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PubMed1990

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: implications for T cell receptor complex formation and activation.

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes.

Animals↗
PubMedDate not supplied

Transfer of chimeric receptor gene made of variable regions of tumor-specific antibody confers anticarbohydrate specificity on T cells.

The antitumor specificity of T cells can be induced by gene transfer using a recently developed therapeutic approach (T body). In this work, we genetically conferred anticarbohydrate specificity onto T cells using the variable regions of monoclonal antibody MLuC1, which binds the Lewis(Y) (LeY) tumor-associated antigen that is overexpressed on several human carcinomas. The variable regions of MLuC1, which are in a single-chain Fv (ScFv) configuration, were cloned and spliced in a eukaryotic expression vector with both the gene encoding the signal-transducing gamma-chain of the human Fcgamma receptor and a flexible hinge domain. The chimeric ScFv-gamma gene was expressed in a murine cytotoxic T-cell hybridoma. Transfectants receiving vector only served as a negative control (mock). Screening for functional transfectants was carried out using a tumor growth inhibition assay. The soluble form of MLuC1 ScFv was recovered from bacteria periplasm and tested for binding to LeY-expressing cells by the fluorescence-activated cell sorter analysis. Despite the low binding ability of the soluble MLuC1 ScFv, 7 of 13 genetically engineered cytotoxic T lymphocyte clones inhibited the growth of LeY-positive cells and did not affect growth of LeY-negative cells. None of the mock clones tested specifically inhibited tumor growth. These data indicate that, by chimeric MLuC1 ScFv-gamma gene transfer, it is possible to confer anticarbohydrate specificity onto T cells and extend the applicability of the T-body approach to tumor-associated antigens that are naturally not recognized by T cells.

Amino Acid Sequence↗
PubMed1991

Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

The T cell co-receptor, CD8, binds to the alpha 3 domain of HLA class I (Salter, R.D., R.J. Benjamin, P.K. Wesley, S.E. Buxton, T.P.J. Garrett, C. Clayberger, A.M. Krensky, A.M. Norman, D.R. Littman, and P. Parham. 1990. Nature [Lond.]. 345:41; Connolly, J.M., T.A. Potter, E.M. Wormstall, and T.H. Hansen. 1988. J. Exp. Med. 168:325; and Potter, T.A., T.V. Rajan, R.F. Dick II, and J.A. Bluestone. 1989. Nature [Lond.]. 337:73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (Ig)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I. Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the Bence-Jones homodimer, REI. Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 alpha-terminal domain and an Ig variable domain.

Amino Acid Sequence↗
PubMed2001

Nucleotide sequence analysis of a human monoclonal antibody TONO-1 with cytotoxic potential for T-leukemia/lymphoma cells.

A human monoclonal antibody (HuMab) TONO-1 (IgM, lambda) recognizes cell surface antigens associated primarily with human T-leukemia/lymphoma cells. In this study, we investigated the reactivity against T-leukemia/lymphoma cells in detail, cytotoxic potential and primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMab TONO-1. Expression of the molecules (TONO-1 Ags) detected by a HuMab TONO-1 was significantly heterogeneous even in the same T-leukemia/lymphoma cell lines HPB-MLT and MOLT-4F. The flow cytometric curves showed an unusual broad-based spread of fluorescence intensity. HuMab TONO-1 was shown to have the ability to kill the T-leukernia/lymphoma cells efficiently in the presence of rabbit complements. However, HuMab TONO-1 did not demonstrate significant antibody-dependent cellular cytotoxic activity. Furthermore, HuMab TONO-1 heavy and light chain variable regions were cloned, sequenced and analyzed. HuMab TONO-1 uses a V(H) gene member of the V(H)IV gene family V(H)71-4, and is productively rearranged with the germ line D(H) gene D(XP')1, and the germ line J(H)5 gene with multiple somatic mutations. HuMab TONO-1 Vlambda belongs to the lambda light chain variable subgroup I family and is derived from the Vlambdalc germ line gene Humlv1042, and germ line gene Jlambda1 without somatic mutations. The results reveal that the production of HuMab TONO-1, with cytotoxic potential for human T-leukemia/lymphoma cells, is achieved by rearrangement of the V(H)71-4/Humlv1042 germ line variable region gene combination, that is associated with the autoimmune repertoire.

Amino Acid Sequence↗
PubMed2001

Spatial structure of lemming populations (Dicrostonyx groenlandicus) fluctuating in density.

The pattern and scale of the genetic structure of populations provides valuable information for the understanding of the spatial ecology of populations, including the spatial aspects of density fluctuations. In the present paper, the genetic structure of periodically fluctuating lemmings (Dicrostonyx groenlandicus) in the Canadian Arctic was analysed using mitochondrial DNA (mtDNA) control region sequences and four nuclear microsatellite loci. Low genetic variability was found in mtDNA, while microsatellite loci were highly variable in all localities, including localities on isolated small islands. For both genetic markers the genetic differentiation was clear among geographical regions but weaker among localities within regions. Such a pattern implies gene flow within regions. Based on theoretical calculations and population census data from a snap-trapping survey, we argue that the observed genetic variability on small islands and the low level of differentiation among these islands cannot be explained without invoking long distance dispersal of lemmings over the sea ice. Such dispersal is unlikely to occur only during population density peaks.

Animals↗
PubMed1990

Heterozygosity and gene flow in populations of Calomys laucha (Rodentia, Cricetidae).

Enzyme polymorphism was studied by means of starch gel electrophoresis on five population samples of Calomys laucha collected in different sites and seasons. C. laucha, a cricetid rodent inhabiting preferentially cultivated fields in the central-eastern plains of Argentina, is a reservoir-host of Junin virus, agent of Argentine hemorrhagic fever. Results show high levels of genic variability in all the samples (P from 62.50 to 77.27; H from 0.118 to 0.163) and low genetic distance among populations (D from 0.0024 to 0.0167). Estimations of gene flow (mean Nm = 10.78) indicate a high migration capability of the species and the possibility of further dispersal of Junin virus.

Animals↗
PubMed2004

Microsatellite variation reveals high levels of genetic variability and population structure in the gorgonian coral Pseudopterogorgia elisabethae across the Bahamas.

The primary mechanism of gene flow in marine sessile invertebrates is larval dispersal. In Pseudopterogorgia elisabethae, a commercially important Caribbean gorgonian coral, a proportion of the larvae drop to the substratum within close proximity to the maternal colony, and most matings occur between individuals in close proximity to each other. Such limited dispersal of reproductive propagules suggests that gene flow is limited in this gorgonian. In this study, we characterized the population genetic structure of P. elisabethae across the Bahamas using six microsatellite loci. P. elisabethae was collected from 18 sites across the Bahamas. Significant deviations from Hardy-Weinberg equilibrium due to deficits of heterozygotes within populations were detected for all 18 populations in at least one of the six screened loci. Levels of genetic structure among populations of P. elisabethae were high and significant. A distance analysis placed populations within three groups, one formed by populations located within Exuma Sound, a semi-isolated basin, another consisting of populations located outside the basin and a third group comprising two populations from San Salvador Island. The patterns of genetic variation found in this study are concordant with the life-history traits of the species and in part with the geography of the Bahamas. Conservation and management plans developed for P. elisabethae should considered the high degree of genetic structure observed among populations of the species, as well as the high genetic diversity found in the San Salvador and the Exuma Sound populations.

Animals↗
PubMed1995

Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines.

In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Genetic variability and differentiation between populations of Rhodnius prolixus and R. pallescens, vectors of Chagas' disease in Colombia.

Enzyme polymorphism in Rhodnius prolixus and R. pallescens (Hemiptera, Reduviidae), principal vectors of Chagas' disease in Colombia, was analyzed using starch gel electrophoresis. Three geographic locations were sampled in order to determine gene flow between populations and to characterize intra- and interspecific differences. Of 25 enzymes assayed 10 were successfully resolved and then used to score the genetic variation. The enzymes PEPD, GPI, PGM and ICD were useful to differentiate these species and PGD, PGM and MDH distinguished between sylvatic and domiciliary populations of R. prolixus. Both polymorphism and heterozygosity indicated greater genetic variability in sylvatic habitats (H = 0.021) compared to domiciliary habitats (H = 0.006) in both species. Gene flow between sylvatic and domiciliary populations in R. prolixus was found to be minimal. This fact and the genetic distance between them suggest a process of genetic isolation in the domiciliary population.

Animals↗
PubMed1992

Analysis of amplified T cell receptor V beta transcripts by a non-isotopic immunoassay.

We have recently described a new colorimetric DNA enzyme immunoassay (DEIA) for detecting specific hybrids of complementary nucleic acids. This technology is based on an antibody that selectively recognizes double, but not single stranded DNA and therefore reveals the hybridization event independently from the DNA sequences. Most importantly, the test has an ELISA format and is very rapid and convenient for processing large numbers of samples. In the present report we have adapted this method to reveal the specificity of amplified T cell receptor V beta transcripts. V beta genes were amplified by polymerase chain reaction, using family specific primers and the specificity of the amplified products was determined by Southern blot and by DEIA. Our data demonstrate that DEIA had the same degree of sensitivity and specificity of conventional Southern hybridization. The possibility of analyzing amplified products with the simplicity of a conventional immunoassay should greatly facilitate the analysis of complex multigenic systems such as the T cell receptor and the immunoglobulin repertoire.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2000

Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor.

In this report, we have analyzed the human T cell repertoire derived in vivo from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-beta sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections in vivo. A similar estimation was obtained in an in vitro murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.

Child, Preschool↗
PubMed1990

Rearrangement and selection of VH11 in the Ly-1 B cell lineage.

One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.

Animals↗
PubMed1992

Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice.

The pattern of VH gene family expression in the primary B cell repertoire of the mouse is strain dependent. In C57Bl/6 mice, the VH J558 family is expressed by more than 45% of the cells, while the expression of VH 7183, VH Q52, and VH 36-60 families together does not exceed 20%. In BALB/c mice, relative expression of VH J558 is lower than 35%, while the sum of the other three families reaches 25%. To assess which genetic loci control strain-specific VH gene family expression, we studied VH gene family usage in splenic B cell repertoires of different congenic strains of mice. Changes in major histocompatibility complex or immunoglobulin (Ig) K light chain genes did not modify VH gene family expression in adult mice. Differences at the IgH locus, however, modified VH gene family usage. In 1-d-old mice, the strain-specific VH gene family expression pattern is determined by the IgH haplotype. In adult mice, the VH gene family expression pattern of resting B cells is independent of the IgH locus and follows the genetic background of the congenic strain, while it is determined by the IgH haplotype among Ig-secreting spleen cells. In F1(B6 x BALB/c) mice, each of the two spleen B cell populations, sorted on the basis of mu heavy chain allotype expression, shows an independent VH gene family expression pattern, determined by the IgH locus. The implications of these results in the control of VH gene family expression, and in the selection of peripheral B cell repertoires are discussed.

Aging↗
PubMed2006

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein.

BACKGROUND: CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. METHODS: The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. RESULTS: The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. CONCLUSION: The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

Amino Acid Sequence↗
PubMed2001

Spatial patterns of genetic diversity in Posidonia oceanica, an endemic Mediterranean seagrass.

Posidonia oceanica is an endemic seagrass species in the Mediterranean Sea. In order to assess levels of genetic structure in this species, the microsatellite polymorphism was analysed from meadows collected in several localities, along the coasts of the Tyrrhenian Sea (Mediterranean Sea). The existence of single population units and the recruitment of seedlings collected in some localities were investigated. Moreover, genetic structure at different spatial scales and biogeographic relationships among populations were also assessed. Our analysis showed the existence of clear patterns of genetic structure in P. oceanica in the area considered in the analysis. P. oceanica, in fact, is present in separate meadows that represent discrete populations, characterized by low genetic diversity. Comparable levels of genetic variability between mature meadows and seedlings were found. Patterns of genetic relatedness among populations seem to be in accord with direction of dominant current flux in the whole area, separating South Tyrrhenian from North Tyrrhenian populations. Moderate levels of gene flow between populations and genetic substructure within populations, together with the finding of the limited role of sexual reproduction in increasing genetic variability, should be a cause for concern for the persistence of this essential resource in the Mediterranean basin.

Genetic Variation↗
PubMed2003

Individualized human scFv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig.

We have developed a method for rapidly producing in plants the idiotype regions of the tumor-specific Ig as single-chain Fv (scFv) proteins for use in the treatment of non-Hodgkin's lymphoma. Variable region gene sequences were generated from either a tumor hybridoma or human tumor biopsy cells, and idiotype domains were joined by a novel linker and cloned into a modified tobacco mosaic virus (TMV) vector designed to secrete the scFv protein in infected Nicotiana benthamiana plants. Thirty-eight out of 44 human scFv proteins showed Coomassie visible material in crude secretory (interstitial fluid, IF) extracts, 21 of those between 100 and 800 microg/ml. Eight of these proteins were tested for appropriate idiotype responses in vaccinated mice. In all eight cases, anti-idiotype immune responses were induced with minimal cross reactivity to irrelevant Ig or scFv proteins. Four out of four anti-scFv sera were also shown to recognize the Ig on human tumor cells by flow cytometry analysis.

Animals↗
PubMedDate not supplied

[Anopheline population genetics].

Population genetic studies of vectors are essential for (i) the determination of their taxonomic status and consequently the definition of their vectorial role in the transmission of pathogenic agents; (ii) the evaluation of the species genetic variability and the estimation of their capacities of adaptation to selection pressure; (iii) an estimation of gene flow among populations in order to evaluate their degree of isolation and gene circulation, especially resistance genes. Among the malaria vectors taken as examples on three continents, Africa, South-East Asia and Latin America, the large majority of the species showed an important polymorphism. The Gambiae Complex, which is by far the most studied one, includes at present 7 species with the recent description of An. quadriannulatus A and B from Ethiopia. An. gambiae s.s. includes itself 5 chromosomal forms. One of them, the Mopti form, should be considered as a species unto itself. For An. arabiensis, a strong differentiation has been observed among the populations from Senegal and the Indian Ocean Islands. The kdr mutation, which confers resistance to pyrethroid knockdown effect, has never been found either in the Mopti form, or An. arabiensis, indicating a restricted gene flow between these latter two and An. gambiae s.s. The speciation process of the Gambiae Complex seems to be a recent phenomenon due to environmental selection pressure. Species of the Funestus Group are distinguishable by morphological characters. The genetic study of An. funestus s.s. did not show the presence of a complex, in spite of high polymorphism and population structure. Anophelines from eastern areas present an important biodiversity. The Minimus Complex includes two species, A and C, which are widely distributed in South-East Asia. Species A is strongly endophilic, on the contrary species C is at once more exophilic and zoophilic. The latter species might have been selected by DDT indoor house spraying. After numerous taxonomic investigations, the Dirus Complex includes now 7 species. In Latin America, An. pseudopunctipennis clustered into three geographic populations which are under a speciation process. One covers North America and Guatemala, the other South America and Belize, whilst the last one is restricted to Grenada Island. On the contrary, An. darlingi showed little morphologic and genetic variability throughout the species geographic range suggesting the existence of a single species. The main objective of these studies is to implement a more selective approach of vector control programs in relation to the incriminated species, their bioecology and their role in malaria transmission. The improvement of efficiency and selectivity of vector control is becoming a major goal in order to make the best out of the available tools and control the impact of interventions on the environment.

Africa↗
PubMed2001

Overcoming multi-drug resistance using an intracellular anti-MDR1 sFv.

We made an intracellular single-chain variable fragment (sFv) from the C219 monoclonal antibody that recognized the intracellular domain of the multidrug resistance (MDR) gene product, P-glycoprotein (P-gp). Immuno-cytochemistry using the FITC conjugated anti-C-myc tag antibody showed that the sFv protein was expressed in the cytoplasm of the cells. Although transfection of the sFv did not result in the down-regulation of P-gp expression in P-gp positive MDR cells as determined by flow cytometry analysis, Adriamycin (ADM) uptake and Rhodamine123 (Rh123) retention were increased by the C219 intra-cellular sFv transfection. The transfected cells exhibited a higher sensitivity to ADM using a 10-day colony formation assay. The conventional 3-day MTT assay showed the drug resistant tendency in C219 sFv transfected cell we tested. The growth rate of C219 sFv transfected cells was delayed in all non-MDR and MDR cells that might be the reason why C219 transfected cells exhibited the drug resistant tendency in the MTT assay. Despite this unexpected effect of C219 sFv on growth rate, our data suggest that the intra-cellular sFv technique could knockout MDR functionally and may offer a means of increasing the effectiveness of tumor chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗