Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Replication of the B19 parvovirus in human bone marrow cell cultures.

The B19 parvovirus is responsible for at least three human diseases. The virus was successfully propagated in suspension cultures of human erythroid bone marrow from patients with hemolytic anemias; release of newly synthesized virus into the supernatants of infected cultures was observed. This culture system allowed study at a molecular level of events associated with the B19 life cycle. The B19 parvovirus replicated through high molecular weight intermediate forms, linked through a terminal hairpin structure. B19 replication in vitro was highly dependent on the erythropoietic content of cultures and on addition of the hormone erythropoietin.

Immune response to B19 parvovirus and an antibody defect in persistent viral infection.

B19 parvovirus has been shown to persist in some immunocompromised patients, and treatment with specific antibodies can lead to decreased quantities of circulating virus and hematologic improvement. A defective immune response to B19 parvovirus in these patients was shown by comparison of results using a capture RIA and immunoblotting. In normal individuals, examination of paired sera showed that the dominant humoral immune response during early convalescence was to the virus major capsid protein (58 kD) and during late convalescence to the minor capsid species (83 kD). In patients with persistent parvovirus infection, variable titers against intact particles were detected by RIA, but the sera from these patients had minimal or no IgG to capsid proteins determined by Western analysis. Competition experiments suggested that this discrepancy was not explicable on the basis of immune complex formation alone and that these patients may have a qualitative abnormality in antibody binding to virus. In neutralization experiments, in which erythroid colony formation in vitro was used as an assay of parvovirus activity, sera from patients with poor reactivity on immunoblotting were also inadequate in inhibiting viral infectivity. A cellular response to purified B19 parvovirus could not be demonstrated using proliferation assays and PBMC from individuals with serologic evidence of exposure to virus. These results suggest that production of neutralizing antibody to capsid protein plays a major role in limiting parvovirus infection in man.

Resin-based sealing of root canals in endodontic therapy.

This study evaluated the effect of the obturation technique on leakage, which may be the primary cause of failure in endodontic treatment. The apical seal and leakage behavior of teeth obturated with a resin-based sealer and gutta-percha alternative were compared to conventionally obturated teeth. Sound premolars (N = 10) were instrumented and treated by conventional root canal obturation. A second group (N = 10) was treated with the Resilon-Epiphany system and the remaining 10 roots were divided into two groups (N = 5) and obturated without sealer. A detector electrode was placed coronally in each root in contact with the obturation system and sealed in place and the apices were left patent. The teeth were immersed in 0.9% sodium chloride with a stainless steel counter electrode. A 20V potential was connected between the stainless steel and each tooth in turn with current flow determined by voltage drop across a standard resistor. Leakage was followed for 30 days and statistically analyzed for differences between groups. All teeth in Groups 1, 3A, and 38 (p > 0.05) leaked at 30 days. In Group 2, four roots showed no leakage, five roots showed minimal leakage, and one root exhibited a leakage current at a greater magnitude than the others in the group. A significant difference (p < 0.005) was found between Groups 1 and 2 but not between Group 1 and Groups 3A and 3B (p > 0.05) or between Group 2 and Groups 3A and 3B.

Gene transfer into vascular cells using adeno-associated virus (AAV) vectors.

OBJECTIVES: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. METHODS: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining. RESULTS: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. CONCLUSIONS: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes using two separate adeno-associated virus vectors.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of enzymes in the biosynthetic pathway for dopamine. Tyrosine hydroxylase (TH) catalyzes the synthesis of L-dopa, which must be converted to dopamine by aromatic L-amino acid decarboxylase (AADC). Since the endogenous AADC activity in the striatum is considered to be low, coexpression of both TH and AADC in the same striatal cells would increase the dopamine production and thereby augment the therapeutic effects. In the present study, the TH gene and also the AADC gene were simultaneously transduced into rat striatal cells, using two separate adeno-associated virus (AAV) vectors, AAV-TH and AAV-AADC. Immunostaining showed that TH and AADC were coexpressed efficiently in the same striatal cells in vitro and in vivo. Moreover, cotransduction with these two AAV vectors resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine (6-OHDA)-lesioned rats, compared with rats receiving AAV-TH alone (p < 0.01). These findings suggest an alternative strategy for gene therapy of PD and indicate that the simultaneous transduction with two AAV vectors can extend their utility for potential gene therapy applications.

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

Nosocomial human parvovirus B19 infection: lack of transmission from a chronically infected patient to hospital staff.

OBJECTIVE: To assess the potential for nosocomial spread of parvovirus B19 from a chronically infected patient. DESIGN: Employees exposed to the index case and control (unexposed) employees were evaluated by baseline and follow up parvovirus B19 serologies and hematologic assessments, and completed baseline and follow up epidemiologic questionnaires. SETTING: A chronically infected patient was hospitalized on a hematology ward in a research referral hospital for 3.5 weeks prior to a diagnosis of parvovirus B19 infection and the institution of isolation precautions. METHODS: Sera were screened for parvovirus B19 DNA (dot blot analysis), and IgG and IgM anti-B19 antibodies (capture immunoassay). Hematologic assessment included CBC, differential, and reticulocyte count. RESULTS: The index case had parvovirus B19 DNA at approximately 10(6) genome copies per ml of serum, elevated IgM and low levels of IgG B19 antibodies. Of the 21 exposed staff, 11 (52%) had IgG B19 antibodies and were immune; of the 8 unexposed staff, 6 (75%) had IgG B19 antibodies. No employees developed IgM B19 antibodies, B19 DNA, hematologic abnormalities, or clinical symptoms. CONCLUSIONS: In contrast to reports of documented nosocomial transmission of B19 parvovirus from patients in transient aplastic crisis, nosocomial transmission did not occur--even in the absence of isolation precautions--presumably from the lower level of B19 viremia in our chronically infected (rather than acutely infected) patient.

Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P < 0.01). Furthermore, the dopaminergic neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

Cotransduction of tyrosine hydroxylase and aromatic L-amino acid decarboxylase genes into cultured striatal cells using adeno-associated virus vectors.

OBJECTIVE: To examine whether tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) genes can be cotransduced into the same target striatal cells using adeno-associated virus (AAV) vectors, and to determine whether the cotransduction would result in better biochemical change than the TH gene alone. METHODS: TH and AADC genes were cotransduced into cultured striatal cells with separate AAV vectors. Expressions of TH and AADC were detected by immunocytochemistry; intracellular catecholamine levels were assayed by high-performance liquid chromatography (HPLC). RESULTS: TH and AADC genes were efficiently cotransduced into the striatal cells. Specifically, the coexpression of TH and AADC resulted in more effective dopamine production compared with the TH gene alone. CONCLUSION: Using AAV vectors, coexpression of TH and AADC in the striatal cells might be a useful approach to gene therapy for Parkinson's disease.

The subperiosteal implant as a viable long-term treatment modality in the severely atrophied mandible: a patient's 40-year case history.

Subperiosteal implants have been a treatment modality for atrophied edentulous mandibles for almost 50 years. A case is presented tracing a patient's 40-year history with treatment of his severely atrophied fully edentulous mandible with a subperiosteal implant and removable implant-borne prosthesis. The subperiosteal implant should be considered as a long-term treatment option in those patients who are unable to wear a lower full denture due to resorption of the ridge and osseous defects that provide inadequate bone for placement of endosseous blade- or root-form implants. This case demonstrates the long-term viability of the subperiosteal implant.

Aesthetic enhancement of anterior dental implants with the use of tapered osteotomes and soft tissue manipulation.

An adequate bone base is usually a prerequisite for functionally and aesthetically optimal reconstruction of the soft tissue architecture around a dental implant. In patients with sufficient bone height but insufficient bone width as a result of tooth loss, a jaw enlargement technique with osteotomes combined with soft tissue manipulation may be utilized to facilitate proper implant placement while concomitantly optimizing the aesthetics of the final implant prosthesis. The learning objective of this article is to familiarize the reader with the principles of ridge expansion with tapered osteotomes combined with periodontal plastic soft tissue surgery to aid in proper implant placement and enhance the aesthetic result of the final prosthesis.

Bar overdentures utilizing the Locator attachment.

Implant-retained overdentures are a restorative option for both the fully and partially edentulous arches. A new attachment, the Locator, which features a reduced interarch requirement and the advantage of built-in guide planes providing precise insertion, is described. The Locator is an advancement in attachment technology, with an improved design combined from the best features of a ball attachment, an ERA attachment, and a cap attachment.

Computerized milled solid implant abutments utilized at second stage surgery.

Single tooth restoration with implants traditionally has been complicated due to the difficulty in achieving ideal emergence profile and crown form. The Atlantis milled abutment overcomes these difficulties, utilizing CAD/CAM technology to develop a customized milled abutment with ideal emergence profile and shape to simplify crown fabrication.

Viruses and bone marrow failure.

Some generalizations can be drawn from a review of virus-associated bone marrow failure. The story of B19 parvovirus illustrates that viral infection may be an occult cause of marrow failure. Although the epidemiology of transient aplastic crisis suggested a viral aetiology, the implication of a single virus was surprising; the sporadic appearance of chronic bone marrow failure in immunosuppressed persons has had none of the features of a viral illness. The incrimination of parvovirus in these cases required development of specific immunological and molecular assays. Human and animal retrovirus studies have shown that small changes in the virus genome can have dramatic effects on the biology of the infectious agent and its pathogenicity in infected hosts. In Epstein-Barr virus infection, the host's immune response may play a more important role in mediating disease than virus cytotoxicity. Finally, the association of aplastic anaemia with hepatitis may be underestimated because of the inability to diagnose virus infection without obvious liver disease. The true spectrum of bone marrow disease due to virus infection is not known.

Fabrication of a single-appointment emergency overdenture using failed fixed prosthetics: a case report.

A single appointment fabrication of an emergency overdenture is described. The technique was used in the treatment of a failed implant-supported fixed bridge that was unable to be reinserted due to fractured components and a lack of passive fit. This technique may also be used for the treatment of a failing natural dentition as well as an implant prosthesis.