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Biomedical subjects

B J Baum

Publications and source records attributed to B J Baum.

At least 55 records · Page 3Linked to original sources
PubMed2000

The impact of tissue engineering on dentistry.

BACKGROUND: Tissue engineering is a novel and highly exciting field of research that aims to repair damaged tissues as well as create replacement (bioartificial) organs. OVERVIEW: The authors provide a general review of the principles underlying key tissue engineering strategies, as well as the typical components used. Several examples of preclinical and clinical progress are presented. These include passive approaches, such as dental implants, and inductive approaches that activate cells with specific molecular signals. PRACTICE IMPLICATIONS: Tissue engineering will have a considerable effect on dental practice during the next 25 years. The greatest effects will likely be related to the repair and replacement of mineralized tissues, the promotion of oral wound healing and the use of gene transfer adjunctively.

Animals↗
PubMed2000

Salivary glands as a model for craniofacial applications of gene transfer.

The potential applications of gene transfer technology to all branches of medicine are increasing. It is quite likely that within the next 10-20 years surgical practice routinely will utilize gene transfer, at least adjunctively. The purpose of this review is to familiarize the oral and maxillofacial surgeon with this technology. Studies performed with salivary glands in animal models are presented as examples of proof of concept.

Animals↗
PubMed1999

Polarized secretion of transgene products from salivary glands in vivo.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Adenoviridae↗
PubMed1999

Re-engineering the functions of a terminally differentiated epithelial cell in vivo.

Because of their easy access, and important role in oral homeostasis, mammalian salivary glands provide a unique site for addressing key issues and problems in tissue engineering. This manuscript reviews studies by us in three major directions involving re-engineering functions of salivary epithelial cells. Using adenoviral-mediated gene transfer in vivo, we show approaches to i) repair damaged, hypofunctional glands and ii) redesign secretory functions to include endocrine as well as exocrine pathways. The third series of studies show our general approach to develop an artificial salivary gland for clinical situations in which all glandular tissue has been lost.

Animals↗
PubMed1999

Salivary gland cytokine expression in NOD and normal BALB/c mice.

The autoimmune diabetes-prone nonobese diabetic (NOD) mouse develops a chronic lymphocytic infiltration of endocrine and exocrine glands. The objectives of this study were to characterize the salivary immune infiltration and cytokine expression of NOD mice and compare these findings to those of normal BALB/c mice. A decline in salivary flow rates in NOD mice began between 8 and 12 weeks of age. At this same time lymphocytic foci are detectable in the salivary glands. Lymphocytic infiltration in the salivary glands of NOD mice increased with age and simultaneously salivary function declined. No lymphocytic infiltration was seen in BALB/c salivary tissues. Messenger RNA expression of several inflammatory cytokines, including interleukin-1beta (IL-1beta), IL-2, IL-10, interferon-gamma, and tumor necrosis factor-alpha was detected in the submandibular glands of both NOD and BALB/c mice by the reverse transcription polymerase chain reaction. IL-4 synthesis was also present in some tissues. Immunohistochemical analysis demonstrated the intense expression of inflammatory cytokines within lymphocytic infiltrates and epithelial cells of all NOD mice. Minimal expression of the same cytokines was detected only occasionally in BALB/c tissues stained in parallel. These results demonstrate cytokine expression in the salivary glands of normal mice and suggest that the overexpression of these inflammatory cytokines is likely involved in the development and progression of the organ-localized autoimmunity in the salivary glands of NOD mice.

Animals↗
PubMed1999

Use of triplex-forming oligonucleotides and adenoviral constructs for studying the regulation of gene expression.

Short synthetic homopyrimidine- or homopurine-rich oligonucleotides can form sequence-specific triplexes with corresponding homopurine-homopyrimidine sites on duplex DNA and block transcription of a target gene in vitro. Such triplex-forming oligonucleotides (TFOs) can be rationally designed to target homopurine/homopyrimidine sequences that are often found in eukaryotic genes and thus used to modulate the expression of these genes. The antigene strategy using TFOs has been successfully applied to a number of genes in vitro. In this article we describe methods used in applying this antigene approach to the rat aquaporin 5 (rAQP5) gene. We specifically focus on the selection of TFOs based on the sequence of the target gene and on a novel method employing adenoviruses for delivery of TFOs to cells in vitro.

Adenoviridae↗
PubMed1999

Extracellular matrix protein-induced changes in human salivary epithelial cell organization and proliferation on a model biological substratum.

We have used a denuded rat tracheal preparation as a biological substratum on which to examine the growth and morphology of a salivary epithelial cell line (HSG) in vitro. In the absence of an additional coating of matrix proteins, HSG cells grew at low density on tracheae. Coating the tracheae with Vitrogen (a commercial collagen I preparation) or fibronectin promoted HSG cell growth and monolayer formation. Conversely, if a coating of Matrigel was applied, cells grew in a more organized fashion, but at low density. Generally similar results were obtained with cells grown on laminin and collagen IV but with less organization. These studies demonstrate the utility of a natural, tubular substratum for testing the influence of different matrix proteins on salivary epithelial cell behavior.

Animals↗
PubMed1999

The need to introduce gene therapy to the dental curriculum.

Recombinant DNA technology is finding its way into many aspects of clinical medicine. No application currently is more dramatic than gene therapy. Proofs of principle have already been established for gene therapy targeted to oral tissues, and more are likely to be demonstrated in the near future. The dental curriculum must begin to include the biological basis of DNA-based therapies and other related biomedical science progress.

Curriculum↗
PubMed1999

Effect of clodronate on macrophage depletion and adenoviral-mediated transgene expression in salivary glands.

Expression of transgenes from adenoviral vectors is short-lived in salivary glands, in part because of immune responses to the virus and/or transgene product. Previous studies demonstrated that depletion of macrophages with multilamellar liposomes containing clodronate (Cl2MBP) increases adenoviral-mediated transgene expression in the liver. This technique was tested in salivary glands. Rats were treated with Cl2MBP-liposomes or control liposomes by femoral vein, intraperitoneal, or carotid artery injections. Thereafter, a recombinant adenovirus, AdCMVluciferase, was instilled intraductally in submandibular glands (SMGs), or delivered to the liver via femoral vein injection. Marked depletion (>94%) of liver macrophages and increased levels of luciferase activity in the liver (45-fold higher than controls) were present in animals receiving Cl2MBP-liposomes. In contrast, the same treatment never depleted more than 41% of SMG macrophages nor increased luciferase activity in SMGs, regardless of the route of administration. In conclusion, while macrophage depletion with Cl2MBP-liposomes is associated with markedly increased adenoviral-mediated transgene expression, this strategy was ineffective for salivary glands.

Adenoviridae↗
PubMed1999

In vivo gene transfer to salivary glands.

Considerable progress has occurred recently in transferring foreign genes to different tissues in vivo. Gene transfer to salivary glands has mirrored progress in the general field. Most salivary studies have utilized replication-deficient, recombinant adenoviruses as gene transfer vectors in rat models. These vectors are able to transduce almost all rat salivary epithelial cell types and lead to relatively high levels of transgene expression. Additionally, successful, though quite modest, gene transfer to salivary glands has been achieved with retroviral vectors and with different plasmid conjugates (liposomes; nonrecombinant adenoviruses). Salivary gland gene transfer has been used for two potential clinical goals: (i) the repair of hypofunctional gland parenchyma, and (ii) the production of secretory transgene products for either systemic or upper gastrointestinal tract pharmaceutical use. Gene transfer can also be used as a powerful tool to alter cellular phenotype in vivo and probe cell biological questions. The current spectrum of studies demonstrates the potential broad and profound influence gene transfer can make on salivary physiology and pathophysiology.

Adenoviridae↗
PubMed1999

Construction and in vivo efficacy of a replication-deficient recombinant adenovirus encoding murine growth hormone.

We have constructed a recombinant, replication-deficient, first-generation adenovirus-encoding mouse GH (mGH), AdCMVmGH. This virus directed mGH production from an epithelial cell line in vitro in a dose-dependent manner. When injected into the quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf mice, AdCMVmGH resulted in the production of significantly elevated serum mGH levels. Furthermore, after i.m. injection, dwarf mice increased in weight by 8% over 4 days and close to 100% by 30 days. When AdCMVmGH was administered to 3- to 4-week-old rats by i.v. injection to assess general metabolic responses, serum mGH, insulin-like growth factor 1, triglycerides and cholesterol levels were significantly elevated. AdCMVmGH should be a valuable experimental tool for the controlled, directed expression of mGH in preclinical mouse model studies.

Adenoviridae↗
PubMed1998

Adenoassociated virus-mediated transfer of a functional water channel into salivary epithelial cells in vitro and in vivo.

Aquaporin 1 (AQP1) is the archetypal member of a family of integral membrane proteins that function as water channels. Previously we have shown that this protein can be expressed transiently from a recombinant adenovirus (AdhAQP1) in vitro in different epithelial cell lines, and in vivo in rat submandibular glands. In the present study we have constructed a recombinant adenoassociated virus (rAAV) containing the human aquaporin 1 gene (rAAVhAQP1). rAAVhAQP1 was produced at relatively high titers. Approximately 10(11)-10(12) particles/ml and approximately 10(8)-10(9) transducing units/ml. We show that the rAAVhAQP1 can transduce in vitro four epithelial cell lines of different origins, at a level sufficient to detect the recombinant hAQP1 protein by either Western blot or confocal microscopic analysis. The recombinant hAQP1 was correctly targeted to the plasma membranes in all cell lines. Function of the recombinant hAQP1 was measured as fluid flow, in response to an osmotic gradient, across a monolayer of transduced epithelial cells. The data show that even at a low level of transduction, typically approximately 10% of the cells in the monolayer, transepithelial fluid movement is enhanced about threefold above basal levels. In addition, we report that rAAVhAQP1 can transduce epithelial cells in the salivary glands and liver of mice in vivo. These results suggest that rAAVs may be useful gene transfer vectors to direct the production of functional transgenes in salivary epithelial cell types.

Animals↗
PubMed1998

Relationship between adenovirus-mediated aquaporin 1 expression and fluid movement across epithelial cells.

AdhAQP1, a recombinant adenovirus encoding the human water channel aquaporin 1 (AQP1), has been shown to be useful for gene therapy of salivary glands rendered hypofunctional following irradiation. Here we utilized AdhAQP1 to examine the relationship between AQP1 expression and fluid movement across a polarized salivary epithelial cell monolayer. In response to a 440 to 340 mosm gradient, net fluid movement across cells infected with AdhAQP1 was approximately 10-fold that seen in uninfected cells or cells infected with a control virus. At a multiplicity of infection (MOI) of 5, fluid movement was linear for 15-30 min. Increasing the osmotic gradient resulted in a proportional increase in fluid movement. At low virus MOIs (0.1-1.0), fluid movement was markedly enhanced relative to that seen at higher MOIs (approximately 5.10), where the level of AQP1 expression and number of cells transduced were considerably greater. We conclude that significant, osmotically-obliged fluid movement in a salivary cell monolayer with low basal water permeability does not require high levels of AQP1 expression.

Adenoviridae↗
PubMed1998

Repetitive adenovirus administration to the parotid gland: role of immunological barriers and induction of oral tolerance.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Adenoviridae↗
PubMed1998

Gamma-irradiation-induced cell cycle arrest and cell death in a human submandibular gland cell line: effect of E2F1 expression.

This study examined the effect of gamma-irradiation (5 and 10 Gy) on the human submandibular cell line (HSG). Radiation treatment (5 Gy and 10 Gy) induced a dose-dependent decrease in cell proliferation, with a G2/M arrest of the cell cycle, and an increase in cell death (cells with <2n DNA increased from 7% in control cells to 34% and 40% in 5 and 10 Gy irradiated cells, respectively). [Ca2+]i measurements demonstrated that the status of internal Ca2+ stores, and muscarinic receptor-mediated Ca2+ mobilization, in irradiated cells was comparable to that in non-irradiated cells. These data suggest that 1) irradiated HSG cells maintain normal physiology and 2) internal Ca2+ store depletion does not account for the decreased cell proliferation. To manipulate the radiation-induced cell cycle arrest, we examined the effect of the transcription factor E2F1, which has been shown to induce cell cycle progression in HSG cells (Lillibridge and O'Connell, 1997, J. Cell. Physiol., 1 72:343-350). The ability of irradiated HSG cells to express and appropriately route proteins was demonstrated by using adenovirus-mediated expression of beta-galactosidase, alpha1-antitrypsin, and aquaporin-1. Infection of HSG cells with an adenoviral vector encoding E2F1, either 12 h before or immediately following irradiation, but not post-irradiation, induced maintenance of cells in the S phase of the cell cycle, reduced the number of cells arrested at G2/M, and decreased the rate of appearance of cells with <2n DNA. While the mechanism of irradiation-induced cell death has not yet been confirmed, these data suggest that expression of the E2F1 gene product in HSG cells can be a useful strategy to manipulate cell cycle events and reduce the initial loss of cells due to radiation.

Adenoviridae↗
PubMed1998

A polarized salivary cell monolayer useful for studying transepithelial fluid movement in vitro.

There are no reported, convenient in vitro models for studying polarized functions in salivary epithelial cells. Accordingly, we examined three often-used salivary cell lines for their ability to form a polarized monolayer on permeable, collagen-coated polycarbonate filters. Only the SMIE line, derived from rat submandibular gland, had this ability. The SMIE cell monolayer exhibited junctional complexes, with a tight-junction-associated protein, ZO-1, localized to cell-cell contact areas. The Na+/K+-ATPase alpha1-subunit was detected predominantly in the basolateral membranes, while the Na+/H+ exchanger isoform 2 appeared primarily in the apical membranes. Using adenovirus-mediated cDNA transfer, SMIE cells were shown to be capable of routing marker proteins (beta-galactosidase +/- a nuclear targeting signal, alpha1-antitrypsin, aquaporin-1) to appropriate locations. Furthermore, this salivary cell monolayer provided a convenient tool for studying aquaporin-1-mediated, osmotically directed, transepithelial fluid movement in vitro. Thus, SMIE cells appear to be a useful experimental model with which to study some polarized functions in a salivary epithelial cell line.

Adenoviridae↗
PubMed1998

Long-term salivary effects of single-dose head and neck irradiation in the rat.

The effect of a single dose of X-irradiation (either 2.5, 5, 7.5, 10 or 15 Gy) to the head and neck region on parotid and submandibular gland function in rats was evaluated for up to 1 year. No animal receiving 15 Gy survived the entire study. Animals receiving > or = 7.5 Gy showed significantly less increase in body weight over time. Average wet weights of both gland types were reduced with as little as 2.5 Gy. Pilocarpine-stimulated parotid salivary flow was diminished significantly at 12 months for each radiation-dose group. Significant salivary flow reductions from submandibular glands were seen at > or = 7.5 Gy at this same time-point. These results show that a single radiation exposure of as low as 2.5 Gy to the head and neck region of rats can cause significant long-term alterations in salivary gland function.

Analysis of Variance↗