Biomedical subjects
Publications and source records attributed to H L Malech.
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Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.
A human disorder caused by mutation in nonmuscle actin has not been reported. We report here a variant of nonmuscle actin in a female patient with recurrent infections, photosensitivity, and mental retardation. She also had abnormalities in neutrophil chemotaxis, superoxide production, and membrane potential response. Two-dimensional PAGE analysis of proteins from neutrophils and other cell types from this patient demonstrated a unique protein spot migrating at 42 kDa with pI shifted slightly to neutral relative to normal beta- and gamma-actin. Digestion peptide mapping and Western blotting showed this spot to be an abnormal actin. A full-length cDNA library was constructed by using mRNA from patient's cells and cDNA encoding the mutant beta-actin molecule was identified by an in vitro translation method. Sequencing of the clones demonstrated a G-1174 to A substitution, predicting a glutamic acid-364 to lysine substitution in beta-actin and eliminating a HinfI DNase restriction site found in normal beta-actin sequence. By HinfI digestion and by sequencing, the mutation in one allele of patient's genomic DNA was confirmed. Though no defect in cell-free polymerization of actin was detected, this defect lies in a domain important for binding to profilin and other actin-regulatory molecules. In fact, the mutant actin bound to profilin less efficiently than normal actin did. Heterozygous expression of mutant beta-actin in neutrophils and other cells of this patient may act in a dominant-negative fashion to adversely affect cellular activities dependent on the function of nonmuscle actin.
In addition to its catalytic roles, the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (H4B) is required for substrate binding and for stabilization of the dimeric structure. We expressed and purified the core of the iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the presence (H4B+) and absence (H4B-) of this cofactor. Both forms bound stoichiometric amounts of heme (>0.9 heme per protein subunit). H4B- CODiNOS was unable to bind arginine, gave an unstable ferrous carbonyl adduct, and was a mixture of monomer and dimer. H4B+ CODiNOS bound arginine, gave a stable ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichiometric titrations.
BACKGROUND: The hyper-IgE syndrome with recurrent infections is a rare immunodeficiency characterized by recurrent skin and pulmonary abscesses and extremely elevated levels of IgE in serum. Associated facial and skeletal features have been recognized, but their frequency is unknown, and the genetic basis of the hyper-IgE syndrome is poorly understood. METHODS: We studied 30 patients with the hyper-IgE syndrome and 70 of their relatives. We took histories, reviewed records, performed physical and dental examinations, took anthropometric measurements, and conducted laboratory studies. RESULTS: Nonimmunologic features of the hyper-IgE syndrome were present in all patients older than eight years. Seventy-two percent had the previously unrecognized feature of failure or delay of shedding of the primary teeth owing to lack of root resorption. Common findings among patients were recurrent fractures (in 57 percent of patients), hyperextensible joints (in 68 percent), and scoliosis (in 76 percent of patients 16 years of age or older). The classic triad of abscesses, pneumonia, and an elevated IgE level was identified in 77 percent of all patients and in 85 percent of those older than eight. In 6 of 23 adults (26 percent), IgE levels declined over time and came closer to or fell within the normal range. Autosomal dominant transmission of the hyper-IgE syndrome was found, but with variable expressivity. Of the 27 relatives at risk for inheriting the hyper-IgE syndrome, 10 were fully affected, 11 were unaffected, and 6 had combinations of mild immunologic, dental, and skeletal features of the hyper-IgE syndrome. CONCLUSIONS: The hyper-IgE syndrome is a multisystem disorder that affects the dentition, the skeleton, connective tissue, and the immune system. It is inherited as a single-locus autosomal dominant trait with variable expressivity.
BACKGROUND: The hyper-IgE (HIE), or Job's, syndrome is a rare, complex disorder characterized by high levels of serum IgE in childhood and chronic dermatitis with recurrent, often severe sinopulmonary and skin infections. Although the etiology of HIE syndrome is unknown, there is evidence that patients with HIE have abnormalities in cellular immune responses, as well as in the production of polyclonal and antigen-specific antibodies. Furthermore, there appears to be a common (but still undefined) mechanism underlying the regulation of IgE and IgG4 in this condition. OBJECTIVE: We sought to assess the role of cytokines or cytokine receptor blockade in regulating IgE and IgG4 production in HIE. METHODS: PBMCs were isolated from patients with HIE (n = 9) and normal individuals (n = 8), and IgE and IgG4 production was assessed spontaneously, in the presence of recombinant IL-4, IL-13, IL-6, IL-8, IL-12, and IFN-gamma, under conditions in which the IL-4R was blocked or when these cytokines were neutralized by specific monoclonal or polyclonal antibodies. RESULTS: In PBMCs from patients with HIE, a significant (P <.01) reduction in the spontaneously produced IgE (and IgG4) was induced by either IFN-gamma or IL-12, although neither cytokine could totally abrogate the immunoglobulin production. Whereas spontaneous IgE (and IgG4) production was not affected by exogenous IL-4 and IL-13, neutralizing antibodies to IL-4 and IL-13 also significantly (P <.01) reduced the production of IgE and IgG4, a finding supported by the observation of increased expression of IgE germline transcripts in these patients. In contrast to the neutralization of IL-4 and IL-13 protein, anti-IL-4R antibodies or soluble IL-4R completely suppressed IgE and IgG4 production in HIE. Similarly, IL-8 or antibodies to IL-6 and TNF-alpha, cytokines known to affect IL-4-dependent IgE production, completely inhibited both IgE and IgG4 production. CONCLUSION: These data show that overproduction of IgE and IgG4 can be regulated by a number of cytokines affecting the IL-4-dependent pathway of IgE/IgG4 production in HIE and suggest new targets for therapeutic intervention.
We compared the cell cycle status and expression of mRNA for the amphotropic retroviral receptor in hematopoietic stem cells isolated from bone marrow and cytokine mobilized peripheral blood. CD34+ cells from six normal volunteers were enriched by immune selection from steady-state bone marrow and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (10 microg/kg/day for 5 days). Cell cycle status of the phenotypically primitive CD34+CD38- hematopoietic stem cell population was analyzed using a four-color flow cytometry technique that distinguished the G0, G1, and S/IG2/M phases of the cell cycle. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to measure mRNA expression of the amphotropic retroviral receptor. Peripheral blood hematopoietic stem cells had 2.6-fold more cells in the G1 phase of the cell cycle compared to steady-state bone marrow. Furthermore, lineage CD34+CD38- cells from G-CSF mobilized peripheral blood had a fourfold higher level of amphotropic retrovirus receptor mRNA. In conclusion, we found that CD34+ CD38- hematopoietic stem cells isolated from G-CSF mobilized peripheral blood differ from those isolated from steady-state bone marrow in that a significant proportion have entered the G1 phase of the cell cycle and express higher levels of amphotropic receptor mRNA. These biologic properties are consistent with the reported rapid recovery of hematopoietic function following transplantation with peripheral blood hematopoietic stem cells and make these cells a preferred target for retroviral-based gene transfer.
The hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent skin abscesses, pneumonia, and highly elevated levels of serum IgE. HIES is now recognized as a multisystem disorder, with nonimmunologic abnormalities of the dentition, bones, and connective tissue. HIES can be transmitted as an autosomal dominant trait with variable expressivity. Nineteen kindreds with multiple cases of HIES were scored for clinical and laboratory findings and were genotyped with polymorphic markers in a candidate region on human chromosome 4. Linkage analysis showed a maximum two-point LOD score of 3.61 at recombination fraction of 0 with marker D4S428. Multipoint analysis and simulation testing confirmed that the proximal 4q region contains a disease locus for HIES.
Progress in development of gene therapy for chronic granulomatous disease (CGD), an inherited defect in leukocyte oxidase deficiency, is reviewed. The use of retrovirus vectors to transfer oxidase enzyme subunit cDNA sequence into hematopoietic progenitors results in correction of oxidase activity in neutrophils differentiating from transduced progenitors. In CGD mouse knockouts (X-linked gp91phox-deficient CGD and autosomal recessive p47phox-deficient CGD), gene therapy correction of the CGD defect resulted in appearance of oxidase-normal neutrophils in the peripheral blood and increased host resistance to challenge with fungi or bacteria. In a phase I clinical trial of ex vivo gene therapy of p47phox-deficient CGD, prolonged production (2-6 months) of a low number (1:5000) of oxidase-normal neutrophils was achieved. This therapy might prove beneficial in a setting of prolonged infection in CGD patients, in which even transient production of autologous gene-corrected neutrophils might augment host defense.
An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence. We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells. Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest. To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-gamma, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs. Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-gamma. DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes. CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-gamma- transduced DCs. In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-gamma secretion. Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection.
Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.
Chronic granulomatous disease (CGD) is a rare inherited disorder of the NADPH oxidase complex in which phagocytes are defective in generating reactive oxidants. As a result, patients with CGD suffer from recurrent bacterial and fungal infections. The most common fungal infections are caused by Aspergillus species. Aspergillus nidulans is a rare pathogen in most patient populations with quantitative or qualitative neutrophil defects. We have reviewed all cases in which A. nidulans was isolated from patients at the National Institutes of Health (Bethesda, MD) between 1976 and 1997. A. nidulans infection occurred in 6 patients with CGD, but was not a pathogen in any other patient group. Aspergillus fumigatus was a more common pathogen in CGD compared with A. nidulans, but A. nidulans was more virulent. A. nidulans was significantly more likely to result in death compared with A. fumigatus, to involve adjacent bone, and to cause disseminated disease. Patients with A. nidulans received longer courses of amphotericin B therapy than patients with A. fumigatus, and were treated with surgery more often. In contrast to A. fumigatus, A. nidulans was generally refractory to intensive antifungal therapy, suggesting that early surgery may be important. These data show that A. nidulans is a distinct pathogen in CGD and its isolation carries more severe implications than that of A. fumigatus.
Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47(phox) deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34(+) peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47(phox). Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bank-compatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.
Differential display polymerase chain reaction is technically challenging at a number of steps, including reliable reamplification of differentially expressed sequences after they are isolated from an acrylamide gel. As the only source of the sequence found to be of interest is the gel from which it was identified as being differentially expressed, failed attempts at reamplification can be particularly frustrating. In our laboratory, attempts to reamplify DNA sequences cut from a denaturing 6% acrylamide gel consistently failed when greater than 5 microliters of eluted product was used as template in a subsequent PCR reaction. If less template was used in subsequent PCR reactions, reamplification was consistently successful. This observation emphasized the importance of using limited amounts of template when reamplifying sequences that are differentially displayed on denaturing acrylamide gels
The p47phox-/- mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus-based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox-/- marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox-/- mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% +/- 0.9% of phorbol myristate acetate-stimulated peripheral blood neutrophils at 4 weeks and 2.6% +/- 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox-/- mice were significantly lower than untreated p47phox-/- mice (0.89 +/- 0.30 colonies v 237.7 +/- 83.6 colonies at 24 hours, P < .02; 4.0 +/- 2.0 colonies v 110.2 +/- 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox-/- mice (P < .001). Thus, stem-cell-directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.
Chronic granulomatous disease (CGD) can result from any of four single gene defects involving the components of the superoxide (O-2) generating phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We show that transduction of peripheral blood CD34+ hematopoietic progenitors from a p67phox deficient CGD patient with replication defective amphotropic retrovirus encoding p67phox (MFGS-p67phox) significantly corrected the CGD functional defect in phagocyte oxidase activity in vitro. Using a chemiluminescence assay of oxidase activity, we showed that transduced patient CD34+ progenitors differentiating to myeloid cells in culture produced 25% of the total superoxide produced by normal CD34+ progenitors differentiating in culture. A flow cytometric assay of oxidase activity used to assess the oxidase function of individual cells in the cultures indicated that up to 32% of maturing granulocytes derived from transduced CD34+ progenitors from the p67phox CGD patient were oxidase positive with the average level of correction per granulocyte of 85% of that seen with granulocytes in similar cultures of CD34+ progenitors from normal volunteers. Nitroblue tetrazolium dye reduction assays of colonies of transduced progenitors in soft agar indicated that in some studies restoration of oxidase activity occurred in myeloid cells within 44% of granulocyte-erythrocyte-monocyte colonies, and within 28% of the combined group of granulocyte colonies/monocyte colonies/granulocyte monocyte colonies. These high correction rates were achieved without any selective regimen to enrich for transduced cells. This study provides a basis for development of gene therapy for the p67phox deficient form of CGD.
We have recently demonstrated that endothelial cells cultured on Gelfoam blocks, but not monolayer matrices can phagocytose and kill Staphylococcus aureus. Experiments determined that penicillin G, included in the endothelial cell growth medium, induces these cells to exhibit the observed bactericidal activity. In this communication, we report on studies aimed at elucidating the mechanism by which penicillin G-induced endothelial cells, cultured on Gelfoam blocks, kill S. aureus. Despite the fact that there is a substantial literature that demonstrates neutrophilic killing of bacteria can be mediated through free radical-dependent and free radical-independent mechanisms, considerably less is known about pathways by which endothelial cells can catalyze similar microbicidal activities. Studies described herein point to the fact that superoxide and products derived from this free radical were not responsible for endothelial killing of S. aureus. Likewise, a possible role for nitric oxide in bacterial killing was explored. As part of this inquiry, we stably transduced a NOS-2 encoding retrovirus into endothelial cells cultured on Gelfoam blocks in the absence of penicillin G. Even though these cells secreted nitric oxide at a rate of 0.5 microM/h per 1 x 10(6) cells, similar to what has been reported for murine macrophages induced with gamma-interferon, in our model, nitric oxide was not found to kill S. aureus. Data presented demonstrate that the microbicidal activity of endothelial cells is mediated through free radical-independent pathways.
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