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In previous animal experiments, we demonstrated that cervical vagus nerve stimulation (VNS) inhibits pancreatic insulin secretion, thereby raises blood glucose levels, and impairs glucose tolerance through afferent signaling. However, there are no reports suggesting that similar effects occur in patients treated with chronic cervical VNS for epilepsy. In contrast to clinical VNS used for epilepsy, where the stimulation is intermittent with cycles of on and off periods, stimulation was continuous in our previous animal experiments. Thus, we hypothesized that the timing of the stimulation on/off cycles is critical to prevent impaired glucose tolerance in epilepsy patients chronically treated with cervical VNS. We conducted a retrospective analysis of medical records from patients with epilepsy. Blood glucose levels did not differ between patients treated with pharmacotherapy only (98 ± 4 mg/dL, n = 16) and patients treated with VNS plus pharmacotherapy (99 ± 3 mg/dL, n = 24, duration of VNS 4.5 ± 0.5 years). However, a multiple linear correlation analysis of patients with VNS demonstrated that during the follow‐up period of 7.9 ± 0.7 years, blood glucose levels increased in patients with long on and short off periods, whereas blood glucose did not change or even decreased in patients that were stimulated with short on and long off periods. We conclude that chronic cervical VNS in patients with epilepsy is unlikely to induce glucose intolerance or hyperglycemia with commonly used stimulation parameters. However, stimulation on times of longer than 25 sec may bear a risk for hyperglycemia, especially if the stimulation off time is shorter than 200 sec.

Background Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response. Methods Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response. Results We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naive and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII. Conclusions These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products. Copyright © 2010 John Wiley & Sons, Ltd.

Expression of the extensive arsenal of virulence factors by Streptococcus pyogenes is controlled by many regulators, of which CovRS is one of the best characterized and can influence ∼15 % of the genome. Animal models have established that mutants of covRS arise spontaneously in vivo resulting in highly invasive organisms. We analysed a pharyngeal and a blood isolate of S. pyogenes recovered from the same individual 13 days apart. The two isolates varied in many phenotypic properties including SpeB production, which were reflected in transcriptomic analyses. PFGE, multilocus sequence typing and partial sequencing of some key genes failed to show any differences except for an 11 bp insert in the covS gene in the blood isolate which caused a premature termination of transcription. Complementation of a fully functional covS gene into the blood isolate resulted in high expression of CovS and expression of speB. These results, showing a pharyngeal and a blood isolate from a single individual differing by a simple insertion, provide evidence for the model that regulatory gene mutations allow S. pyogenes to invade different niches in the body.

Clinical success for gene therapy of hemophilia A will be judged by achievement of sustained, therapeutic levels of coagulation factor VIII (fVIII). Previous clinical trials have suffered from transient, subtherapeutic expression of human fVIII transgenes. Porcine fVIII contains sequence elements that enable more efficient biosynthesis than human fVIII due to enhanced posttranslational transit through the secretory pathway. In this study, we evaluated ex vivo retroviral gene transfer of a high-expression porcine fVIII transgene into bone marrow–derived stromal and hematopoietic stem/progenitor cells (MSCs and HSCs, respectively) and transplantation into genetically immunocompetent hemophilia A mice. Both MSCs and HSCs demonstrated high-level expression of porcine fVIII in vivo. However, following transplantation of gene-modified MSCs, fVIII activity levels rapidly returned to baseline due to the formation of anti–porcine fVIII–neutralizing antibodies. Alternatively, transplantation of HSCs into myeloablated and nonmyeloablated hemophilia A mice resulted in high-level fVIII expression despite low-level hematopoietic reconstitution by gene-modified cells. FVIII expression was sustained beyond 10 months, indicating that immunologic tolerance to porcine fVIII was achieved. Furthermore, transplantation of bone marrow from primary recipients into naive secondary recipients resulted in sustained, high-level fVIII expression demonstrating successful genetic modification and engraftment of HSCs.

Posttranscriptional regulation of endothelial nitric oxide synthase (eNOS) expression is an important mechanism by which endothelial cells respond to various physiological and pathophysiological stimuli. Previously, we showed that eNOS expression was dramatically altered by the state of cell growth and that the mechanism responsible for this regulation was entirely posttranscriptional, occurring via changes in eNOS mRNA stability. The present study identifies a role for actin cytoskeleton organization in the posttranscriptional regulation of eNOS during cell growth and examines the relationship between the state of actin polymerization and eNOS expression. We identified monomeric actin (globular [G]-actin) as the major component of a 51-kDa ribonucleoprotein that binds to the eNOS mRNA 3′ untranslated region in UV-crosslinking analysis. Binding activity of the ribonucleoprotein complex correlated with the relative concentration of G-actin versus filamentous actin (F-actin). ENOS transcripts colocalized with cytoplasmic G-actin in cells subjected to fluorescence in situ hybridization and G-actin fluorescence staining. In subcellular fractionation studies, eNOS transcripts were enriched in the free polysomal fraction of nonproliferating cells and enriched in the cell matrix-associated polysomal fraction of proliferating cells. Furthermore, an inverse relationship between the concentration of G-actin and eNOS expression was observed in endothelial cells subjected to pharmacological alteration of their cytoskeleton; lower G/F-actin ratios correlated with increased eNOS expression. Our findings provide some insight into how endothelial cells may use the dynamic organization of the actin cytoskeleton to regulate expression of an enzyme that is crucial to vascular homeostasis.

Articular chondrocytes from nine arthritic patients, five infants, and Balb/c neonatal mice were analyzed for the presence of various cytokine mRNAs by a reverse transcriptase polymerase chain reaction (RT-PCR). Four cytokine mRNAs, interleukin (IL)-6, IL-8, IL-11, and macrophage colony stimulating factor (M-CSF), were detected in all human chondrocytes, regardless of source. IL-10, IL-12p35, and tumor necrosis factor alpha (TNF-alpha) transcripts were found in at least 12 of the 14 human samples. IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and TNF-beta mRNAs were found more predominantly in infant samples and in samples from patients with rheumatoid arthritis (RA) compared with samples from patients with osteoarthritis (OA). Another group of cytokine mRNAs, IL-1 (alpha, beta), IL-4, IL-5, and IL-7, were only weakly expressed in some human samples. The cytokine transcripts that were not found were IL-2, IL3, and interferon gamma (IFN-gamma). Because of the large array of cytokine transcripts detected, human chondrocyte preparations were further purified by reacting them with a monoclonal antibody specific to chondrocyte differentiation antigen and subjecting them to fluorescent-activated cell sorting. A similar array of cytokines was found between the sorted and unsorted chondrocytes, although TNF-alpha, G-CSF and GM-CSF transcripts appeared to be upregulated during the sorting process. Human chondrocytes that dedifferentiated into fibroblasts (a 40-day and a 77-day culture) no longer expressed mRNAs for IL-1, G-CSF, GM-CSF, and TNF-alpha, but all other cytokine mRNAs remained detectable. Although certain phenotypic characteristics were lost, including reactivity to chondrocyte-specific monoclonal antibodies and morphological features, chondrocytes in long-term culture still expressed cytokine mRNAs. As expected, more consistent results were obtained when seven preparations of chondrocytes from neonatal Balb/c mice were examined using available cytokine primers. They contained IL-1, IL-5, IL-6, IL-7, IL-12, GM-CSF, M-CSF, transforming growth factor beta (TGF-beta), TNF-alpha, and TNF-beta mRNAs but lacked IL-2, IL-3, IL-4, IL-10, and IFN-gamma mRNAs. Future experiments to define conditions by which these cytokine protein products are expressed are needed to help assess their roles in chondrocyte biology and in disease states.

Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown that the major limitations of HSC gene therapy are similar to those encountered when using non-hematopoietic cells as targets and include (1) an inability to genetically modify sufficient numbers of target cells, (2) the loss of transgene function over time, and (3) potential complications due to vector integration. With continued improvements in transduction protocols, murine HSC transduction and transplantation are now routine with transduction efficiencies50% easily achievable and even90% feasible. However, attaining high-level engraftment of gene-modified cells after transplantation is still problematic. Basic transduction conditions entail cytokine stimulation of HSC populations, such as stem cell antigen-1 positive (Sca-1(+)) cells isolated from bone marrow, in serum-free media followed by multiple additions of recombinant retrovirus. Analysis of peripheral blood 12 weeks post transplantation of transduced cells into lethally irradiated recipients shows genetic marking in all hematopoietic lineages. Transduction of HSCs is then confirmed by transplanting bone marrow cells harvested from primary transplant recipients into lethally irradiated secondary recipients. Analysis of these mice shows that recombinant retroviruses transduce murine HSCs efficiently and stably and that the genetically modified cells are capable of completely repopulating the hematopoietic system.

Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown that the major limitations of HSC gene therapy are similar to those encountered when using non-hematopoietic cells as targets and include (1) an inability to genetically modify sufficient numbers of target cells, (2) the loss of transgene function over time, and (3) potential complications due to vector integration. With continued improvements in transduction protocols, murine HSC transduction and transplantation are now routine with transduction efficiencies >50% easily achievable and even >90% feasible. However, attaining high-level engraftment of gene-modified cells after transplantation is still problematic. Basic transduction conditions entail cytokine stimulation of HSC populations, such as stem cell antigen-1 positive (Sca-1(+)) cells isolated from bone marrow, in serum-free media followed by multiple additions of recombinant retrovirus. Analysis of peripheral blood 12 weeks post transplantation of transduced cells into lethally irradiated recipients shows genetic marking in all hematopoietic lineages. Transduction of HSCs is then confirmed by transplanting bone marrow cells harvested from primary transplant recipients into lethally irradiated secondary recipients. Analysis of these mice shows that recombinant retroviruses transduce murine HSCs efficiently and stably and that the genetically modified cells are capable of completely repopulating the hematopoietic system.

Insufficient expression of factor VIII (fVIII) is a major hurdle in the development of successful nucleic acid treatments for hemophilia. However, we recently showed that under myeloablative and reduced-intensity total body irradiation (TBI) conditioning, transplantation of hematopoietic stem cells (HSCs) transduced with recombinant retroviruses containing B domain–deleted porcine fVIII (BDDpfVIII) sequences provides curative fVIII levels in a hemophilia A mouse model. In the current study, we tested BDDpfVIII activity after nonmyeloablative conditioning with busulfan, cyclophosphamide, or fludarabine and immunosuppressive agents CTLA4-Ig + anti-CD40L or anti-(murine)thymocyte serum (ATS). ATS is similar in action to anti-(human)thymocyte globulin (ATG), which is used clinically with busulfan in bone marrow transplantations to increase donor cell engraftment. Mice conditioned with busulfan + ATS and that received a transplant of BDDpfVIII-transduced stem-cell antigen 1-positive cells exhibited moderate levels of donor cell chimerism (between 20% and 60%) and achieved sustained fVIII levels more than 1 U/mL. Similar results were observed in mice preimmunized with human fVIII and conditioned with 5 Gy TBI + ATS or busulfan + ATS. These data demonstrate that it is possible to achieve sufficient fVIII expression after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pretransplantation conditioning with targeted immunosuppression, potentially even in the context of preexisting inhibitors.

To examine the genotypic and phenotypic characteristics of a Micronesian kindred with autosomal dominant precocious osteoarthritis (OA).We reviewed records and radiographs of 3 index patients and their parents, administered questionnaires to 16 additional kindred members, performed whole-genome scans of 24 family members, and sequenced relevant genes from 16 family members.The kindred displayed early onset OA, enlarged epiphyses, platyspondyly, and brachydactyly with dysplastic findings consistent with mild spondyloepiphyseal dysplasia. Genetic analysis revealed an arginine to cysteine substitution at position 75 of the collagen 2A1 gene, a mutation that has been described in 4 other geographically distinct families. The major phenotypic differences among the families were in height (ranging from short to tall) and hearing loss noted in 3 of the 5 families.The presence of the COL2A1 Arg75Cys mutation in 5 geographically distinct areas helps to confirm a potential mutational hotspot. The diverse phenotypic spectrum suggests that modifier genes and environmental factors play a role in the expression of this mutation.

Regulation of allogenic immune response could play a very significant role in establishing stable mixed chimerism for the treatment of several hematological diseases. Indoleamine 2, 3 dioxygenase (IDO) is a rate-limiting enzyme in tryptophan degradation along the kynurenine catabolic pathway and has been shown to regulate allogenic immune responses across the placental barrier. Also, engagement of CTLA4 with CD80/86 on antigen presenting cells has been shown to up-regulate IDO expression and play a significant role in immune regulation. Further, IDO responsive T-cells have been shown to be more susceptible to Fas (CD95) induced apoptosis. Fas ligand (FasL) is another protein that plays an important role in immune homeostasis, inducing apoptosis through interaction with its receptor, Fas. In this context, we generated a novel chimeric protein linking the carboxy terminus of IDO to the N-terminus of FasL with a (Gly) 4 (Ser) 3 repeat linker sequence using standard PCR cloning techniques. The cDNA encoding the chimeric protein was cloned into an MSCV retroviral vector co-expressing either eGFP or N-acetyl transferase for selection with puromycin. Control vectors encoding only IDO or FasL were also generated. The retroviral constructs were confirmed by sequencing of the entire transgene and by in vitro analysis of transfected NIH3T3 cells. Recombinant MSCV retrovirus pseudotyped with an ecotropic envelope was generated by transient transfection of GP2-293T producer cells. NIH3T3 cells were transduced with concentrated virus at an MOI of 1, and harvested after 48 hours for FasL and IDO expression. FasL and IDO-FasL modified fibroblasts showed a comparable marked reduction in their growth rate compared to IDO transduced or unmodified NIH 3T3 cells. Flow cytometric analysis of cells modified with IDO-FasL/GFP showed that all GFP expressing NIH3T3 cells were FasL positive. The slower growth rate of the fibroblasts was shown to be the result of Fas expression on NIH3T3 cells. Using the IDO-FasL/puro construct, murine bone marrow cells were transduced and plated in methylcellulose. Colony counts of the IDO-FasL gene modified cells were comparable with that of nontransduced or IDO only transduced cells, indicating expression of the chimeric protein does not change the normal growth of hematopoietic progenitor cells. IDO activity assays, determined spectrophotometrically for kynurenine production, showed similar activities between cells transduced with the IDO-FasL construct and IDO only construct, further indicating the chimeric protein is functional. To study the application of these constructs in an allotransplant setting, non-lethally irradiated mice have been transplanted with bone marrow cells transduced to express IDO, FasL, or the chimeric protein and are being followed for alloengraftment.

Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al., Blood2007). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of >100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (>2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

A major barrier to successful gene therapy for hemophilia A using bone marrow cells is achieving engraftment of a sufficient number of gene-modified cells to result in therapeutic factor VIII (fVIII) levels under conditions of acceptable patient risk. Previously, we demonstrated sustained levels of a high-expression porcine (HEP)-fVIII construct, at greater than 1 unit/mL for over one year, in hemophilia A mice following myeloablative HSC transplantation (HSCT) thus overcoming the barrier of low fVIII expression (Gangadharan et al, Blood, 2006). In the current study, we tested HEP-fVIII expression after several non-myeloablative and reduced-intensity stem cell transplantation (RIST) protocols incorporating a clinically-relevant dose of genetically-modified hematopoietic stem cells transduced at low multiplicity of infections of ≤ 2. Recipient fVIII exon 16-knockout mice received 3 × 105 HEP-fVIII-MSCV transduced, congenic stem cell antigen-1+ cells. Mice were conditioned using one of the following regimens: 5.5 Gy total body irradiation (TBI), 2 Gy TBI, 2 Gy TBI + 200 mg/kg cyclophosphamide (Ctx), 200 mg/kg Ctx, 200 mg/kg Ctx + 90 mg/kg fludarabine (Flu), 35 mg/kg busulfan (Bu), 35 mg/kg Bu + 200 mg/kg Ctx, 35 mg/kg Bu + costimulation blockade (0.5mg/day CTLA4-Ig and α-CD40L on days 0, 2 post-transplantation). Resulting donor cell engraftment, HEP-fVIII activity, and inhibitory anti-fVIII antibody levels were followed. No fVIII activity was seen in the 2 Gy, 2 Gy + Ctx, Ctx only, or Ctx + Flu cohorts. The Bu and Bu/Ctx treated animals expressed therapeutic levels of HEP-fVIII (0.05 ± 0.07 U/mL and 0.81 ± 0.37 U/mL, respectively) at 1 wk post-HSCT, but returned to baseline in all mice by 2 wks with inhibitor formation in 3 of 7 mice in the Bu group and 0 of 4 mice in the Bu/Ctx cohort. In order to suppress inhibitor formation following conditioning with Bu, recipient mice were administered a low dose course of costimulation blockade post-HSCT. Four of 10 mice in this group demonstrated therapeutic levels of HEP-fVIII activity on day 34-post HSCT (0.11 ± .09 U/mL). All animals demonstrated donor cell engraftment in peripheral blood mononuclear cells at day 23 post-HSCT (18.4 ± 12.1%) and at 34 days post-HSCT, none of the animals had detectable levels of fVIII inhibitors in a modified Bethesda assay. Additionally, we were able to identify specific hematopoietic lineages expressing HEP-fVIII in recipient mice using a novel enzyme-linked immunospot (ELISPOT) assay developed in our laboratory. These data demonstrate that it is possible to achieve sufficient fVIII expression under conditions of i) low MOI transduction with an optimized high-expression fVIII transgene, ii) mild pretransplantation conditioning, iii) limited genetically-modified HSC engraftment, and iv) mild post-HSCT immunosuppression. Furthermore, our results show that clinically-significant fVIII activity levels can be maintained without inhibitor formation suggesting that the current protocol may be on the threshold, in terms of the level of immunosuppression, of promoting tolerance to HEP-fVIII.