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Biomedical subjects

M W Moore

Publications and source records attributed to M W Moore.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cardiotrophin-1. Biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex.

Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and oncostatin M. These cytokines induce a pleiotropic set of growth and differentiation activities via receptors that use a common signaling subunit, gp130. In this work we determine the activity of CT-1 in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-1 is inactive in stimulating the growth of the hybridoma cell line, B9 and inhibits the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the differentiation of mouse embryonic stem cells. CT-1 and LIF cross-compete for binding to M1 cells, Kd [CT-1] approximately 0.7 nM, and this binding is inhibited by an anti-gp130 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M1 cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-1 binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-1 has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp130 signaling subunit.

Amino Acid Sequence↗
PubMed1994

Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice.

Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.

Animals↗
PubMed1994

Thrombocytopenia in c-mpl-deficient mice.

Thrombopoietin (TPO) is a cytokine that is involved in the regulation of platelet production. The receptor for TPO is c-Mpl. To further investigate the role and specificity of this receptor in regulating megakaryocytopoiesis, c-mpl-deficient mice were generated by gene targeting. The c-mpl-/- mice had an 85 percent decrease in their number of platelets and megakaryocytes but had normal amounts of other hematopoietic cell types. These mice also had increased concentrations of circulating TPO. These results show that c-mpl specifically regulates megakaryocytopoiesis and thrombopoiesis through activation by its ligand TPO.

Animals↗
PubMed1994

Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.

Interleukin-8 (IL-8) is a proinflammatory cytokine that specifically attracts and activates human neutrophils. A murine gene with a high degree of homology to the two known human IL-8 receptors was cloned and then deleted from the mouse genome by homologous recombination in embryonic stem (ES) cells. These mice, although outwardly healthy, had lymphadenopathy, resulting from an increase in B cells, and splenomegaly, resulting from an increase in metamyelocytes, band, and mature neutrophils. Thus, this receptor may participate in the expansion and development of neutrophils and B cells. This receptor was the major mediator of neutrophil migration to sites of inflammation and may provide a potential therapeutic target in inflammatory disease.

Animals↗
PubMed1994

Acute rejection of marrow grafts in mice. Dependence on and independence of functional TCR in the rejection process.

The question of how irradiated mice acutely reject marrow grafts has remained controversial, and evidence in support of T cell- and natural killer cell-mediated rejection mechanisms has been provided. Here we show in support of previous data that CB17 severe combined immunodeficiency mice acutely reject allogeneic marrow but the specificity of rejection cannot be mapped within the MHC. It is shown that a similar rejection specificity is also expressed in normal CB17 mice and that it is caused by CD3- or CD3+ effector cells that do not utilize TCR. In search of TCR-independent rejection mechanisms in other mouse strains, use is made of TCR transgenic mice expressing a defect in recognizing H-2Dd. It is shown that, although marrow graft rejection is impaired in these mice, pointing to participation of TCR in the rejection process, residual resistance does exist. This resistance maps to the MHC, cannot be shown to involve TCR, and appears to be expressed by NK1+ CD3+ cells. It is concluded that acute marrow graft rejection in normal mice can be mediated by both TCR-mediated and NK cell receptor-dependent effector mechanisms, depending on the particular mouse strains.

Acute Disease↗
PubMed1992

Both intrathymic and peripheral selection modulate the differential expression of V beta 5 among CD4+ and CD8+ T cells.

Murine T cells expressing V beta 5 are characterized by (a) intrathymic deletion in the presence of I-E and products of endogenous mouse mammary tumor viruses, and (b) a greater representation in CD8+ relative to CD4+ peripheral T cells, thought to be due to more efficient intrathymic positive selection on class I rather than class II major histocompatibility complex antigens. We have engineered mice that are transgenic for a rearranged gene encoding a V beta 5+ beta chain of the T cell receptor for antigen. Deletion is not predicted in I-E- V beta 5+ transgenic mice, and until the age of 2 wk, the CD4/CD8 ratio of peripheral T cells is > 3:1 and indistinguishable between transgenic and nontransgenic mice. Transgenic mice then show a rapid, age-dependent decline in the ratio of CD4+ to CD8+ T cells in the lymphoid periphery, reaching a low of 1:10 by 7 mo of age. Furthermore, the percent of peripheral CD4+ cells that express the transgene drops with age, reaching a low of about 60% at 7 mo, while the percent of CD8+ cells that express V beta 5 remains greater than 95% at all ages. The lymphoid periphery is implicated in this selection against CD4+ V beta 5+ T cells as it occurs more rapidly in thymectomized transgenic mice, and can be delayed in mice whose peripheral T cells are replaced by recent thymic emigrants after depletion by in vivo treatment with anti-Thy-1 antibodies. These results indicate that the relative expression of V beta 5 in T cell subsets can be influenced not only intrathymically in I-E+ V beta 5+ transgenic mice, but also by events in the periphery, in the absence of I-E expression.

Animals↗
PubMed1992

Transcription and recombination of the murine RS element.

The deletion of C kappa is a frequent event in lambda-producing B cells in both mice and humans. Deletions of the murine C kappa gene are mediated by recombination events that involve the RS (recombining segment) element located downstream of the C kappa gene. RS recombinations appear to be mediated by the same mechanisms involved in Ig and TCR gene rearrangement. It has been suggested that RS recombinations might activate a factor that is involved in the initiation of lambda gene rearrangement in maturing pre-B cells. We have identified a unique RNA transcript derived from the recombined RS element present in some pre-B cell lines. However, gene transfer studies indicate that this RS transcript is not sufficient to induce lambda gene recombination in pre-B cell lines. We also find that recombination of the RS element in pre-B cell lines is closely correlated with changes in chromatin structure and transcriptional activation. Thus, recombination of the RS element in pre-B cells appears to be regulated in a manner similar to the regulation of antibody gene VDJ joining.

Amino Acid Sequence↗
PubMed1992

T cell receptor alpha-chain pairing determines the specificity of residue 262 within the Kb-restricted, ovalbumin257-264 determinant.

Ovalbumin-specific, Kb-restricted T cells recognize the minimal fully active synthetic peptide ovalbumin (OVA)257-264. This sequence coincides with the eight residue, allele-specific peptide binding motif previously predicted from direct sequencing of naturally occurring Kb-associated peptides (Falk, K., Rotzscke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351:290, 1990). T cell recognition of a panel of analogs with single residue substitutions between the two putative Kb anchor residues at OVA261 and OVA264 suggested that at least one residue, Glu at position 262, is involved in TCR interaction. OVA-specific cytotoxic T lymphocytes (CTL) derived from TCR beta-chain transgenic mice, where the beta-chain originates from an OVA-specific, Kb-restricted CTL B3, showed that differences in TCR alpha-chain pairing determined the specificity for OVA residue 262. These data support the notion that residue 262 of the OVA T cell determinant, corresponding to position 6 within the Kb-binding motif, represents a contact site for TCR. This residue interacts directly with the TCR alpha-chain or with a site on the TCR beta-chain whose conformation is affected by TCR alpha-chain pairing.

Amino Acid Sequence↗
PubMed1991

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.

Animals↗
PubMed1989

Characterization of the subset of immature thymocytes which can undergo rapid in vitro differentiation.

Recently, we reported that thymocytes expressing the CD8 molecule on their surface can give rise to CD4+CD8+ double-positive and CD4+ single-positive progeny following intrathymic transfer into an irradiated host mouse. Thymcoytes expressing a high density of CD8, referred to as CD8hi, and those expressing a low density of the molecule, CD8lo, were both able to differentiate in vivo. In this study we examined the ability of these CD8+ thymocytes populations and of CD4-CD8- double-negative thymocytes to change their phenotype during brief in vitro culture. CD8+ thymocytes were prepared by anti-CD4 plus complement lysis followed by positive selection of the survivors on anti-CD8-coated plates. After 16 h of culture, greater than 60% of CD8+ thymocytes became double-positive. Both CD8hi and CD8lo cells were able to show this in vitro change: about 30% of the former and about 80% of the latter became double-positive. In contrast to this, double-negative thymocytes which had been depleted of cells expressing low densities of CD8 did not show such a phenotypic conversion in vitro. Further panning experiments suggested that all of the CD8+ thymocytes actually express a low surface density of the CD4 molecule which is undetectable in our cytofluorometric assays.

Animals↗
PubMed1989

Analysis of T cell receptor gamma chains from adult CD4- CD8- thymocytes.

The role of T cell receptor (TcR) gamma genes in T cell development has not been determined. To extend our understanding of the repertoire of TcR gamma expression, we prepared a cDNA library from CD4-CD8- adult BALB/c thymocytes and cloned and sequenced 15 TcR gamma genes from this cDNA library. We found that two clones were transcripts of the unrearranged C gamma 2 gene and that three clones terminated in the J gamma 2 region. Nine of the remaining clones were V gamma 1.2 J gamma 2 C gamma 2 genes and five of these were in frame. Only one clone corresponded to C gamma 1 and resulted from an in-frame V gamma 2 J gamma 1 C gamma 1 join. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gamma chain proteins from the surface of both BALB/c and C57BL/6 adult CD4-CD8- thymocytes did not detect the 32-kDa V gamma 1.2 C gamma 2 protein, but did detect the 35-kDa V gamma C gamma 1 protein. These results suggest that despite the abundance of full-length functional V gamma 1.2 C gamma 2 transcripts in the thymocyte subset, the protein product is not expressed on the cell surface as the predicted 32-kDa gamma protein. Finally, our analysis of the V-J joining of the gamma genes reveals both flexibility at the V-J junction and extensive N-region nucleotide addition that lead to diversity of the predicted protein sequence.

Amino Acid Sequence↗
PubMed1989

T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential.

Immature CD8-CD4- double-negative (DN) thymocytes differentiate intrathymically into CD8+CD4- and CD8-CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double-positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)alpha chain, unlike the less mature interleukin 2 receptor-positive DN thymocytes. Surface expression of the TcR-associated CD3 molecule occurs on approximately 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (approximately 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%-34% on day 1 and 48%-68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR alpha chain genes and express TcR alpha/beta heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.

Animals↗
PubMed1988

Introduction of soluble protein into the class I pathway of antigen processing and presentation.

In order to investigate how peptides associate with class I major histocompatibility complex (MHC) glycoproteins intracellularly, we generated cytotoxic T lymphocytes (CTL) specific for a readily available soluble protein in association with class I. C57BL/6 (H-2b) mice immunized against a syngeneic tumor cell transfected with chicken ovalbumin (OVA) cDNA gave rise to H-2Kb-restricted CTL specific for the OVA258-276 peptide. This synthetic peptide and CNBr fragments of OVA (242-285 and 242-273) were able to target H-2b cells for lysis by the CTL in a 3 hr assay. Cells incubated with native OVA for up to 24 hr did not become sensitized for recognition and lysis. However, when OVA was introduced directly into the cytoplasm of cells by the osmotic lysis of pinosomes, the Kb restricted determinant formed readily.

Animals↗
PubMed1988

Induction of cytotoxic T lymphocytes by primary in vitro stimulation with peptides.

Antigen-specific cytotoxic T cells can be generated by primary in vitro stimulation of spleen cells from C57BL/6 mice with appropriate peptide fragments. This response can be elicited without prior in vivo immunization. Chicken OVA fragmented with either cyanogen bromide (CN OVA) or trypsin (T OVA) was used as a source of mixed peptides. A synthetic peptide, NP365-380, representing the sequence 365-380 from influenza virus A/PR/8 nucleoprotein, was also used, since this contains the main determinants recognized by CTL generated from H-2b mice infected with A/PR/8 virus. The primary in vitro cytotoxic T cell response was peptide specific, since targets were lysed only in the presence of appropriate peptide antigens. Native OVA could not elicit primary effectors in vitro nor could it sensitize targets for lysis by OVA digest-specific CTL. A synthetic peptide corresponding to residues 111-122 within the OVA sequence could sensitize targets for lysis by effectors induced against T OVA. Effectors generated by in vitro stimulation were CD8+, CD4-, and H-2Db-restricted for NP365-380 and T OVA recognition. CN OVA-specific effectors were also CD8+, CD4-, but surprisingly, were able to lyse a range of H-2-different targets in an antigen-specific manner. These effectors failed to lyse a tumor line that does not express class I MHC molecules. This broad MHC restriction pattern was also apparent at the clonal level. In all cases, the antipeptide CTL generated by primary in vitro stimulation were inefficient in lysing target cells expressing endogenous forms of antigens, such as influenza virus-infected cells or cells transfected with the OVA cDNA. However, cytotoxic T cell lines generated in vitro against the NP365-380 peptide did contain a minor population of virus-reactive cells that could be selectively expanded by stimulation with A/PR/8-infected spleen cells. These results are discussed in terms of class I-restricted T cell stimulation in the absence of antigen processing by high surface densities of peptide/MHC complexes.

Animals↗
PubMed1987

The human kappa deleting element and the mouse recombining segment share DNA sequence homology.

We have used cloned mouse and human DNA probes to identify regions of conserved homology between the human and murine DNA segments, (termed kappa deleting element (kde) and recombining segment (RS) respectively) which are frequently recombined in lambda-producing B cells. Heteroduplex analysis indicated extensive homology in the region immediately downstream of the recombination site of both segments. This was confirmed by Southern and direct nucleotide sequence analyses. Fifty percent homology was detected within the 500 nucleotides that neighbour the recombination points in the kde and RS segments. These results indicate that the kde and RS sequences are evolutionarily conserved and may be functionally relevant to normal B cell development.

Animals↗
PubMed1985

Deletions of kappa chain constant region genes in mouse lambda chain-producing B cells involve intrachromosomal DNA recombinations similar to V-J joining.

We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse lambda light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the kappa light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two lambda chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region kappa light chain gene (V kappa) joined directly to RS sequences. Our results suggest that the deletion of the kappa light chain constant region (C kappa) exon in many lambda chain-producing B cells is the result of RS recombination and that C kappa deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.

Animals↗