prepare a set of cDNA clones which direct the synthesis of functional KIAA gene products. However, since the KIAA cDNAs were derived from long mRNAs (> 4 kb), it was not expected that all of them were full-length. Thus, as the first step toward preparing these clones, we evaluated the integrity of protein-coding sequences of KIAA cDNA clones through comparison with homologous protein entries in the public database. As a result, 1141 KIAA cDNAs had at least one homologous entry in the database, and 619 of them (54%) were found to be truncated at the 5' and/or 3' ends. In this study, 290 KIAA cDNA clones were tailored to be full-length or have considerably longer sequences than the original clones by isolating additional cDNA clones and/or connected parts of additional cDNAs or PCR products of the missing portion to the original cDNA clone. Consequently, 265, 8, and 17 predicted CDSs of KIAA cDNA clones were increased in the amino-, carboxy-, and both terminal sequences, respectively. In addition, 40 cDNA clones were modified to remove spurious interruption of protein-coding sequences. The total length of the resultant extensions at amino- and carboxy-terminals of KIAA gene products reached 97,000 and 7,216 amino acid residues, respectively, and various protein domains were found in these extended portions.

Cloning, Molecular↗
PubMed1999

To clone or not to clone--whither the law?

The cloning of Dolly the lamb from adult cells by scientists at the Roslin Laboratories near Edinburgh in February 1997 has startled the world because it now opens the way to clone adult human beings. The reaction to Ian Wilmut's breakthrough has been instant and largely negative. Bills were rushed into both the US Senate and House of Representatives aimed at banning the cloning of human beings. Human cloning is premature at this stage, but there are many positive spin-offs of cloning in the field of genetic engineering, such as the production of human proteins such as blood clotting factors which aid in healing wounds. Progress by means of cloning can also be made into devising a cure for Parkinson's Disease amongst others. No lesser ethicist than John C. Fletcher of the University of Virginia foresees circumstances in which human cloning is acceptable e.g. to enable a couple to replace a dying child, to enable a couple, one of whom is infertile, to clone a child from either partner. Extensive regulation of cloning by the law is inevitable but, in doing so, the legislation should be careful not to outlaw research in this area which could be beneficial to mankind.

Adult↗
PubMed1992

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen.

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.

Animals↗
PubMed1993

Distinction of mouse CD8+ suppressor effector T cell clones from cytotoxic T cell clones by cytokine production and CD45 isoforms.

Mouse CD8+ T cell clones could be subdivided into two subgroups by the pattern of cytokine production and CD45 isoforms that exactly corresponded with the functionally assigned cytotoxic (CTL) and suppressor (Ts) subsets. All the Ts clones invariably produced IL-10 after stimulation with immobilized anti-CD3, whereas none of CTL clones expressed mRNA of IL-10 by the same stimulation. All the CTL clones were positive for CD45RA Ag, a product of exon 4 of CD45 gene, whereas none of the Ts clones were positive for this Ag. The results are analogous to the situation within CD4+ T cell clones in which Th1 and Th2 subtypes can be distinguished by CD45 isoforms and cytokines they produce. Ts clones could suppress the proliferative responses of both Th1 and Th2 type CD4+ clones by the production of two different cytokines, IL-10 and IFN gamma, respectively, but none of the CTL clones could suppress the proliferation of CD4+ T cells. The participation of an additional nonspecific molecule in the suppression of antibody response is suggested.

Animals↗
PubMed1996

[Dose dependence of the frequency of micronucleated clone cells and division-related median clone size difference. II].

PURPOSE: Following irradiation of the progenitor cells the clone growth of CHO cells decreases as a result of cell losses. Lethally acting expressions of micronuclei are produced by heritable lethal mutations. The dependency of the frequency of micronucleated binucleated clone cells and of the median clone sizes difference on the radiation dose was measured and compared to non-irradiated controls. METHODS: Using the cytokinesis-block-micronucleus-method binucleated cells with micronuclei were counted as ration of all binucleated cells within a clone size distribution. This ratio (shortened: micronucleus yield) was determined for all clone size distributions, which had been exposed to different irradiation doses and incubation times. The micronucleus yields were compared to the corresponding median clone sizes differences. RESULTS: The micronucleus yield is linearly dependent on the dose and is independent of the incubation time. The same holds true for the division related median clone sizes difference, which as a result is also linearly dependent on the micronucleus yield. CONCLUSIONS: Due to the inevitably errors of the cell count of micronucleated binucleated cells, an automatic measurement of the median clone sizes differences is the preferred method for evaluation of cellular radiation sensitivity for heritable lethal mutations. This value should always be determined in addition, if clone survival fractions are used as predictive test because it allows for an estimation of the remission probability of surviving cells.

Animals↗