Differences in fairness and trust between lean and corpulent men.

BACKGROUND: Employment disparities are known to exist between lean and corpulent people, for example, corpulent people are less likely to be hired and get lower wages. The reasons for these disparities between weight groups are not completely understood. We hypothesize (i) that economic decision making differs between lean and corpulent subjects, (ii) that these differences are influenced by peoples' blood glucose concentrations and (iii) by the body weight of their opponents. METHODS: A total of 20 lean and 20 corpulent men were examined, who performed a large set of economic games (ultimatum game, trust game and risk game) under euglycemic and hypoglycemic conditions induced by the glucose clamp technique. RESULTS: In the ultimatum game, lean men made less fair decisions and offered 16% less money than corpulent men during euglycemia (P=0.042). During hypoglycemia, study participants of both weight groups accepted smaller amounts of money than during euglycemia (P=0.031), indicating that a lack of energy makes subjects to behave more like a Homo Economicus. In the trust game, lean men allocated twice as much money to lean than to corpulent trustees during hypoglycemia (P<0.001). Risk-seeking behavior did not differ between lean and corpulent men. CONCLUSION: Our data show that economic decision making is affected by both, the body weight of the participants and the body weight of their opponents, and that blood glucose concentrations should be taken into consideration when analyzing economic decision making. When relating these results to the working environment, the weight bias in economic decision making may be also relevant for employment disparities.

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.

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.

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.

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.

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.

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.

Gene transfer into the mouse retina mediated by an adeno-associated viral vector.

Gene transfer to photoreceptor cells may provide a means for arresting the retinal degeneration that is characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). However, transduction of photoreceptors has to date been inefficient, and further limited by toxicity and immune responses directed against vector-specific proteins. An alternative vector system based on adeno-associated virus (AAV) may obviate these problems, and may be useful for transduction of neuronal cells. In this study we have demonstrated successful transduction of all layers of the neuroretina as well as the retinal pigment epithelium (RPE) following subretinal injection of recombinant AAV particles encoding lac Z. Furthermore, the efficiency of transduction of photoreceptors is significantly higher than that achieved with an equivalent adenoviral vector. This is the first report showing that AAV is capable of transducing photoreceptor cells and supports the use of this vector system for gene therapy of retinal diseases such as RP.

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.

Bone marrow gene transfer in three patients with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency results in severe combined immune deficiency disease (SCID), which is fatal without treatment. Allogeneic bone marrow transplantation (BMT) is the treatment of choice if an HLA-identical sibling bone marrow donor is available, resulting in almost 100% cure rate. BMT-related mortality is high in patients lacking such a donor. For these patients, efficient transfer of a recombinant ADA gene into hematopoietic stem cells is a therapeutic option if it results in the outgrowth of a 'genetically repaired' lymphoid system. Based on successful gene transfer studies in monkeys, we performed retrovirus-mediated gene transfer into CD34+ bone marrow cells of three patients with ADA deficiency. Two patients received bovine ADA conjugated to polyethylene glycol (PEG-ADA); in the third patient, PEG-ADA was started 4 months after gene transfer. Gene transfer resulted in a 5-12% transduction frequency of in vitro colony forming cells (CFU-Cs). No toxicity was observed during and after infusion of the graft. Following infusion of the transduced CD34+ cells, transduced granulocytes and mononuclear cells persisted in the circulation for 3 months. In addition, the gene was present in the marrow of one of the patients at 6 months after gene transfer. Expression of the gene was not detected. After this period, the gene could not be detected. In monkey studies we showed that myeloablation, which was not performed in the patients, may enhance engraftment of genetically modified cells. We hypothesize that lack of myeloablation, administration of bovine ADA and low numbers of transduced progenitor cells all may have contributed to the relative low numbers of transduced cells in the patients. Under these conditions, no selective advantage of the genetically corrected progenitor cells was observed.

Screening for mutations causing X-linked severe combined immunodeficiency in the IL-2R gamma chain gene by single-strand conformation polymorphism analysis.

Mutations in the common gamma chain (gamma c or IL2RG) of the interleukin-2, -4, -7, -9 and -15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the gamma c proteins produced as a result of these mutations are discussed.

Generation of recombinant adeno-associated virus (rAAV) from an adenoviral vector and functional reconstitution of the NADPH-oxidase.

The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).

The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase.

X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain.

Functional reconstitution of the NADPH-oxidase by adeno-associated virus gene transfer.

Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.

Gene transfer to primary chronic granulomatous disease monocytes.

For somatic gene therapy to become a realistic therapeutic strategy for chronic granulomatous disease (CGD), we have to be able to assign the molecular lesion to a specific component of the NADPH oxidase and to confirm that transfer of a functional copy of the corresponding defective gene will result in correction of the cellular defect. We used an adenovirus vector expressing p47phox to transduce monocytes from patients with CGD. We showed by nitroblue-tetrazolium staining that NADPH-oxidase activity was restored to these cells. This technique offers a rapid means for molecular diagnosis. In the short term, this approach may have therapeutic potential.

The protein defective in X-linked agammaglobulinemia, Bruton's tyrosine kinase, shows increased autophosphorylation activity in vitro when isolated from cells in which the B cell receptor has been cross-linked.

X-linked agammaglobulinemia is a primary inherited immunodeficiency resulting in a lack of or dramatic reduction in the number of mature B lymphocytes and, thus, greatly reduced levels of serum immunoglobulin. The defect results from mutations in the gene for Bruton's tyrosine kinase (Btk). Using rabbit antisera generated against Btk, we have demonstrated an increase in the level of in vitro kinase activity present in anti-Btk immunoprecipitates from B cells following stimulation with anti-immunoglobulin antibody. This increase in immune complex kinase activity is detectable 1 to 2 min following stimulation and remains elevated for over 30 min. A similar increase was not seen with two late pre-B cell lines investigated in the same way. This stimulation of activity may suggest a role for Btk in signalling through the B cell receptor or associated proteins, in mature B cells.

Expression of Bruton's tyrosine kinase protein within the B cell lineage.

Defects in the gene encoding Bruton's tyrosine kinase (Btk), normally expressed in B cells, cause X-linked agammaglobulinemia (XLA). The phenotype of XLA is characterized by a lack of circulating B cells and immunoglobulin. It has been suggested that B cell maturation from the pre-B cell stage to more mature stages is dependent on the appropriate expression of this gene. The Btk mRNA is expressed in B cells and myeloid cells, but protein expression in relation to B cell maturation has not been determined. Moreover, expression of the Btk protein has so far only been investigated in human Epstein-Barr virus-transformed B cell lines, and in murine splenocytes and B cell lines. We have developed an antiserum which recognizes the human Btk protein and shown that normal human tonsillar B cells, peripheral blood monocytes and myeloid cells express the protein, whereas tonsil-derived T cells do not. We also show that the protein is present in early and mature human B cell lines, but is absent in terminally differentiated plasma cell lines. Furthermore, expression is reduced or absent in three B lineage cell lines derived from two patients with defined genetic mutations in Btk and suffering from XLA.

Physical mapping in the region of the Bruton's tyrosine kinase and alpha-galactosidase A gene loci in proximal Xq22.

We have produced physical maps of the proximal part of Xq22, containing the Bruton's tyrosine kinase (BTK) and alpha-galactosidase A (GLA) gene loci, using long range physical mapping techniques and yeast artificial chromosomes (YACs). These maps reveal five previously unidentified CpG islands which could indicate the presence of other genes in this region.