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Biomedical subjects

C Kinnon

Publications and source records attributed to C Kinnon.

At least 19 recordsLinked to original sources
PubMed2003

Improved antitumour immunity in murine neuroblastoma using a combination of IL-2 and IL-12.

Neuroblastoma immunotherapy using cytokine-modified tumour cells has been tested in clinical trials. However, because of the complex nature of antitumour immune responses, a number of therapies may be required for complete tumour eradication and generation of systemic immunity. We report here the improved antitumour effect of two cytokines, interleukin-2 (IL-2) and interleukin-12 (IL-12), when coexpressed by neuroblastoma cell lines. Initially, transfection of human and mouse neuroblastoma cell lines resulted in high expression levels of biologically active IL-2 and IL-12 in vitro. These cytokines when expressed by transfected Neuro-2A cells completely abolished their in vivo tumorigenicity in a syngeneic neuroblastoma model. Vaccination of established tumours with IL-12-producing cells exhibited a clear effect with reduced tumour growth in the presence of IL-2. In vivo depletion studies showed that CD4(+) and CD8(+) T cells mediate the response against cytokine-producing cells. These results suggest that IL-2 and IL-12, when cotransfected in tumour cells, are effective against established disease and provide a promising immunotherapeutic approach for the treatment of neuroblastoma.

Adjuvants, Immunologic↗
PubMed2002

T cell transduction and suicide with an enhanced mutant thymidine kinase.

Retroviral transfer of Herpes simplex virus thymidine kinase to T cells has been used to confer sensitivity to the antiviral agent ganciclovir. This has allowed therapeutic approaches to be developed in which T cells mediating graft-versus-host disease after bone marrow transplantation can be selectively eliminated by the administration of ganciclovir. Although the strategy has been shown to be generally successful in early clinical trials, there are concerns about possible resistance to ganciclovir and the risk of myelosuppressive side-effects at the doses required to induce T cell suicide. We have incorporated the enhanced mutant HSV-TKSR39 into retroviral vectors tailored to exhibit high levels of expression in T cells and have used protocols optimized for the transduction and selection of primary lymphocytes. We demonstrate that leukemic and primary T cells can be efficiently transduced and highly enriched under conditions that should be readily adaptable for clinical use. T cells carrying HSV-TKSR39 were inhibited by exposure to ganciclovir at concentrations an order of magnitude below those required for wild-type HSV-TK. The less toxic agent aciclovir also eliminated T cells transduced with HSV-TKSR39 (but not HSV-TK), underlining the increased therapeutic potential of the mutant suicide gene system in the bone marrow transplantation setting.

Acyclovir↗
PubMed2001

Defective expression of the interleukin-2/interleukin-15 receptor beta subunit leads to a natural killer cell-deficient form of severe combined immunodeficiency.

Development of T and natural killer (NK) cells is critically dependent on cytokine signaling, and defects in cytokine receptor complex subunits have been shown to result in severe combined immunodeficiency (SCID) syndromes in humans and in murine models. An infant boy had typical clinical features of SCID and was found to lack NK cells in his peripheral circulation. Molecular analysis did not reveal abnormalities in his gammac or JAK-3 genes, and he was investigated for defects in the interleukin-15 (IL-15) receptor complex because functional IL-15 signaling is essential for NK cell development. Expression of the IL-2R/IL-15Rbeta chain was significantly reduced in the patient's peripheral blood mononuclear cells (PBMCs) by immunoblot, flow cytometry, and Northern blot analysis. Furthermore, IL-2 stimulation of PBMCs showed only minimal tyrosine phosphorylation of JAK-3. These data demonstrate that defects in IL-2R/1L-15Rbeta expression can lead to a unique NK-deficient SCID immunophenotype. (Blood. 2001;98:877-879)

Exons↗
PubMed2001

Cutting edge: the Wiskott-Aldrich syndrome protein is required for efficient phagocytosis of apoptotic cells.

Phagocytosis of apoptotic cells by macrophages and dendritic cells is necessary for clearance of proinflammatory debris and for presentation of viral, tumor, and self Ags. While a number of receptors involved in the cognate recognition of apoptotic cells by phagocytes have been identified, the signaling events that result in internalization remain poorly understood. Here we demonstrate that clearance of apoptotic cells is accompanied by recruitment of the Wiskott-Aldrich syndrome (WAS) protein to the phagocytic cup and that it's absence results in delayed phagocytosis both in vitro and in vivo. Therefore, we propose that WAS protein plays an important and nonredundant role in the safe removal of apoptotic cells and that deficiency contributes significantly to the immune dysregulation of WAS. The efficiency of apoptotic cell clearance may be a key determinant in the suppression of tissue inflammation and prevention of autoimmunity.

Animals↗
PubMed2001

Rapid protein-based assays for the diagnosis of T-B+ severe combined immunodeficiency.

The severe combined immunodeficiencies (SCID) are a heterogeneous group of conditions arising from a variety of molecular defects. The X-linked form of SCID (X-SCID) is caused by defects in the common gamma chain (gammac), and is characterized by a T-B+NK- immunophenotype. This lymphocyte profile is seen in an autosomal recessive form of SCID caused by mutations in the JAK3 molecule. Thus, X-SCID and JAK3-deficient SCID are clinically and immunologically indistinguishable. Knowledge of the precise molecular defect is essential for antenatal diagnosis, carrier testing and for treatment using somatic gene therapy. To identify the molecular defect in children presenting with a T-B+NK- form of SCID, we have developed rapid assays based on flow cytometric analysis of gammac, immunoblotting for JAK3 and gammac, and detection of interleukin-2 (IL-2)-induced tyrosine phosphorylation of JAK3. Sixteen T-B+NK- SCID patients from 15 families were examined. Nine had no detectable gammac, four had abnormal gammac expression and no IL-2-induced JAK3 tyrosine phosphorylation, and one had normal gammac expression but no IL-2-induced JAK3 tyrosine phosphorylation, although JAK3 was present. All these patients had mutations identified in their gammac gene. Two patients exhibited normal gammac expression, but JAK3 was not detected by immunoblotting and these patients were confirmed as having JAK3 gene mutations. Thus, these protein-based assays have led to rapid molecular diagnoses in T-B+ SCID that have subsequently been confirmed by genetic analysis.

B-Lymphocytes↗
PubMed2001

Protein assays for diagnosis of Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

Mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) give rise to Wiskott-Aldrich syndrome (WAS), a condition that exhibits a wide spectrum of clinical severity. Patients may develop mild thrombocytopenia or suffer from a wide range of associated disorders including eczema, immune dysfunction, autoimmune disease and malignancy. The clinical diagnosis of Wiskott-Aldrich syndrome (WAS) can be difficult and is usually supported by the detection of WASp gene mutations using genetic analysis. Recently, protein-based assays have been used to demonstrate the absence of WASp in patients known to have WASp gene mutations. We have now reversed this approach and report on the use of immunoblot assays to rapidly diagnose WAS in 13 patients. There was a complete absence of WASp in 10 out of 13 patients and an abnormal protein form was detected in the remaining three patients. In all cases, subsequent genetic analysis confirmed the presence of a WASp gene mutation. We believe that protein-based assays should be employed as the first line of investigation in the diagnosis of WAS spectrum disorders.

Aged↗
PubMed2000

Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region.

In the mammal, definitive hematopoietic stem cells (HSCs) are first derived from mesodermal cells within a region of the embryonic para-aortic splanchnopleura known as the aorta-gonad-mesonephros (AGM). Within this region, HSCs are thought to arise from hemangioblast precursors located in the ventral wall of the dorsal aorta. However, the factors that regulate HSC development in vivo are still largely unknown. Bone morphogenetic protein (BMP)-4, a member of the transforming growth factor beta (TGF-beta) superfamily of growth factors, is a potent ventralizing factor and has been implicated in the commitment of embryonic mesodermal cells to a hematopoietic fate in a number of systems. In the human AGM, we find that BMP-4 is expressed at high levels, and with striking polarity, in a region of densely packed cells underlying intra-aortic hematopoietic clusters. In contrast, TGF-beta1 is expressed predominantly by hematopoietic cells within the clusters. These findings implicate both BMP-4 and TGF-beta1 in the initiation and regulation of hematopoiesis in the human AGM. Furthermore, the distribution of BMP-4 expression is highly suggestive of a direct role in the specification of human hematopoietic cells from embryonic mesoderm in vivo. (Blood. 2000;96:1591-1593)

Bone Morphogenetic Protein 4↗
PubMed2000

Wiskott-Aldrich syndrome protein is necessary for efficient IgG-mediated phagocytosis.

Interactions between the Wiskott-Aldrich (WAS) protein (WASp), small GTPases, and the cytoskeletal organizing complex Arp2/3 appear to be critical for the transduction of signals from the cell membrane to the actin cytoskeleton in hematopoietic cells. This study shows that Fcgamma-receptor (FcgammaR)-mediated phagocytosis is impaired in WASp-deficient peripheral blood monocytes, and that in macrophages, formation of the actin cup and local recruitment of tyrosine phosphorylated proteins is markedly attenuated. Results also show that, in normal macrophages, WASp itself is actively recruited to the cup, suggesting that assembly of this specialized cytoskeletal structure is dependent on its expression. (Blood. 2000;95:2943-2946)

Actins↗
PubMed2000

A defined window for efficient gene marking of severe combined immunodeficient-repopulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector.

We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.

Animals↗
PubMed2000

Intrinsic defects of B cell function in X-linked severe combined immunodeficiency.

The cytokine receptor common gamma chain mutation in X-linked SCID results in a failure of T and NK cell development and an as yet undefined defect of B cells. Using immunoglobulin isotype-specific reverse transcription-PCR we show that although hematopoietic stem cell transplantation restores a diverse repertoire of class-switched B cell clones, on further analysis these are almost all of donor origin. This suggests that host B cells, which predominate after unconditioned transplantation, are still defective even in the presence of normal T cells. These studies imply that effective humoral reconstitution can only be achieved by the engraftment of normal donor B cells.

B-Lymphocytes↗
PubMed2000

Diagnosis of X-linked lymphoproliferative disease by analysis of SLAM-associated protein expression.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency in which affected boys show abnormal responses to Epstein-Barr virus infection. The gene defective in XLP has been identified and designated SH2D1A and encodes a protein termed SLAM-associated protein (SAP). Mutation analysis in individuals with typical XLP presentations and family histories has only detected abnormalities in approximately 60% of patients. Thus, genetic analysis alone cannot confirm a diagnosis of XLP We have developed a SAP expression assay that can be used as a diagnostic indicator of XLP We show that SAP is constitutively expressed in normal individuals, in patients with severe sepsis and in patients with other primary immunodeficiencies. In six XLP patients, four with classical and two with atypical presentations, SAP expression was absent. In the latter two, who were previously assigned as having common variable immunodeficiency (CVID), the diagnosis of XLP was initially made using the protein expression assay. In two further patients in whom no mutation could be detected by genetic analysis, lack of SAP expression strongly suggests that these individuals have XLP. We therefore suggest that XLP should be suspected in certain boys previously diagnosed as having CVID and recommend that patients are investigated both by genetic analysis of SH2D1A and by expression of SAP protein.

Agammaglobulinemia↗
PubMed2000

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

Animals↗
PubMed2000

An integrin-targeted non-viral vector for pulmonary gene therapy.

Gene therapy offers potential for the treatment of severe respiratory diseases. However, the vectors which are currently available have drawbacks limiting their therapeutic application. Here we report on an integrin-targeted, non-viral gene delivery system for pulmonary gene transfer. We demonstrate that this vector can deliver the lacZ reporter gene to the lung, transfecting bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome. In addition, vector administration can be repeated leading to further gene expression without inducing inflammation. The advantages of this novel gene delivery system provide considerable potential for targeted gene therapy in vivo. Gene Therapy (2000) 7, 393-400.

Animals↗
PubMed2000

Kinase mutant Btk results in atypical X-linked agammaglobulinaemia phenotype.

X-linked agammaglobulinaemia (XLA) is a B cell humoral abnormality arising from mutations in the gene encoding Bruton's tyrosine kinase (Btk). The phenotype of XLA can be variable, with some individuals having a less severe immunophenotype, although in most cases this cannot be correlated with the Btk mutation or expression of Btk protein. In this study we describe clinical and immunological heterogeneity within the same pedigree. Analysis of the genetic defect identified a missense mutation in the kinase domain of Btk which, unusually, preserved Btk protein expression but at reduced levels, and also considerably diminished autophosphorylation activity. Structural analysis of the effect of this mutation on the kinase domain suggests that this mutation is not an integral part of the ATP or substrate binding domains but may affect the interaction of the kinase domain with its own kinase domain and other substrates. Together, these data may provide an explanation for the variable XLA phenotype.

Adult↗
PubMed2000

Normal development of human fetal hematopoiesis between eight and seventeen weeks' gestation.

OBJECTIVE: The aim of this study was to compare the hematologic compositions of fetal blood and liver and to phenotypically quantify the hematopoietic stem and progenitor cells during early human gestation. STUDY DESIGN: Fifty fetal blood samples and 50 fetal livers were collected at 10 to 17 weeks' gestation and 8 to 17 weeks' gestation, respectively. Investigations included fetal blood cell counts, determinations of red blood cell index values, and flow cytometric analyses of mononuclear cells. RESULTS: Fetal red blood cell, white blood cell, and platelet counts all increased with gestation, reflecting hematologic development. The proportion of normoblasts decreased dramatically with gestation. Individual mature red blood cells were larger and contained more hemoglobin during early gestation. Circulating and hepatic T lymphocytes increased in number shortly before the 13th week of gestation, which reflected thymic maturation. As a proportion fetal liver contained fewer T lymphocytes than did fetal blood (2.5% vs 18.6%; P =.003) but more CD34(+) hematopoietic stem and progenitor cells (17.5% vs 4.3%; P =. 004). As a proportion, fetal liver contained more of the primitive CD34(+) and CD38(-) hematopoietic stem and progenitor cells than did fetal blood (32% vs 17%; P =.04). CONCLUSION: Both fetal blood and liver provide a rich source of hematopoietic stem and progenitor cells. Fetal liver provides a richer source of more primitive hematopoietic stem and progenitor cells than does fetal blood. For stem cell transplantation we suggest that fetal livers be collected before the 13th week of gestation, because T lymphocytes are present in much greater numbers in the fetal liver after this stage of gestation. Further, we suggest that in utero stem cell transplantations in fetuses with normal immune development should be performed before the 13th week of gestation.

ADP-ribosyl Cyclase↗
PubMed1999

High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.

Animals↗