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PubMed1990

The effect of mutations in the MHC class I peptide binding groove on the cytotoxic T lymphocyte recognition of the Kb-restricted ovalbumin determinant.

The H-2Kb-restricted cytotoxic T lymphocyte (CTL) response directed against ovalbumin (OVA) is specific for a region contained within the sequence OVA253-276. In this study we have characterized this response by examining the class I-restricted presentation of OVA peptides by the naturally occurring Kb mutant (Kbm) glycoproteins Kbm1, Kbm3, Kbm5, Kbm8, Kbm10, Kbm11 and Kbm23. To facilitate this study we derived a series of somatic cell hybrid targets expressing the various Kbm class I molecules. Experiments using bulk OVA-specific CTL from C57BL/6 mice demonstrated that all the Kbm molecules except for Kbm1 and Kbm8 could present OVA peptides for effective T cell recognition. Clonal analysis revealed a more complex and relatively diverse pattern of CTL recognition of the Kbm/peptide combinations. This diversity is unlikely to result from the existence of multiple, independent Kb-restricted T cell determinants within OVA, since all CTL tested were specific for a single region between residues 259 and 273. Examination of the fine specificity of Kbm presentation identified individual changes at residues 77, 80 and 116 which affected T cell recognition. The results imply that these changes do not inhibit peptide binding since some clones could recognize peptide presented by a particular Kbm molecule, while other clones could not. All three residues reside within the peptide-binding cleft of the class I protein and are not expected to directly contact the T cell receptor. Although we did not formally demonstrate that OVA binding by Kbm vs. Kb is quantitatively identical, our results are best explained by postulating that the changes at residues 77, 80 and 116 indirectly affect T cell recognition by altering peptide conformation. Taken together our results suggest that changes within the class I binding site can profoundly modify peptide presentation without significantly inhibiting peptide-class I association.

Animals↗
PubMed1992

Specificity, T cell receptor diversity and activation requirements of CD4+ and CD8+ clones derived from human melanoma-infiltrating lymphocytes.

To try to understand the functional significance of human melanoma-infiltrating lymphocytes (TIL), a clonal analysis of the specificity, T cell receptor (TcR) diversity and activation requirements of these lymphocytes isolated from four different tumors was carried out. Supporting the presence of in vivo primed tumor-specific T lymphocytes in these four tumors, a high frequency of the Cd8+ and CD4+ clones, obtained from the TIL cultured for a few days with recombinant interleukin (rIL)-2 and autologous tumor cells, exhibited a restricted lysis or proliferation in response to the autologous tumor cell line. In contrast, no tumor-specific clone was obtained from freshly extracted TIL, suggesting that the frequency of tumor-specific effectors remained low in these tumors. Only the CD8+ clones lysed the autologous tumor cells and their activity was major histocompatibility complex MHC class I restricted. Significant expansion of CD4+ and CD8+ tumor-specific clones required regular restimulation by autologous melanoma cells but also the addition of exogenous IL-2 and of Epstein-Barr virus-transformed B feeder cells. Five different tumor-specific clones, three CD8+ and two CD4+ clones were identified in a single tumor on the basis of their TcR gene configuration. Together, these data suggest that a spontaneous and diverse immune response, mediated by tumor-specific CD4+ as well as CD8+ T lymphocytes, arises in most MHC-bearing human melanomas but that antigen-MHC complex presentation by tumor cells does not, at least in vitro, allow a significant proliferation of these lymphocytes.

CD4 Antigens↗
PubMed1994

Allelotype of head and neck paragangliomas: allelic imbalance is confined to the long arm of chromosome 11, the site of the predisposing locus PGL.

Paragangliomas of the head and neck region are usually slow growing, benign tumors. A considerable fraction has a positive family history, and the predisposing locus, PGL, has recently been assigned to 11q22-q23. The inheritance pattern of the disease suggests that PGL undergoes maternal genomic imprinting. We have investigated 26 tumor samples from 22 patients with head and neck paragangliomas for the occurrence of loss of heterozygosity (LOH) on all non-acrocentric autosome arms. LOH was found only on chromosome 11, with a marked clustering on the distal half of the q-arm. However, in many cases the resulting allelic imbalance relative to normal DNA was weak, suggesting that only part of the tumor showed this abnormality. In all eight cases where we were able to determine the parental origin, the allele undergoing loss was maternally derived. Clonality analysis with a polymorphic marker for the X-chromosome indicated that two of three informative female cases were polyclonal, although a number of tumors carry aneuploid stemlines in DNA flow cytometry. We conclude that either tumor heterogeneity or polyclonality may explain the partial allele loss events seen in certain cases.

Alleles↗
PubMed2004

Movement of calmodulin between cells in the ovary and embryo of drosophila.

Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. We used null mutations of the Drosophila CaM gene (Cam) in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos-a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism.

Animals↗
PubMed2004

Olig genes are expressed in a heterogeneous population of precursor cells in the developing spinal cord.

Recent results from multiple laboratories have identified Olig genes as important in regulating glial differentiation. Here we show that Olig2 expression at early stages of development (prior to E16.5) identifies a domain in the developing spinal cord, which contains a heterogeneous population of progenitors that includes stem cells and glial progenitors. We show that Nkx2.2 and Olig2, which are present initially in nonoverlapping domains, are coexpressed at later stages, likely due to a second wave of Olig expression. We find that Olig1, like Olig2, is present in cells that coexpress astrocytic and radial glial markers and that Olig1/2 double knockouts lead to a loss of oligodendrocytes with preservation of NG2 expression. These results coupled with previously published data indicate that Olig1/2 and Nkx2.2, while clearly important in regulating early progenitor cell differentiation, do not unambiguously demonstrate the existence of an oligodendrocyte-neuron precursor or negate the existing retroviral lineage and clonal analysis data that suggest the existence of other types of precursors such as oligodendrocyte-astrocyte precursors or neuronal precursors.

Animals↗
PubMed1991

Tracing glial cell lineages in the mammalian forebrain.

Astrocytes and oligodendrocytes emerge in late gestational and early post-natal development in the mammalian CNS. The nature, and number, of progenitors for each glial type is a central question. This review will focus upon several unresolved issues relating to glial cell lineages and describe new methods to try to illuminate these issues further: 1) How can developmental patterns by which immature neuroectodermal cells give rise to classes of neurons and glia be understood in the context of lineage? 2) What are the lineage relationships among the various cell classes, how many glial lineages are there in the developing CNS, and how can recent methods of clonal analysis using stable markers be used to clarify lineage patterns? 3) Do patterns of gliogenesis vary in different regions of the CNS? 4) How do patterns of gliogenesis observed in vitro relate to those in vivo?

Animals↗
PubMed1986

Acquired resistance towards immune defense during metastatic progression represents a secondary phenomenon.

The role of natural immune defense in the control of metastatic spread of tumor cells was evaluated by adapting an s.c.-grown, antigenic, but non-immunogenic rat fibrosarcoma (Bsp6S) to ascitic growth (BSp6A) in the strain of origin (BDX), where peritoneal cells display a high level of NK and macrophage activity. Parallel tests were performed to determine whether tumor cells can selectively adapt to non-specific immune defense, i.e. whether antigenicity of the non-immunogenic tumor remains unaltered, and whether this is accompanied by metastatic progression. During adaptation to ascitic growth the tumor line gradually lost susceptibility to NK cells and macrophages. This was due to the appearance of an increasing number of resistant clones (BSp6S, none; BSp6A, 50% of clones), which had lost binding structures for NK cells and macrophages. No alteration could be observed in antigenicity of the BSp6A variant as revealed by clonal analysis using LD of CTL. Neither BSp6S nor BSp6A cells metastasized. When the ascitic variant was retransplanted s.c. (BSp6AS), susceptibility to NK cells and macrophages was further decreased, in the sense that all clones of the BSp6AS variant became resistant. Furthermore, the BSp6AS variant had lost some of the tumor-associated antigens (TAAs) found on BSp6S and A variants. More important, upon s.c. transplantation BSp6AS regularly metastasized to the draining LN, contrary to BSp6S and BSp6A. When locally growing tumors were excised, rats implanted with BSp6AS frequently died with metastatic tumor burdens in LNs and lung, while other animals survived after excision of BSp6S or BSp6A. The data indicate a correlation between resistance to lysis, morphology and metastatic capacity. But the initial loss of susceptibility to NK cells and macrophages during adaptation to ascitic growth, which can be considered as an escape mechanism, was not accompanied by increased metastatic capacity. Hence, we suppose that with respect to metastatic progression of the BSp6AS variant, resistance to lysis by loss of binding structures represents only a secondary, but not a causative, element.

Animals↗
PubMed1992

MHC-restricted responses of CD8+ and CD4+ T-cell clones from regional lymph nodes of melanoma patients.

Regional lymph-node (LN) lymphocytes may constitute an important defence against the spread of human melanoma beyond regional LNs. The present study was directed to clonal analysis of lymphocytes cultured either directly from the LNs or after stimulation in cultures with autologous melanoma (MLTC). T-cell clones derived from MLTC reactions had either CD4+ or CD8+ phenotypes. Inhibition studies with monoclonal antibodies (MAbs) suggested that the CD8+ cytotoxic T-cell (CTL) clones had MHC-class-I-restricted cytotoxic activity against the autologous and a proportion of HLA-class-I-compatible allogeneic melanomas. The pattern of cytotoxicity against a panel of HLA-typed melanoma cells and inhibition by (polyclonal) HLA-typing sera suggested the CD8+ CTL were restricted by HLA-A3. The CD4+ T-cell clones had weak cytotoxic activity which appeared restricted by HLA-DR2. T cells cultured from unstimulated lymphocytes were all CD4+. One of the clones exhibited cytotoxic activity against both the autologous and HLA-DR2-compatible allogeneic melanoma cells, whereas another 2 had cytotoxic activity only against a HLA-DR2-compatible allogeneic melanoma established from a primary melanoma. IL-2 production by a 4th non-cytotoxic clone had similar specificity. These results suggest that HLA-A3 and DR2 may act as restricting elements in recognition of melanoma antigens by T cells from LNs and that they may have recognized at least 2 different antigens on the melanoma cells.

Aged↗
PubMed1995

IGF-2 autocrine stimulation in tumorigenic clones of a human colon-carcinoma cell line.

Clonal analysis has shown that the SW613-S human colon-carcinoma cell line is heterogeneous: some cell clones display a high level of amplification of the c-myc gene and are tumorigenic in nude mice, whereas others have a small number of copies of this gene and are non-tumorigenic. Tumorigenic clones can proliferate in a chemically defined serum-free medium, whereas non-tumorigenic clones cannot. Suramin, like anti-insulin-like growth factor (IGF) or anti-IGF-I receptor antibodies, efficiently inhibits the growth of tumorigenic clones in defined medium. Inhibition by suramin or by anti-IGF antibodies can be reversed by pure IGF-I or IGF-2. Pure IGF-1 or IGF-2 or culture medium conditioned by tumorigenic clones can stimulate DNA synthesis in cells of non-tumorigenic clones. Co-culture with cells of tumorigenic clones sustains the growth of non-tumorigenic clones in defined medium. Cells of both tumorigenic and non-tumorigenic clones express high-affinity IGF-1 receptors at their surface but tumorigenic clones produce on average 5 times more IGF-1 and 25 times more IGF-2 than non-tumorigenic ones. These results indicate that autocrine growth stimulation of tumorigenic clones by IGFs through the IGF-1 receptor is essential for their ability to grow in defined medium. Since cells of tumorigenic clones produce IGF-2 at levels 80 times higher than IGF-1 and since an antibody strictly specific for IGF-1 has no effect on DNA synthesis in cells of tumorigenic clones grown in defined medium, IGF-2 is very likely the main effector in the autocrine loop.

Cell Division↗
PubMed1985

Photoregulated expression of a pea rbcS gene in leaves of transgenic plants.

A 2.4-kb pea genomic fragment, containing a member (rbcS-E9) of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase, was inserted into a non-oncogenic, Ti-plasmid vector and introduced into the genomes of Petunia hybrida (Mitchell) and Nicotiana tabacum (SR1) plants by in vitro transformation. Petunia and tobacco plants containing the introduced pea rbcS-E9 gene were regenerated from protoplasts. In these transgenic plants the rbcS-E9 gene is transcribed accurately using its own promoter and its expression is light-induced and organ-specific. A deletion mutant with 352 bp of 5'-upstream sequence still retains photoinducibility and leaf-specific expression. Clonal analysis of independent transgenic petunia plants revealed that chromosomal positions in the recipient plant genome affect the quantitative but not qualitative aspects of rbcS-E9 expression.

Journal Article↗
PubMed1982

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole.

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.

Animals↗
PubMed1989

Cell enlargement: one possible mechanism underlying cellular senescence.

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding greater than 32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.

Animals↗
PubMed1987

Myoblast differentiation is induced by nerve transplanted to chick embryo legs.

Chick embryos were denervated early in development in order to disrupt the normal inductive interactions between the nervous system and developing populations of mononucleated myoblasts and their precursors in the leg. Neural tissue, either spinal cord or ciliary ganglion, was transplanted to one leg of the denervated embryos; the other leg remained aneural. Clonal analysis of cell populations in the transplant-containing legs showed that ectopic nerve tissue can recapitulate some of the neuromuscular interactions that occur in normally developing embryos. Chief among these was the observation that transplantation induced the appearance of the CMR-III myoblast class in the leg muscle. Since the process by which CMR-III myoblasts are produced from a precursor is dependent on nerve both in vivo and in vitro (Bonner, P.H. and T.R. Adams, Dev. Biol., 90:175-184, 1982), it was concluded that transplanted nerve tissue can also induce myoblast differentiation.

Animals↗
PubMed2004

High prevalence of mixed genotype infections in hepatitis B virus infected intravenous drug users.

The clinical relevance of hepatitis B virus (HBV) genotypes has been documented; however, the prevalence of mixed HBV genotype infections in at-risk groups remains controversial. The HBV genotypes were determined in 325 HBV-infected intravenous drug users (IVDU) who were at a greater risk of multiple exposures to different HBV genotypes by using a newly developed line probe assay. The distribution of HBV genotype was as follows: genotype A alone in 2 (0.6%); genotype B alone in 256 (78.8%); genotype C alone in 10 (3.1%); mixed genotype A and B in 18 (5.5%); genotype B and C in 30 (9.2%); genotype B and D in 1 (0.3%); genotype A and C in 1 (0.3%); and mixed infections of genotype A, B, and C in 3 (0.9%). Clonal analysis confirmed further the existence of mixed genotype infection and recombination between different genotypes. Compared with our previous data, the line probe assay seemed more sensitive than polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay in identifying HBV genotype (98.8% vs. 65.0%) and detecting mixed genotype infections (16.3% vs. 0%). In conclusion, the prevalence of mixed HBV infections is substantially higher in IVDU in endemic areas, and the line probe assay is a useful method for rapid genotyping of HBV, with particular reference to the detection of mixed genotype infections.

Adult↗
PubMed2005

Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm.

Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs.

Animals↗
PubMed1986

Normal and dystrophic hamster myoblast and fibroblast growth in culture.

Normal and dystrophic hamster myoblasts and fibroblasts were compared for characteristic indicators of growth and differentiation. Clonal analysis of myoblast cultures indicated that 80% of colonies judged to be fusion-competent had differentiated. Dystrophic myoblasts were identical to normal in terms of their morphology, fusion potential (81.4%), and myokinase activity (59.6-49.1 mU/mg at 2-7 days), but displayed a significantly higher plating efficiency (normal: 52.6%; dystrophic: 82.1%), a longer doubling time (normal: 21.7 hours; dystrophic: 33.3 hours), and a lower day-7 creatine kinase activity (normal: 60.7 mU/mg; dystrophic: 41.3 mU/mg). Dystrophic fibroblasts were indistinguishable from normal ones in terms of their morphology, plating efficiency (90.4%), and doubling time (32.5 hours), but displayed a significantly lower day-2 creatine kinase activity (normal 58.3 mU/mg; dystrophic: 35.8 mU/mg) and day-7 myokinase activity (normal: 52.7 mU/mg; dystrophic: 39.9 mU/mg). The results are suggestive of an early and differential expression of the primary defect in dystrophic hamster myoblasts and fibroblasts in culture.

Adenylate Kinase↗
PubMed1993

Distribution of pluripotent neural crest cells in the embryo and the role of brain-derived neurotrophic factor in the commitment to the primary sensory neuron lineage.

Many early migratory neural crest cells are pluripotent in the sense that their progeny are able to generate more than one differentiated phenotype (Sieber-Blum and Cohen, 1980, Dev. Biol. 80:95-106; Baroffio, Dupin, and Le Douarin, 1988, Proc. Natl. Acad. Sci. USA 85:5325-5329; Bronner-Fraser and Fraser, 1988, Nature 335:161-164; Sieber-Blum, 1989a, Science 243:1608-1611; Ito and Sieber-Blum, 1991, Dev. Biol. 148:95-106). At trunk levels, the neural crest contains two classes (Sieber-Blum and Cohen, 1980) and at posterior rhombencephalic levels, three different classes of pluripotent cells (Ito and Sieber-Blum, 1991). We investigated cell differentiation by in vitro clonal analysis to determine when in development the pool of pluripotent neural crest cells becomes exhausted. The data suggest that different classes of pluripotent cells, precursor cells with more restricted developmental potentials, and apparently committed cells, exist at sites of advanced migration (posterior branchial arches) and even at target sites of neural crest cell differentiation [posterior branchial arches, dorsal root ganglia (DRG), sympathetic ganglia (SG), and epidermal ectoderm]. Some putative classes of pluripotent cells persist well into the second half of embryonic development. These observations have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation. They support the idea that cues originating from the microenvironment affect differentiation of pluripotent neural crest cells. One such signal appears to be brain-derived neurotrophic factor (BDNF). In the presence of BDNF, but not nerve growth factor (NGF), there is a significant increase in the number of neural crest cells per colony that express a sensory neuron-specific marker. Because this increase is not accompanied by a corresponding increase in the total number of cells per colony, this suggests that BDNF plays a role in cell type specification.

Animals↗
PubMed1996

Cloning differentially expressed genes by linker capture subtraction.

We have developed a simple and effective method, designated linker capture subtraction (LCS), for cloning differentially expressed genes between two cell types or between cells treated in two different ways. In the first step of the method, two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for PCR amplification. In the second step, the linkered DNA (tester) from one mRNA population is hybridized to an excess of the unlinkered DNA (driver) from the other mRNA population, followed by incubation with mung bean nuclease which digests single-stranded DNA specifically. This leaves only tester-tester homohybrids to be amplified by PCR in the following step, so as to achieve an enrichment of tester-specific sequences. The amplified PCR products are then used as tester for another round of subtraction. The process of subtraction is carried out three times, and the final PCR products are inserted into a vector for clonal analysis. We have used the strategy to begin to clone and identify the genes expressed differentially between the human prostate cancer cell lines LNCaP and PC-3, which have different tumorigenic and metastatic potentials. We demonstrated strong enrichment of target sequences. We also report the identities of two of the genes expressed differentially in these cell lines. One is prostate-specific antigen (PSA) which is known to be expressed in LNCaP but not in PC-3. The other is vimentin, the differential expression of which has not been reported previously in these prostate cancer cells.

Base Sequence↗