Biomedical subjects

D W Metzger

Publications and source records attributed to D W Metzger.

At least 19 recordsLinked to original sources

PubMed2002

Novel current-conducting composite substrates for exposing osteoblasts to alternating current stimulation.

The present study demonstrates that novel nanocomposites consisting of blends of polylactic acid and carbon nanotubes effectively can be used to expose cells to electrical stimulation. When osteoblasts cultured on the surfaces of these nanocomposites were exposed to electric stimulation (10 microA at 10 Hz) for 6 h/day for various periods of time, there was a 46% increase in cell proliferation after 2 days, a 307% increase in the concentration of extracellular calcium after 21 consecutive days, and upregulation of mRNA expression for collagen type-I after both 1 and 21 consecutive days. These results provide evidence that electrical stimulation delivered through novel, current-conducting polymer/nanophase composites promotes osteoblast functions that are responsible for the chemical composition of the organic and inorganic phases of bone. Furthermore, this evidence elucidates aspects of the cellular/molecular-level mechanisms involved in new bone formation under electrical stimulation.

Animals↗

PubMed2001

Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response.

BACKGROUND: Porcine small intestinal submucosa (SIS) is an acellular, naturally derived extracellular matrix (ECM) that has been used for tissue remodeling and repair in numerous xenotransplantations. Although a vigorous immune response to xenogeneic extracellular matrix biomaterials is expected, to date there has been evidence for only normal tissue regeneration without any accompanying rejection. The purpose of this study was to determine the reason for a lack of rejection. METHODS: Mice were implanted s.c. with xenogeneic tissue, syngeneic tissue, or SIS, and the graft site analyzed histologically for rejection or acceptance. Additionally, graft site cytokine levels were determined by reverse transcriptase polymerase chain reaction and SIS-specific serum antibody isotype levels were determined by ELISA. RESULTS: Xenogeneically implanted mice showed an acute inflammatory response followed by chronic inflammation and ultimately graft necrosis, consistent with rejection. Syngeneically or SIS implanted mice, however, showed an acute inflammatory response that diminished such that the graft ultimately became indistinguishable from native tissue, observations that are consistent with graft acceptance. Graft site cytokine analysis showed an increase in interleukin-4 and an absence of interferon-gamma. In addition, mice implanted with SIS produced a SIS-specific antibody response that was restricted to the IgG1 isotype. Reimplantation of SIS into mice led to a secondary anti-SIS antibody response that was still restricted to IgG1. Similar results were observed with porcine submucosa derived from urinary bladder. To determine if the observed immune responses were T cell dependent, T cell KO mice were implanted with SIS. These mice expressed neither interleukin-4 at the implant site nor anti-SIS-specific serum antibodies but they did accept the SIS graft. CONCLUSIONS: Porcine extracellular matrix elicits an immune response that is predominately Th2-like, consistent with a remodeling reaction rather than rejection.

Animals↗

PubMed2001

Fc receptor-mediated phagocytosis makes a significant contribution to clearance of influenza virus infections.

Fc receptors for IgG expressed on macrophages and NK cells are important mediators of opsonophagocytosis and Ab-dependent cell-mediated cytotoxicity. Phagocyte-mediated opsonophagocytosis is pivotal for protection against bacteria, but its importance in recovery from infection with intracellular pathogens is unclear. We have now investigated the role of opsonophagocytosis in protection against lethal influenza virus infection by using FcR gamma(-/-) mice. Absence of the FcR gamma-chain did not affect the expression of IFN-gamma and IL-10 in the lungs and spleens after intranasal immunization with an influenza subunit vaccine. Titers of serum and respiratory Abs of the IgM, IgG1, IgG2a, and IgA isotypes in FcR gamma(-/-) mice were similar to levels seen in FcR gamma(+/+) mice. Nevertheless, FcR gamma(-/-) mice were highly susceptible to influenza infection, even in the presence of anti-influenza Abs from immune FcR gamma(+/+) mice. NK cells were not necessary for the observed Ab-mediated viral clearance, but macrophages were found to be capable of actively ingesting opsonized virus particles. We conclude that Fc receptor-mediated phagocytosis plays a pivotal role in clearance of respiratory virus infections.

Animals↗

PubMed2001

IL-12-mediated increases in protection elicited by pneumococcal and meningococcal conjugate vaccines.

Interleukin-12 (IL-12) may be a beneficial adjuvant for augmenting vaccine efficacy against encapsulated bacteria such as Streptococcus pneumoniae and Neisseria meningitidis since it can stimulate production of interferon-gamma (IFN-gamma) and secretion of antibody isotypes that are efficient at mediating complement fixation and opsonophagocytosis. In this study, we demonstrate the ability of IL-12 to enhance murine antibody responses, particularly IgG2a levels, to both pneumococcal and meningococcal conjugate vaccines. Transfer of immune serum from mice immunized with the meningococcal conjugate vaccine and IL-12 resulted in increased survival times, whereas transfer of serum from mice immunized with the pneumococcal conjugate and IL-12 resulted in protection from death upon bacterial challenge. Although treatment with vaccine and IL-12 increased levels of IFN-gamma mRNA, IL-12-mediated enhancement of antibody responses still occurred in IFN-gamma(-/-) mice. The results demonstrate the effectiveness of IL-12 as an adjuvant for polysaccharide conjugate vaccines, especially the pneumococcal conjugate vaccine.

Animals↗

PubMed2001

IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection.

IgA is considered to be the principal Ab involved in defense against pathogens in the mucosal compartment. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have examined the precise role of IgA in protective anti-influenza responses after intranasal vaccination. IgA(-/-) mice immunized intranasally with soluble hemagglutinin (hemagglutinin subtype 1) and neuraminidase (neuraminidase subtype 1) vaccine in the absence of adjuvant were found to be more susceptible to influenza virus infection than IgA(+/+) mice (13 vs 75% survival after virus challenge). Inclusion of IL-12 during immunization restored the protective efficacy of the vaccine to that seen in IgA(+/+) animals. IgA(-/-) mice had no detectable IgA expression, but displayed enhanced serum and pulmonary IgM and IgG Ab levels after IL-12 treatment. Assessment of T cell function revealed markedly depressed splenic lymphoproliferative responses to PHA in IgA(-/-) animals compared with IgA(+/+) mice. Furthermore, IgA(-/-) animals displayed impaired T cell priming to the H1N1 subunit vaccine, with concomitant reduction in recall memory responses due to a defect in APC function. Collectively, these results provide evidence that a major role of IgA is to facilitate presentation of Ag to mucosal T cells. IL-12 treatment can overcome IgA deficiency by providing adequate T cell priming during vaccination.

Administration, Intranasal↗

PubMed2001

Frequency- and duration-dependent effects of cyclic pressure on select bone cell functions.

The present study demonstrated unique correlations between characteristic parameters of mechanical loading and osteoblast functions. Specifically, osteoblast proliferation was dependent on the frequency and on the duration of the applied cyclic pressure stimulus: decreased cell proliferation was only observed when these cells were exposed to cyclic pressure at 1.0-Hz (but not at 0.25-Hz) frequency for 1 h (but not for 20 min) daily for 5 days. In contrast, endothelial cells were not responsive to cyclic pressure, whereas fibroblast proliferation increased under similar test conditions. Most important, cyclic pressure affected various osteoblast genes differently: exposure of osteoblasts to cyclic pressure (at 1.0-Hz frequency for 1 h daily) resulted in enhanced transcription and translation of alkaline phosphatase after 5 days; the same mechanical stimulus, however, did not affect osteopontin mRNA expression during the same time periods. These findings provide cellular and molecular level information, which is not only important in elucidating the correlation between mechanical loading and bone homeostasis, but can be useful in development of new technology in skeletal tissue engineering.

Animals↗

PubMed2001

Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a major pathogen in humans that enters the host primarily through the respiratory tract. Targeting mucosal surfaces directly may therefore be an optimal approach for vaccination to prevent bacterial colonization and invasive disease. We have previously demonstrated the effectiveness of interleukin-12 (IL-12) delivered intransally (i.n.) as an antiviral respiratory adjuvant. In this study, we examined the effects of i.n. IL-12 treatment on induction of protective humoral immunity against S. pneumoniae. Immunization i.n. with pneumococcal surface protein A (PspA) and IL-12 resulted in enhanced lung IL-10 mRNA expression and marked augmentation of respiratory and systemic immunoglobulin G1 (IgG1), IgG2a, and IgA antibody levels compared to those in animals receiving PspA alone. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Flow cytometric analysis revealed a threefold increase in antibody-mediated, complement-independent opsonic activity in the sera of PspA- and IL-12-treated animals, which was mainly contributed by IgG2a and, to a lesser extent, IgA. Passive transfer of these immune sera conferred complete protection from death upon systemic pneumococcal challenge. These findings demonstrate the effectiveness of combining PspA and IL-12 at mucosal sites to achieve optimal antibody-mediated opsonization and killing of S. pneumoniae.

Adjuvants, Immunologic↗

PubMed2001

Fcgamma-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-alpha levels.

The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-alpha (TNF-alpha) levels. The present study evaluated the role of Fcgamma-receptor (FcgammaR) signaling and complement activation in the effect of EIgG on the TNF-alpha response to LPS. The role of FcgammaR was determined using FcR gamma-chain knockout mice that lack functional FcgammaRI and FcgammaRIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-alpha response to LPS, whereas there was no augmentation in the FcgammaR-deficient animals. Heat-damaged erythrocytes also augmented the TNF-alpha response to LPS. This effect was absent in FcgammaR-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-alpha levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that FcgammaR signaling primarily mediates the augmented serum TNF-alpha response to LPS caused by EIgG.

Animals↗

PubMed2000

Interleukin-12 enhances clinical experimental autoimmune myasthenia gravis in susceptible but not resistant mice.

Experimental autoimmune myasthenia gravis (EAMG) is induced by antibodies against the nicotinic acetylcholine receptor (AChR). Studies indicate a role for interferon-gamma (IFN-gamma) in EAMG. We examined the effect of IL-12, a major inducer of IFN-gamma production, on EAMG in C57BL/6 mice. Five doses of IL-12 accelerated and enhanced clinical disease in AChR-immunized mice. Control B6 mice, IFN-gamma gene-knockout mice, and EAMG-resistant bm12 mice showed no enhancement of disease. Shifting to a Th1-type antibody isotype distribution was insufficient to cause disease. Other factors, such as direct effects of Th1 cytokines on muscle tissue, may be involved in EAMG susceptibility.

Animals↗

PubMed2000

Neonatal administration of IL-12 enhances the protective efficacy of antiviral vaccines.

Neonates are highly susceptible to infectious agents and are known to display polarized expression of Th2-like cytokines and Abs. This neonatal immune bias has important implications for the development of vaccine strategies, particularly against viral infections. We now report that coadministration of IL-12 and an influenza subunit vaccine at birth enhances the protective efficacy of antiviral vaccination. Immunization and treatment with IL-12 within 24 h of birth resulted in elevated expression of IFN-gamma, IL-10, and IL-15 mRNA in the spleens of newborn mice compared with animals exposed to vaccine only. In addition, these animals showed dramatic increases in IFN-gamma-, IL-2-, and IL-4-secreting cells, and in IgG2a Ab levels upon adult challenge compared with mice primed with vaccine alone. Most importantly, animals vaccinated and simultaneously treated with IL-12 at birth displayed enhanced survival after lethal challenge with infectious influenza virus as adults compared with infected animals that had been primed with vaccine alone. This augmented protection required B cells and could be transferred to naive mice by immune serum. Collectively, these results provide evidence that administration of IL-12 to neonates induces a Th1-like response in newborns and elicits protective antiviral immune memory.

Adjuvants, Immunologic↗

PubMed2000

A pivotal role for interferon-gamma in protection against group A streptococcal skin infection.

Administration of exogenous recombinant interleukin-12 (rIL-12) either prophylactically or therapeutically provides significant protection against lethal group A streptococcal skin infection in a mouse model. Treatment of mice with rIL-12 before infection with group A streptococci induced expression of interferon-gamma (IFN-gamma) at the infection site. In vivo neutralization of IFN-gamma increased susceptibility to lethal infection and completely abrogated the protective effects of rIL-12. IFN-gamma knockout mice were also more susceptible to lethal infection. Although IL-12 treatment provided protection, higher doses induced significantly elevated levels of IFN-gamma transcription that were associated with increased susceptibility to lethal infection. These results support the hypothesis that IFN-gamma at the infection site is critical for protection but suggest that increased systemic levels are detrimental to survival after infection with group A streptococci.

Animals↗

PubMed1999

Modulation of mucosal and systemic immunity by intranasal interleukin 12 delivery.

Interleukin-12 (IL-12) is an important mediator of both cell-mediated and humoral immunity. We have now utilized a noninvasive intranasal (i.n.) delivery system to evaluate the ability of IL-12 to modulate both mucosal and systemic components of the immune system. Mice immunized i.n. with dinitrophenyl conjugated to ovalbumin (DNP-OVA) in combination with cholera toxin B subunit and IL-12 were found to have elevated levels of IFN-gamma and IL-10 mRNA transcripts in both lungs and spleens compared with mice not receiving IL-12. In addition, expression of lung IL-5 mRNA was inhibited. Analysis of bronchoalveolar lavage fluid after IL-12 treatment revealed a significant increase in IgG2a and unaltered IgG1 and IgA anti-OVA antibody levels. Serum IgG2a, IgG2b and IgG3 anti-DNP antibody levels were significantly increased by IL-12 given i.n., while serum IgG1 antibody levels were suppressed, results that are similar to those seen after systemic antigen plus IL-12 administration. Delivery of IL-12 i.n. also enhanced faecal IgG2a and suppressed IgA levels, in contrast to parenteral treatment which increased both faecal IgG2a and IgA antibody expression. These results provide evidence that i.n. IL-12 treatment can effectively modulate antigen-specific immune responses and enhance immunization strategies for mucosal vaccines.

Adjuvants, Immunologic↗

PubMed1999

IL-12 is a potent neonatal vaccine adjuvant.

Neonatal animals show generally poor responsiveness to foreign antigens and are known to display polarized expression of Th2-like cytokines and antibody responses. We now report that newborn mice display a reduction in peripheral expression of the Th1-inducing cytokine, IL-12. Attempts to overcome this decrease by immunization and treatment with IL-12 within 24 h of birth resulted in elevated levels of IFN-gamma and IL-10 mRNA in the spleens of mice compared to animals exposed to antigen only. Moreover, such animals showed dramatic enhancement of IgG2a and IgG2b antibody levels upon adult challenge compared to mice primed with antigen alone. These effects appeared to be due to induction of neonatal B cell memory. IgG1 antibody levels, a measure of Th2 activity, were unaffected or even somewhat enhanced by neonatal IL-12 treatment. Taken together, these results provide evidence that IL-12 administration induces a Th1-like cytokine response in newborns and causes priming for heightened memory antibody responses in vivo. Our findings suggest the use of IL-12 as a vaccine adjuvant in neonates for inducing protection against common childhood pathogens.

Adjuvants, Immunologic↗

PubMed1999

Intranasal interleukin-12 is a powerful adjuvant for protective mucosal immunity.

The use of interleukin (IL)-12 as a new vaccine adjuvant for stimulating protective antiviral mucosal immunity has been examined. Mice were immunized intranasally (in) with an influenza vaccine consisting of soluble hemagglutinin (H1) and neuraminidase (N1) plus IL-12. This treatment resulted in elevated levels of lung and splenic interferon-gamma and IL-10 mRNA. Total and IgG2a anti-H1N1 antibody levels in serum were significantly elevated, as were total, IgG1, IgG2a, and secretory IgA antibody levels in bronchoalveolar lavage (BAL) fluids compared with animals receiving vaccine alone. Mice immunized in with vaccine and IL-12 also exhibited decreased weight loss and dramatically enhanced survival after lethal challenge with infectious influenza virus. Protection was dependent upon the presence of B cells and could be transferred to naive mice by inoculation of either serum or BAL fluid from IL-12-treated mice. These findings show for the first time that soluble IL-12 delivered in serves as a powerful respiratory adjuvant for protective antiviral immunity.

Adjuvants, Immunologic↗

PubMed1998

IL-12 enhances antibody responses to T-independent polysaccharide vaccines in the absence of T and NK cells.

Polysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-gamma knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-gamma. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.

Adjuvants, Immunologic↗

PubMed1998

Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments.

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 microg/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomerdimer equilibrium mixture after 30 days at 4 degrees C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr approximately 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-IG10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.

Amino Acid Sequence↗

PubMed1997

Interleukin-12 acts as an adjuvant for humoral immunity through interferon-gamma-dependent and -independent mechanisms.

Interleukin-12 (IL-12) is a pivotal cytokine that has dramatic effects on cell-mediated immunity. It is now becoming increasingly recognized that IL-12 also strongly controls humoral immunity. We have investigated the mechanism by which IL-12 induces alterations in antibody isotype expression by determining the influence of IL-12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL-12 plus lipopolysaccharide or anti-CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL-12. IL-12 treatment of spleen cell cultures induced expression of gamma2a germ-line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL-12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon-gamma (IFN-gamma). However, in the absence of IFN-gamma, IL-12 still had significant effects on Ig secretion. Specifically, IL-12 enhanced IgG1 and IgG2b anti-DNP antibody levels in mice containing specific disruptions in the IFN-gamma gene. Our results suggest that IL-12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN-gamma which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL-12 has direct or indirect effects on B cells that are independent of IFN-gamma. The IFN-gamma-independent effects may include enhancement of Ig expression by post-switched cells.

Adjuvants, Immunologic↗

PubMed1997

Primary human immune response to Neisseria meningitidis serogroup C in interleukin-12-treated severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes.

Lack of primary immune response in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL) has limited the applicability of this model. Use of human cytokines, in particular interleukin (IL)-12, was studied in the hu-PBL-SCID model. SCID mice were treated with IL-12 and reconstituted with hu-PBL in T replacement factor. The hu-PBL-SCID mice were immunized with serogroup C meningococcal polysaccharide (MCPS). The MCPS-specific antibody response was determined by ELISA. Thirteen of the 15 immunized, IL-12-treated hu-PBL-SCID mice demonstrated a primary human antibody response to MCPS ranging from 0.25 to 3.3 microg/mL, while no MCPS-specific antibody response was detectable in the 18 controls. Expression of cross-reactive idiotypic markers found on human anti-MCPS antibodies in the immunized hu-PBL-SCID mice was similar to that observed in immunized volunteers.

Adult↗