Biomedical subjects
Publications and source records attributed to J M Wilson.
A 10-year-old girl sustained closed fractures of the distal radius and ulna. This was manipulated and she was treated in an above-elbow plaster for 4 weeks. Two weeks later she was discharged, only to have a second injury to the same forearm. X-ray showed a new fracture distal to undisrupted callus.
Currently, rAAV appears to be one of the most promising vectors for gene therapy applications. Attractive features of the vector include nonpathogenicity, the ability to infect nondividing cells, escape from host immune responses, and integration into the host genome. Tremendous progress has been made in the production of this vector, which makes it possible to start to examine the vector performance in large animals and to implement the transition to phase I human clinical trials with a variety of target tissues and therapeutic genes. However, some major challenges remain to be addressed by more extensive studies. These include the current inability to provide rAAV vectors in sufficient quantity and purity for large-scale clinical human applications, lack of site-specific integration, and lack of efficient transduction in some tissues such as airway epithelial cells. There is a limited transgene capacity in recombinant virus particles, and repeated administration of the vectors may be necessary to treat patients with chronic forms of genetic disease. Nevertheless, it is reasonable to assume that significant refinements will be made in all these areas in the relatively near future. This will promote the potential for successful therapeutic applications in humans, using rAAV-mediated gene transfer for a variety of different diseases.
The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin-Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.
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One of the primary limitations of adenoviral (Ad) -mediated gene therapy is the generation of anti-Ad inflammatory responses that can induce clinical toxicity and impair gene transfer efficacy. The effects of immunosuppression on these inflammatory responses, transgene expression, and toxicity have not yet been systematically examined in humans undergoing Ad-based gene therapy trials. We therefore conducted a pilot study investigating the use of systemic corticosteroids to mitigate antivector immune responses. In a previous phase I clinical trial, we demonstrated that Ad-mediated intrapleural delivery of the herpes simplex virus thymidine kinase gene (HSVtk) to patients with mesothelioma resulted in significant, but relatively superficial, HSVtk gene transfer and marked anti-Ad humoral and cellular immune responses. When a similar group of patients was treated with Ad.HSVtk and a brief course of corticosteroids, decreased clinical inflammatory responses were seen, but there was no demonstrable inhibition of anti -Ad antibody production or Ad-induced peripheral blood mononuclear cell activation. Corticosteroid administration also had no apparent effect on the presence of intratumoral gene transfer. Although limited by the small numbers of patients studied, our data suggest that systemic administration of steroids in the context of Ad-based gene delivery may limit acute clinical toxicity, but may not inhibit cellular and humoral responses to Ad vectors.
One of the current limitations of adenoviral gene therapy is a vector-induced humoral immune response that blocks effective re-administration of the vector. In an animal model of the inborn error of urea synthesis ornithine transcarbamylase (OTC) deficiency, the sparse fur (spf/y) mouse, we tested a strategy to transiently block the CD4 mediated immune response at the time of virus administration using an anti-CD4 monoclonal antibody (GK1.5). The co-administration of GK1.5 resulted in a significantly diminished production of neutralizing antibody to the adenovirus vector, but minimally prolonged metabolic correction. A second infusion of the same virus in GK1.5 treated spf/y mice led to a complete normalization of liver OTC activity at day 3 after infection and a significant metabolic correction of urinary orotate and plasma glutamine. In contrast, there was no evidence of enhanced OTC expression or metabolic correction (measured by normalization of plasma glutamine and urinary orotate) after the second infusion of virus in spf/y mice not treated with GK1.5. Furthermore, when co-administered with two consecutive doses of adenovirus, the anti-CD4 treatment allowed improved transgene expression upon a third administration of virus and a partial normalization of the metabolic abnormalities, compared with mice that did not receive anti-CD4 treatment. The level of OTC expression from the third viral infusion, however, was lower than that from the second viral infusion. Passive transfer experiments suggest that low levels of neutralizing antibodies developing over repeated viral administration was the likely cause of the reduced transgene expression. Together, these findings demonstrated that the host immune system can be modulated to permit effective transgene expression at therapeutic levels by re-administered adenoviral vectors.
Recombinant adenovirus, expressing the CS protein of Plasmodium yoelii, AdPyCS, was shown to induce a comparable degree of T cell-mediated protection against malaria as a single dose of irradiated P. yoelii sporozoites, causing inhibition of liver stage development. We now report that differently from sporozoite-induced immunity, interferon (IFN)-gamma does not mediate the protective immunity induced by AdPyCS, since a similar degree of protection was observed in AdPyCS immunized mice lacking IFN-gamma-/- and the IFN-gamma receptor (IFN-gammaR-/-) compared to that in wild-type mice. Depletion of CD8+ T cells from these immunized mice almost completely abolished the AdPyCS-induced immunity, indicating that the immunization with AdPyCS induces CD8+ T cell-mediated protective anti-malaria immunity, which is independent of IFN-gamma.
BACKGROUND/PURPOSE: The authors' have shown pulmonary alveolarization (capillary and alveolar growth) both after fetal tracheal occlusion and postnatal pulmonary distension. The trophic and developmental mechanisms responsible for this growth remain largely unknown; however, experimental systems have defined an enhanced expression of angiogenic proteins in response to tissue stretch. The authors hypothesize that the stimulation of pulmonary alveolarization after stretch is secondary to upregulation of the potent endothelial cell mitogen vascular endothelial growth factor (VEGF) and that the endothelial cell represents the central stimulus of parenchymal growth. METHODS: A mixed primary pulmonary cell culture obtained by enzymatic digestion of fetal, neonatal, and adult mouse lung was plated on Bioflex elastomer bottom plates, grown to confluence, rendered quiescent, and subjected to continuous cycles of stretch-relaxation with nonstretched cells as controls. Cells were harvested at time-points 0, 30 minutes, 2 hours, 4 hours, 8 hours, and 24 hours. RNA was extracted and VEGF gene expression analyzed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Similar cell groups were harvested, processed, and analyzed utilizing Western Blot techniques. VEGF PCR of mRNA isolated from fetal sheep subjected to surgical creation of diaphragmatic hernia both with (DH-TL) and without (DH) tracheal ligation also was analyzed. RESULTS: VEGF mRNA isoforms 120, 164, and 188 showed increased expression in all stretched groups, which was noted by 30 minutes with maximal expression seen at 2 to 4 hours and a return to baseline expression by 24 hours. VEGF protein was similarly elevated in all stretched cell groups. In preliminary studies, DH/TL sheep showed upregulation of VEGF compared with DH sheep alone. CONCLUSIONS: These data show in an in vitro system that "pulmonary stretch" upregulates VEGF mRNA and protein expression supporting the role of angiogenesis in the stretch-induced pulmonary alveolarization. The authors speculate that such angiogenic activity is a rate-limiting factor in stimulating alveolar epithelial development, and as a treatment modality, therapeutic angiogenesis may provide a noninvasive method with which to treat pulmonary hypoplasia.
The infant born with congenital diphragmatic hernia (CDH) remains one of the most complex patients to manage. Pulmonary hypoplasia and immaturity of the CDH lung are well recognized as the definitive limitation leading to the high mortality rates. Based on the knowledge that CDH is more a physiological disease than a surgical disease, we have shifted our management strategy from immediate repair to delayed repair and stabilization. The associated pulmonary hypertension and right-to-left shunt are common and rarely the cause of death and as such may be largely ignored. Extracorporeal membrane oxygen has been shown to salvage some of the most severely affected neonates. Other advanced and experimental respiratory therapies merit investigation in properly conducted prospective randomized multi-center trials. Survivors of CDH have predictable pulmonary, gastrointestinal and nutritional problems which, when identified and treated early, are correctable. CDH survival is close to 90% at most advanced centers. Uniform standards for CDH management do not exist however. Therefore a minimal set of practice standards should be developed from evidenced-based scientific review.
EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.
A number of pathogens of the upper respiratory tract express an unusual prokaryotic structure, phosphorylcholine (ChoP), on their cell surface. We tested the hypothesis that ChoP, also found on host membrane lipids in the form of phosphatidylcholine, acts so as to decrease killing by antimicrobial peptides that target differences between bacterial and host membranes. In Haemophilus influenzae, ChoP is a phase-variable structure on the oligosaccharide portion of the lipopolysaccharide (LPS). There was a bactericidal effect of the peptide LL-37/hCAP18 on a nontypeable H. influenzae strain, with an increasing selection for the ChoP(+) phase as the concentration of the peptide was raised from 0 to 10 microgram/ml. Moreover, constitutive ChoP-expressing mutants of unrelated strains showed up to 1,000-fold-greater survival compared to mutants without ChoP. The effect of ChoP on resistance to killing by LL-37/hCAP18 was dependent on the salt concentration and was observed only when bacteria were grown in the presence of environmental choline, a requirement for the expression of ChoP on the LPS. Further studies established that there is transcription of the LL-37/hCAP18 gene on the epithelial surface of the human nasopharynx in situ and inducible transcription in epithelial cells derived from the upper airway. The presence of highly variable amounts of LL-37/hCAP18 in normal nasal secretions (<1.2 to >80 microgram/ml) was demonstrated with an antibody against this peptide. It was concluded that ChoP alters the bacterial cell surface so as mimic host membrane lipids and decrease killing by LL-37/hCAP18, an antimicrobial peptide that may be expressed on the mucosal surface of the nasopharynx in bactericidal concentrations.
Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.
Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.
The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.
Teleost fishes, living in fresh water, engage in active ion uptake to maintain ion homeostasis. Current models for NaCl uptake involve Na(+) uptake via an apical amiloride-sensitive epithelial Na(+) channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or alternatively by an amiloride-sensitive Na(+)/H(+) exchange (NHE) protein, and apical Cl(-) uptake mediated by an electroneutral, SITS-sensitive Cl(-)/HCO(3-) anion-exchange protein. Using non-homologous antibodies, we have determined the cellular distributions of these ion-transport proteins to test the predicted models. Na(+)/K(+)-ATPase was used as a cellular marker for differentiating branchial epithelium mitochondria-rich (MR) cells from pavement cells. In both the freshwater tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), V-ATPase and ENaC-like immunoreactivity co-localized to pavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical anion-exchanger-like immunoreactivity is found in the MR cells. Thus, a freshwater-type MR chloride cell exists in teleost fishes. The NHE-like immunoreactivity is associated with the accessory cell type and with a small population of pavement cells in tilapia.
The branchial epithelium of the mudskipper Periophthalmodon schlosseri is densely packed with mitochondria-rich (MR) cells. This species of mudskipper is also able to eliminate ammonia against large inward gradients and to tolerate extremely high environmental ammonia concentrations. To test whether these branchial MR cells are the sites of active ammonia elimination, we used an immunological approach to localize ion-transport proteins that have been shown pharmacologically to be involved in the elimination of NH(4)(+) (Na(+)/NH(4)(+) exchanger and Na(+)/NH(4)(+)-ATPase). We also investigated the role of carbonic anhydrase and boundary-layer pH effects in ammonia elimination by using the carbonic anhydrase inhibitor acetazolamide and by buffering the bath water with Hepes, respectively. In the branchial epithelium, Na(+)/H(+) exchangers (both NHE2- and NHE3-like isoforms), a cystic fibrosis transmembrane regulator (CFTR)-like anion channel, a vacuolar-type H(+)-ATPase (V-ATPase) and carbonic anhydrase immunoreactivity are associated with the apical crypt region of MR cells. Associated with the MR cell basolateral membrane and tubular system are the Na(+)/K(+)-ATPase and a Na(+)/K(+)/2Cl(-) cotransporter. A proportion of the ammonia eliminated by P. schlosseri involves carbonic anhydrase activity and is not dependent on boundary-layer pH effects. The apical CFTR-like anion channel may be serving as a HCO(3)(-) channel accounting for the acid-base neutral effects observed with net ammonia efflux inhibition.
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Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol and other lipids in the lysosomal compartment. In this report, we use subcellular fractionation and microscopy to determine the localization of the murine Niemann-Pick C1 (NPC1) protein. Fractionation of mouse liver homogenates indicates that some NPC1 cosediments with lysosome-associated membrane protein 1 (LAMP1)-containing membranes. However, a significant amount of NPC1 is also found in membranes that do not contain LAMP1. Moreover, fractionation of liver membranes and fibroblasts in the presence of a nonionic detergent showed that a fraction of NPC1 cosediments with caveolin-1 in rafts. Immunofluorescence microscopy of cultured mouse fibroblasts showed that NPC1 is found in two morphologically distinct structures. The first population is characterized by large punctate structures that do not colocalize with major organelle protein markers, but do colocalize with filipin and a small fraction of caveolin-1. Examination of these large NPC1-containing compartments using electron microscopy shows that these structures contain extensive internal membranes. The second population is represented by smaller, more diffuse structures, a fraction of which colocalize with LAMP1-positive compartments. Incubation of fibroblasts with low density lipoprotein (LDL) increases colocalization of NPC1 with LAMP1-containing compartments. This colocalization can be further enhanced by treating fibroblasts with progesterone or chloroquine. The results indicate that NPC1 is associated with an unique vesicular compartment enriched with cholesterol and containing caveolin-1, and that NPC1 cycles to LAMP1-positive compartments, presumably to facilitate the processing of LDL-derived cholesterol.