PubMed Health⌕ Search

Biomedical subjects

C Kinnon

Publications and source records attributed to C Kinnon.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer.

The definitive long-term repopulating human hematopoietic stem cell, which seeds the adult blood system, was previously thought to derive from the extra-embryonic yolk sac. However, there is now considerable evidence that in both avian and murine systems, yolk sac hematopoietic cells are largely a transient, embryonic population and the definitive stem cell, in fact, derives from a distinct region within the embryonic mesoderm, the aorta-gonad-mesonephros region. In the human embryo, an analogous region has been found to contain a cluster of cells distinct from, but closely associated with, the ventral endothelium of the dorsal aorta, the appearance of which is restricted both spatially and temporally. We have used antibodies recognising hematopoietic regulatory factors to further characterise this region in the human embryo. These studies indicate that all factors examined, including vascular endothelial growth factor and its receptor FLK-1, Flt-3 ligand and its receptor STK-1, and stem cell leukemia transcription factor, are expressed by both hematopoietic cells in the cluster and endothelial cells. However, there is some discontinuity in cells directly underlying the cluster. Furthermore, we have identified a morphologically distinct region of densely-packed, rounded cells in the mesenchyme directly beneath the ventral wall of the dorsal aorta, and running along its entire length. In the preumbilical AGM region, directly underlying the hematopoietic cluster, but not at more rostral and caudal levels, this region of mesenchyme expresses tenascin-C, an extracellular matrix glycoprotein known to facilitate cell-cell interactions and migration. This region of cells may therefore provide the microenvironmental support for the intraembryonic development of definitive hematopoietic stem cells, a process in which tenascin-C may play a pivotal role.

Antigens, CD34↗
PubMed1999

The SH3 domain of Bruton's tyrosine kinase displays altered ligand binding properties when auto-phosphorylated in vitro.

Btk is a member of the Tec family of protein tyrosine kinases expressed in B cells. It is stimulated following cross-linking of the B cell receptor which leads to the autophosphorylation of a specific residue in the SH3 domain, Y223. Previous work using Btk-derived fusion proteins has shown that the Btk SH3 domain binds to c-Cbl and Wiskott-Aldrich syndrome protein (WASP) in vitro. We show here that when the Btk SH3 domain fusion protein is autophosphorylated, its ability to take part in protein interactions is altered as compared to the non-phosphorylated fusion protein. Although the phosphorylated Btk SH3 domain still binds c-Cbl, it no longer binds WASP and instead acquires a high affinity for kinase-active Syk. The region of Syk responsible for this interaction is contained within its C terminus, suggesting a novel mechanism by which these proteins may interact.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1998

Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Amino Acid Sequence↗
PubMed1998

Adeno-associated virus gene transfer to mouse retina.

Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.

Animals↗
PubMed1998

Intrinsic dendritic cell abnormalities in Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immune deficiency, eczema, and microthrombocytopenia. Biochemical evidence indicates that the Wiskott-Aldrich syndrome protein (WASp) is involved in regulating the actin cytoskeleton. Here we report that WAS dendritic cells (DC) have an immunophenotype very similar to normal DC. However, as a consequence of an intrinsically abnormal cytoarchitecture, they are unable to polarize normally and have severely reduced translocational motility in vitro. These findings indicate that WASp is an essential effector for Cdc-42-mediated polarization of primary hematopoietic cells, and suggest that a significant component of the clinical phenotype of WAS could arise from peripheral DC dysmotility and aberrant immune cell trafficking in vivo. Intrinsic dysfunction of the DC population may also have an important role in the pathogenesis of other primary immunodeficiency syndromes, while induced changes in DC cytoskeletal signaling pathways may contribute to the initiation of acquired immunological and inflammatory disorders.

Actins↗
PubMed1998

Absence of expression of the Wiskott-Aldrich syndrome protein in peripheral blood cells of Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is usually associated with thrombocytopenia and eczema. The very variable phenotype of WAS results from defects in the WAS protein (WASP), the function of which is not well understood. In many cases causative mutations have now been identified in the WAS gene. Attempts have been made to correlate the nature of the mutations with the severity of the disease. In this study we investigated mutations in 13 patients with WAS and analyzed the expression of WASP in patient blood samples by immunoblot analysis. We found that despite extensive variation in the nature of the mutations in patients with severe WAS symptoms, none express the protein. However, in 1 patient with a mild clinical phenotype WASP expression was detected. Such an analysis could be used as an initial screening procedure for the diagnosis of WAS prior to genotypic analysis.

Base Sequence↗
PubMed1998

Chemotaxis of macrophages is abolished in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare disease characterized by microthrombocytopenia, eczema and immune deficiency. In this study a direct-viewing chemotaxis chamber was used to analyse chemotactic responses of WAS neutrophils and macrophages in stable linear concentration gradients. In five patients with classic WAS, chemotaxis of macrophages but not of neutrophils was found to be abolished, whereas the speed of random motility of both cell types was found to be indistinguishable from control cells. This supports the existence of an essential functional link, previously suggested by biochemical studies, between Cdc42, WAS protein (WASp) and the actin cytoskeleton in primary human macrophages. Moreover, these data suggest that Cdc42-WASp-mediated filopodial extension is a requirement for chemotaxis but not for chemokinesis in these cells. Abnormal directional cell motility of macrophages and related antigen-presenting cells may play a significant part in the immune deficiency and eczema of WAS.

Chemotaxis↗
PubMed1998

Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain.

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life-spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor gamma chain (gamma c) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)-deficient strain produced mice doubly homozygous for the gamma c and RAG2 null alleles (gamma c-/RAG2-). These mice have a stable phenotype characterized by the absence of all T lymphocyte. B lymphocyte and NK cell function. Injection of human B-lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz-scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz-scid mice. Levels of engraftment with peripheral-blood-derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.

Animals↗
PubMed1998

Bruton's tyrosine kinase expression and activity in X-linked agammaglobulinaemia (XLA): the use of protein analysis as a diagnostic indicator of XLA.

Mutations in the Bruton's tyrosine kinase (BTK) gene result in XLA. Despite the large numbers of BTK mutations reported, no correlation can be made between the clinical phenotype and the gene defects. Analysis of Btk protein expression and activity in individuals with XLA was performed to characterize the relationship between a particular mutation, the resultant Btk protein and the clinical phenotype. In most patients studied, including those with atypical phenotypes, there was complete absence of protein expression and activity. Furthermore, in two undiagnosed individuals with a clinical phenotype suggestive of XLA, lack of protein expression was used to confirm an abnormality in Btk. These results underline the importance of protein analysis prior to speculating on protein structure and function based on the gene mutation. Lack of Btk expression in atypical phenotypes suggests that there is redundancy in B lymphocyte signalling such that alternative signalling molecules, or mechanisms, can compensate for the lack of Btk. We also suggest that analysis of Btk expression can be used as an indicator of XLA. These rapid assays may be used to screen a wider spectrum of individuals with humoral immunodeficiency in order to characterize fully the extent of Btk deficiency.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene.

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice. Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1997

B-cell-negative severe combined immunodeficiency associated with a common gamma chain mutation.

Severe combined immunodeficiency (SCID) is caused by a variety of underlying defects. Approximately 40% of cases are thought to be of the X-linked type (SCIDX1), which is phenotypically characterised by the absence, or very low numbers, of T cells, but normal or even high B cell numbers. The gene responsible for SCIDX1 is that coding for the common gamma chain (gamma c), a component of multiple cytokine receptors. Mutations in this gene have been demonstrated in a large number of boys affected by typical SCIDX1. We describe a sporadic case of a boy who had SCID with absent B cells and absent T cells, but in whom a mutation in the gamma c gene has been demonstrated. In the absence of a typical X-linked pedigree, the phenotype in this boy suggested an autosomal recessive form of SCID and the family would usually have been counselled accordingly. This family raises the question of the true frequency of SCIDX1 amongst sporadic male cases of SCID and highlights the need to screen these boys for gamma chain mutations.

Antigens, CD↗
PubMed1996

Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways.

Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling.

Amino Acid Sequence↗
PubMed1996

Efficient retroviral transduction of human bone marrow progenitor and long-term culture-initiating cells: partial reconstitution of cells from patients with X-linked chronic granulomatous disease by gp91-phox expression.

The primary immunodeficiencies are attractive candidates for the development of gene therapy approaches based on the transduction of hematopoietic cells. We have constructed a high-titer recombinant retrovirus for expression of gp91-phox, deficiencies of which cause the X-linked form of chronic granulomatous disease (X-CGD). We have used this vector to transduce human bone marrow, using either unfractionated mononuclear cells or purified CD34+ cells as targets and evaluated several infection protocols. Efficient gene transfer to progenitors and long-term culture-initiating cells (LTC-IC) was obtained for each target population. Importantly for potential clinical application, this could be achieved without the use of exogenous cytokines or polybrene. Progenitors representing each of the lineages detectable in vitro were transduced at equal efficiencies. The vector was shown partially to restore gp91-phox deficiency and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in transduced cells derived from X-CGD patients. These data demonstrate that it is possible to transduce primitive human hematopoietic cells efficiently and reconstitute NADPH oxidase.

Base Sequence↗
PubMed1996

Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis.

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.

Amino Acid Sequence↗
PubMed1996

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 225 entries from 189 unrelated families showing 148 unique molecular events. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites forming arginine residues. A decreased frequency of missense mutations was found in the TH, SH3 and upper lobe of the kinase domain. The putative structural implications of all the missense mutations are given in the database.

Agammaglobulinemia↗