Biologic Drugs for the Treatment of Noninfectious Uveitis.

ABSTRACT: The management of noninfectious uveitis is constantly evolving. A new "biologic era" in treatment began after the effectiveness of tumor necrosis factor-alpha blocking drugs was demonstrated in rheumatologic inflammatory diseases. The goal of specific immunomodulation with a biologic drug is to target inflammation at the molecular level with a low rate of serious adverse events. The purpose of this review is to summarize current knowledge of biologic drugs in the treatment of noninfectious uveitis by describing clinical studies and recent pharmacological developments.

Perspectives on Feeding and Nutrition.

Many palliative care patients have reduced oral intake during their illness. Managing inadequate intake through appetite stimulation and/or artificial hydration and nutrition poses many clinical, ethical, and logistical dilemmas. This article aids the health care team in making appropriate recommendations regarding assisted nutrition and hydration for palliative care and terminal patients. It provides a decision-making framework, including an ethical approach to determining appropriate use of assisted feeding and hydration methods in pets at the end of life. It also summarizes various clinical and logistical approaches to treating decreased food/water consumption, including potential benefits and burdens, should intervention be deemed appropriate.

Regenerative Endodontics: Burning Questions.

Pulp regeneration and its clinical translation into regenerative endodontic procedures are receiving increasing research attention, leading to significant growth of the published scientific and clinical literature within these areas. Development of research strategies, which consider patient-, clinician-, and scientist-based outcomes, will allow greater focus on key research questions driving more rapid clinical translation. Three key areas of focus for these research questions should include cells, signaling, and infection/inflammation. A translational pathway is envisaged in which clinical approaches are increasingly refined to provide regenerative endodontic protocols that are based on a robust understanding of the physiological processes and events responsible for the normal secretion, structure, and biological behavior of pulpal tissue.

Dental Pulp Defence and Repair Mechanisms in Dental Caries.

Dental caries is a chronic infectious disease resulting from the penetration of oral bacteria into the enamel and dentin. Microorganisms subsequently trigger inflammatory responses in the dental pulp. These events can lead to pulp healing if the infection is not too severe following the removal of diseased enamel and dentin tissues and clinical restoration of the tooth. However, chronic inflammation often persists in the pulp despite treatment, inducing permanent loss of normal tissue and reducing innate repair capacities. For complete tooth healing the formation of a reactionary/reparative dentin barrier to distance and protect the pulp from infectious agents and restorative materials is required. Clinical and in vitro experimental data clearly indicate that dentin barrier formation only occurs when pulp inflammation and infection are minimised, thus enabling reestablishment of tissue homeostasis and health. Therefore, promoting the resolution of pulp inflammation may provide a valuable therapeutic opportunity to ensure the sustainability of dental treatments. This paper focusses on key cellular and molecular mechanisms involved in pulp responses to bacteria and in the pulpal transition between caries-induced inflammation and dentinogenic-based repair. We report, using selected examples, different strategies potentially used by odontoblasts and specialized immune cells to combat dentin-invading bacteria in vivo.

Isolation of adipose and bone marrow mesenchymal stem cells using CD29 and CD90 modifies their capacity for osteogenic and adipogenic differentiation.

Mesenchymal stem cells isolated from rats are frequently used for tissue engineering research. However, considerable differences have been identified between rat mesenchymal stem cells and those derived from humans, and no defined panel of markers currently exists for the isolation of these cells. The aim of this study was to examine the effects of cell sorting for CD29(+)/CD90(+) cells from rat adipose and bone marrow tissues on their differentiation and expression of stem cell-associated genes. Flow cytometry showed 66% and 78% CD29(+)/CD90(+) positivity within passage 1 of adipose and bone marrow cultures, respectively. CD29(+)/CD90(+) cells showed a reduction in both osteogenic and adipogenic differentiation when compared with unsorted cells, as determined by alizarin red and Oil Red-O staining, respectively. These findings could not entirely be explained by fluorescence-activated cell sorting-induced cell injury as sort recovery was only modestly affected in adipose-derived cells. Maintaining cells in fluorescence-activated cell sorting buffer did not affect adipose-derived cell viability, but a significant (p < 0.05) reduction was found in bone marrow-derived cell viability. Additionally, CD29(+)/CD90(+) selection was associated with a significant decrease in the expression of Lin28, Sox2, Nanog and CD73 in adipose-derived cell cultures, whereas differences in stem cell-associated gene expression were not observed in sorted bone marrow-derived cell cultures. In summary, this study demonstrated that fluorescence-activated cell sorting had differential effects on adipose-derived cells and bone marrow-derived cells, and both CD29(+)/CD90(+) cells displayed a significantly reduced capacity for osteogenic/adipogenic differentiation. In conclusion, we identify that maintaining heterogeneity within the mesenchymal stem cell population may be important for optimal differentiation.

Dentin matrix proteins (DMPs) enhance differentiation of BMMSCs via ERK and P38 MAPK pathways.

Dentin, the predominant mineralized tissue of the tooth, comprises an extracellular matrix of collagen and a heterogeneous mixture of non-collagenous components, many of which have cellular signaling properties. These properties may be important in signaling stem cell involvement in tissue regeneration following injury and the present study investigates their morphogenic effects on differentiation of Bone Marrow Stromal Stem Cells (BMMSCs) in vitro. Non-collagenous dentin matrix proteins (DMPs) were isolated from healthy human teeth and their effects on BMMSCs behavior examined during in vitro culture. In vitro, DMPs enhanced alkaline phosphatase activity and mineralization in BMMSCs cultures as well as increasing the expression of dentinogenic and osteogenic differentiation markers (including runt-related transcription factor 2, osterix, bone sialoprotein, dentin sialophosphoprotein and osteocalcin) at both transcript and protein levels, with 10 µg/mL DMPs being the optimal stimulatory concentration. Expression of phosphor-ERK/phosphor-P38 in BMMSCs was up-regulated by DMPs and, in the presence of the ERK1/2- and p38-specific inhibitors, the differentiation of BMMSCs was inhibited. These data indicate that DMPs promote the dentinogenic/osteogenic differentiation of BMMSCs via the ERK/p38 MAPK pathways.

Lipopolysaccharide enhances Wnt5a expression through toll-like receptor 4, myeloid differentiating factor 88, phosphatidylinositol 3-OH kinase/AKT and nuclear factor kappa B pathways in human dental pulp stem cells.

INTRODUCTION: Lipopolysaccharide (LPS) has been implicated in mesenchymal stem cell differentiation processes. Wnt5a, one of the "non-canonical" Wnt family members, is important in signaling stem cell differentiation and in the inflammatory responses of immune cells. Here we studied whether LPS can regulate the expression of Wnt5a in human dental pulp stem cells (hDPSCs) and investigated the intracellular signaling pathways activated by LPS. METHODS: Wnt5a mRNA and protein expression changes in hDPSCs were investigated by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay. In addition, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and luciferase activity assays were used to determine whether toll-like receptor 4 (TLR4), myeloid differentiating factor 88 (MyD88), nuclear factor kappa B (NF-kB), or the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathways are involved in LPS-induced Wnt5a expression. The activation of PI3K and AKT in hDPSCs was measured by Western blot analysis. RESULTS: Wnt5a mRNA and protein expression was rapidly increased in response to LPS in a time- and dose-dependent manner. LPS-induced Wnt5a expression was effectively attenuated by administration of a TLR4 neutralizing antibody, MyD88 inhibitory peptide, PI3-kinase inhibitors (LY294002 and wortmannin), an AKT inhibitor, or NF-κB inhibitor (pyrrolidine dithiocarbamate), IκBa phosphorylation inhibitor (Bay 117082), or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). Treatment of hDPSCs with LPS activated PI3-kinase (p85) and AKT signaling in a time-dependent manner. Moreover, LPS-mediated increases in κB-luciferase activity were diminished by the overexpression of dominant negative mutants of TLR4, MyD88, p85, AKT, and IκBa. CONCLUSIONS: These results demonstrated that LPS-induced Wnt5a expression was mediated through the TLR4/MyD88/PI3-kinase/AKT pathway, which then initiated NF-κB activation in hDPSCs.

Human stem cells from the apical papilla response to bacterial lipopolysaccharide exposure and anti-inflammatory effects of nuclear factor I C.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) are important for tooth root development and may be candidates for regenerative endodontic procedures involving immature teeth. The potential use of SCAPs for clinical applications requires a better understanding of their responses to bacterial challenge. We have investigated the effects of exposure of these cells to lipopolysaccharide (LPS). Inflammatory responses arising from bacterial challenges can constrain postinjury tissue regeneration and the effects of nuclear factor I C (NFIC), which plays a critical role in tooth root development. NFIC has been explored for its anti-inflammatory action in the context of endodontic treatment of immature teeth where continued root development is an important outcome. METHODS: SCAPs were exposed to LPS, and the expression of Toll-like receptor-4 (TLR4), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF-α) were assessed by real-time polymerase chain reaction (RT-PCR). The pLenti6.3/v5-NFIC plasmid encoding the full-length NFIC or NFIC silencing by si-RNA (small interfering RNA) in SCAPs were measured by Western blotting or RT-PCR; the effects of NFIC on IL-6, IL-8, and TNF-α were analyzed by RT-PCR. The protein levels were subsequently measured by enzyme-linked immunoassay. RESULTS: LPS induced the synthesis of IL-6, IL-8, and TNF-α in SCAPs in a time-dependent manner. Pretreatment with a TLR4 inhibitor significantly inhibited LPS-induced IL-6, IL-8, and TNF-α expression. Knockdown of NFIC increased the expression of IL-6, IL-8, and TNF-α, whereas the overexpression of NFIC resulted in the suppression of the inflammatory response stimulated by 1 µg/mL LPS, especially for IL-8. Together, these data show that LPS is recognized by the transmembranous receptor TLR4 to mediate inflammatory responses in SCAPs and NFIC overexpression can suppress LPS-initiated innate immune responses. CONCLUSIONS: The anti-inflammatory effects of NFIC overexpression provide a valuable target to dampen inflammatory responses in the infected pulp to allow tissue regeneration to occur.

Differentiation of BMMSCs into odontoblast-like cells induced by natural dentine matrix.

OBJECTIVE: To assess the odontogenic potential of bone marrow mesenchymal stem cells (BMMSCs) to differentiate into odontoblast-like cells under the morphogenetic influence of dentine matrix as a possible basis for new stem cell-mediated therapeutic approaches to pulp diseases. DESIGN: BMMSCs were harvested from the whole bone marrow and cells at passages 3-5 were used for subsequent experiments. For in vitro studies, 1×10(4) cells were seeded on the surface of dentine slabs and co-cultured for 2 weeks in 24-well plates, then fixed, decalcified, embedded in paraffin and serial sections were processed for analyses. Haematoxylin-eosin (HE) staining was used for the morphological analysis of BMMSCs on the dentine slabs. The protein expression of dentine sialoprotein (DSP) in co-cultured BMMSCs was detected by immunohistochemical (IHC) staining. For in vivo studies, 5×10(6) cells were collected as cell pellets, seeded onto dentine slices and transplanted into renal capsules for 6 weeks. Histological analyses of harvested tissues were performed as described for the in vitro studies. Total RNA and protein were extracted from harvested tissues and Dspp/DSP expression was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. RESULTS: After 2 weeks of co-culture with dentine slabs, BMMSCs demonstrated good viability in terms of morphological appearance and some showed polarization and extension of their cytoplasmic processes into dentine tubules with DSP expression. In vivo study demonstrated similar morphological changes and DSP expression in cells adjacent to dentine. RT-PCR and Western blot also demonstrated that the expression of Dspp/DSP in the co-cultured BMMSCs groups was higher than in the control groups. CONCLUSION: Dentine matrix can signal morphogenic induction of differentiation of BMMSCs into odontoblast-like cells in vivo and in vitro.

Hepatocyte growth factor is sequestered in dentine matrix and promotes regeneration-associated events in dental pulp cells.

Extracellular matrix of dentine contains a rich cocktail of soluble cytokines and growth factors which mediate wound repair of the dentine-pulp complex. Hepatocyte growth factor (HGF) is a mesenchyme derived growth factor regulating a broad range of physiological processes including tissue development and regeneration. In this study, we have investigated the sequestration of HGF in the dentine matrix and analysed its action as a chemokine in the induction of differentiation and mineral deposition in pulp derived cells in vitro. Using ELISA, the presence of HGF was demonstrated in solubilised fractions of dentine matrix released by the therapeutic pulp repair materials of white and grey mineral trioxide aggregate. HGF was shown to be a chemo-attractant for primary rat dental pulp cells (RDPCs) in transwell assays highlighting its potential in progenitor cell recruitment during dentine-pulp tissue repair. Transcription factors Osterix and Runx2, and genes encoding for Osteopontin and Osteocalcin, were up-regulated in HGF-exposed RDPC cultures compared with controls. Adenoviral-mediated expression of HGF in RDPCs or exposure to recombinant HGF induced mineral secretion in RDPCs which was significantly greater than controls. The receptor of HGF, c-Met was also detected within human dental pulp indicating the potential for HGF released from dentine matrix to contribute to cellular signalling events following tissue injury. Combined, these data suggest that HGF is important in the repair of the dentine-pulp complex potentially participating in several aspects of wound healing.

Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.

INTRODUCTION: Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses. METHODS: VPA (0.125-5 mmol/L) and TSA (12.5-400 nmol/L) were applied to a pulp-derived cell population and compared with unsupplemented controls. Cell proliferation and viability were evaluated by trypan blue staining and cell counting, whereas cell cycle and apoptosis were analyzed by immunocytochemical staining with antibodies for p53, phosphorylated p53, Bcl-2 homologous antagonist/killer (BAK), caspase-3 and p21(WAF1/CIP), and DNA staining with Hoechst 33342. For mineralization analysis, cultures were stained with Alizarin red and quantified spectrophotometrically. Relative gene expression levels of mineralization associated markers were analyzed using reverse-transcriptase polymerase chain reaction. One-way analysis of variance and Tukey post hoc tests were applied to the data (P < .05). RESULTS: VPA and TSA reduced cell proliferation dose dependently with no significant effect on cell viability except at 400 nmol/L TSA. The transcription factor p21(WAF1/CIP) was significantly increased at the highest concentration of TSA but not VPA. Significant increases (P < .05) in the apoptosis marker protein active caspase-3 and cell cycle alterations were only evident at the maximum concentrations of TSA/VPA, whereas HDACi-induced mineralization per cell was stimulated dose dependently with a significant increase in the expression of the dentinogenic-associated transcript, dentine matrix protein-1. CONCLUSIONS: These results indicate that HDACis are capable of epigenetically modulating pulp cell behavior, signifying their therapeutic potential for augmenting biomaterials, and stimulating regenerative responses in the damaged pulp.

Cumulative mechanisms of lymphoid tissue fibrosis and T cell depletion in HIV-1 and SIV infections.

The hallmark of HIV-1 and SIV infections is CD4(+) T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-ß1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-ß, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.

Effects of morphogen and scaffold porogen on the differentiation of dental pulp stem cells.

INTRODUCTION: Dental pulp tissue engineering is an emerging field that can potentially have a major impact on oral health. However, the source of morphogens required for stem cell differentiation into odontoblasts and the scaffold characteristics that are more conducive to odontoblastic differentiation are still unclear. This study investigated the effect of dentin and scaffold porogen on the differentiation of human dental pulp stem cells (DPSCs) into odontoblasts. METHODS: Poly-L-lactic acid (PLLA) scaffolds were prepared in pulp chambers of extracted human third molars using salt crystals or gelatin spheres as porogen. DPSCs seeded in tooth slice/scaffolds or control scaffolds (without tooth slice) were either cultured in vitro or implanted subcutaneously in immunodefficient mice. RESULTS: DPSCs seeded in tooth slice/scaffolds but not in control scaffolds expressed putative odontoblastic markers (DMP-1, DSPP, and MEPE) in vitro and in vivo. DPSCs seeded in tooth/slice scaffolds presented lower proliferation rates than in control scaffolds between 7 and 21 days (p < 0.05). DPSCs seeded in tooth slice/scaffolds and transplanted into mice generated a tissue with morphological characteristics similar to those of human dental pulps. Scaffolds generated with gelatin or salt porogen resulted in similar DPSC proliferation. The porogen type had a relatively modest impact on the expression of the markers of odontoblastic differentiation. CONCLUSIONS: Collectively, this work shows that dentin-related morphogens are important for the differentiation of DPSC into odontoblasts and for the engineering of dental pulp-like tissues and suggest that environmental cues influence DPSC behavior and differentiation potential.

The MAP kinase pathway is involved in odontoblast stimulation via p38 phosphorylation.

INTRODUCTION: We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. METHODS: We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. RESULTS: Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. CONCLUSIONS: Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis.

Adrenomedullin is expressed during rodent dental tissue development and promotes cell growth and mineralization.

BACKGROUND INFORMATION: ADM (adrenomedullin) has pleiotropic effects, including regulation of inflammation, infection, angiogenesis, mineralized-tissue formation and development. Recently, we demonstrated up-regulation of the ADM transcript in diseased pulpal tissue while the protein is sequestered within the dentine extracellular matrix during dentinogenesis. The present study aimed to characterize ADM localization during rodent dental tissue development and determine its potential effects on dental cells. Finally, we sought to profile ADM transcript levels in adult organs and tissues to compare its expression in teeth relative to other tissues. RESULTS: Immunohistochemical analysis of developmental rat oral tissues indicated that, at E16 (embryonic day 16), ADM was present in dental epithelium and, by E18, ADM localized to the dental papilla and inner and outer dental epithelia. By E20, ADM was detected in secretory odontoblasts and ameloblasts and exhibited a similar expression profile to that of the key dentinogenesis signalling molecule, TGF-beta1 (transforming growth factor-beta1). Cell growth analysis in the dental MDPC-23, OD-21 and control 3T3 cell lines exposed to ADM (range 10(-15)-10(-7) M) together with EDTA-extracted DMPs (dentine matrix proteins) (range 0.00001-1000 mg/ml) containing comparable concentrations of ADM demonstrated that ADM stimulated a biphasic response in dental cell growth, comparable with that of DMPs, with peak stimulation observed at approximately 10(-11) M. For mineralization analysis, cell lines were exposed to combinations of 50 microg/ml ascorbic acid, 10 mM beta-G (beta-glycerophosphate), 10(-8) M DEX (dexamethasone) and ADM (range 10(-15)-10(-7) M). The results demonstrated that ADM could substitute for DEX to stimulate mineralization. Postnatally, multiple tissue expression profiling indicated abundant ADM levels in tongue and pulpal tissues. CONCLUSIONS: During oral and dental tissue development ADM initially localizes to epithelial tissue, whereas during later stages it is present in mineralized secreting cells, including odontoblasts. ADM may regulate proliferation and mineralization processes during development, whereas, in adulthood, it may be important for maintaining dental tissue homoeostasis.

Tooth slice-based models for the study of human dental pulp angiogenesis.

Treatment of avulsed young permanent teeth aims to revascularize the dental pulp. The study of therapeutic strategies for avulsed teeth has been hindered by the scarcity of experimental models. The purpose of this work is to characterize two model systems to study dental pulp revascularization. Tooth slices from human third molars were prepared with a sterile diamond saw. The tooth slices were cultured in vitro for up to 7 days. Immunohistochemical staining with Factor VIII showed an increase in microvascular density in pulps treated with 50 ng/mL rhVEGF(165) as compared with untreated controls (p < 0.05). Alternatively, tooth slices were prepared and immediately implanted subcutaneously in immunodeficient mice. Pulp vitality and vascularization were confirmed by histological analysis and terminal deoxynucleotide transferase dUTP nick end labeling assays 7 days after implantation. The models presented here may be valuable in the assessment of angiogenesis-based therapeutic strategies for the dental pulp.

Chain-terminating dinucleoside tetraphosphates are substrates for DNA polymerization by human immunodeficiency virus type 1 reverse transcriptase with increased activity against thymidine analogue-resistant mutants.

Nucleoside reverse transcriptase inhibitors are an important class of drugs for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Resistance to these drugs is often the result of mutations that increase the transfer of chain-terminating nucleotides from blocked DNA termini to a nucleoside triphosphate acceptor, resulting in the generation of an unblocked DNA chain and synthesis of a dinucleoside polyphosphate containing the chain-terminating deoxynucleoside triphosphate analogue. We have synthesized and purified several dinucleoside tetraphosphates (ddAp4ddA, ddCp4ddC, ddGp4ddG, ddTp4ddT, Ap4ddG, 2'(3')-O-(N-methylanthraniloyl)-Ap4ddG, and AppNHppddG) and show that these compounds can serve as substrates for DNA chain elongation and termination resulting in inhibition of DNA synthesis. Thymidine analogue-resistant mutants of reverse transcriptase are up to 120-fold more sensitive to inhibition by these compounds than is wild-type enzyme. Drugs based on the dinucleoside tetraphosphate structure could delay or prevent the emergence of mutants with enhanced primer unblocking activity. In addition, such drugs could suppress the resistance phenotype of mutant HIV-1 that is present in individuals infected with resistant virus.

In vitro differentiation and mineralization of human dental pulp cells induced by dentin extract.

In this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], beta-glycerophosphate [beta-GP]) and MS + DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS + DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, beta-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations.

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3'-azido-3'-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [(32)P]ddAMP-terminated DNA primer/template gave rise to (32)P-labeled adenosine 2',3'-dideoxyadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)ddA), ddATP, Gp(4)ddA, and Ap(3)ddA, corresponding to the transfer of [(32)P]ddAMP to ATP, PP(i), GTP, and ADP, respectively. Incubation with [(32)P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous (32)P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PP(i) levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4(+) or CD8(+) T cells, while PP(i) was present at 7 to 15 microM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4(+) or CD8(+) T cells contained 1.4 to 2.7 mM ATP and 55 to 79 microM PP(i). These cellular PP(i) concentrations are lower than previously reported; nonetheless, the PP(i)-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PP(i)-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.

Passive transfer of Sjogren's syndrome IgG produces the pathophysiology of overactive bladder.

OBJECTIVE: The presence, in patients with primary and secondary Sjogren's syndrome (SS), of autoantibodies that acutely inhibit M(3) muscarinic receptor (M3R)-mediated bladder contractions is difficult to reconcile with the fact that symptoms of detrusor overactivity and other features of cholinergic hyperresponsiveness occur in this disease. This study was undertaken to examine the in vivo effects of these autoantibodies on bladder function by examining bladder responsiveness and compliance following passive transfer of patient IgG to mice. METHODS: Contractile responses of isolated bladder strips both to the muscarinic agonist carbachol and to electrically evoked acetylcholine release were measured 48 hours after injection of mice with patient or control IgG. A whole bladder assay with intact neuronal pathways was developed to assess bladder wall compliance on filling cystometry. Expression of M3R in bladders from IgG-injected mice was assessed by immunohistochemistry. RESULTS: Passive transfer of SS IgG with inhibitory anti-M3R activity produced a paradoxical increase in contractile responses of detrusor strips to cholinergic stimulation. Cystometry of whole bladders revealed a corresponding decrease in bladder wall compliance and phasic detrusor contractions upon filling, replicating the urodynamic features of an overactive bladder. The features of cholinergic hyperresponsiveness were associated with increased postsynaptic M3R expression and were reproduced by injecting mice with a rabbit antibody against the second extracellular loop of M3R. CONCLUSION: These findings are consistent with the notion that there is initial inhibition of parasympathetic neurotransmission by antagonistic autoantibodies to M3R, which produces a compensatory increase in M3R expression in vivo. The enhanced cholinergic responses during bladder distention result in detrusor overactivity. We conclude that the overactive bladder associated with SS is an autoantibody-mediated disorder of the autonomic nervous system, which may be part of a wider spectrum of cholinergic hyperresponsiveness.