Characterisation of miRNA Expression in Dental Pulp Cells during Epigenetically-Driven Reparative Processes.

Within regenerative endodontics, exciting opportunities exist for the development of next-generation targeted biomaterials that harness epigenetic machinery, including microRNAs (miRNAs), histone acetylation, and DNA methylation, which are used to control pulpitis and to stimulate repair. Although histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) induce mineralisation in dental pulp cell (DPC) populations, their interaction with miRNAs during DPC mineralisation is not known. Here, small RNA sequencing and bioinformatic analysis were used to establish a miRNA expression profile for mineralising DPCs in culture. Additionally, the effects of a HDACi, suberoylanilide hydroxamic acid (SAHA), and a DNMTi, 5-aza-2'-deoxycytidine (5-AZA-CdR), on miRNA expression, as well as DPC mineralisation and proliferation, were analysed. Both inhibitors increased mineralisation. However, they reduced cell growth. Epigenetically-enhanced mineralisation was accompanied by widespread changes in miRNA expression. Bioinformatic analysis identified many differentially expressed mature miRNAs that were suggested to have roles in mineralisation and stem cell differentiation, including regulation of the Wnt and MAPK pathways. Selected candidate miRNAs were demonstrated by qRT-PCR to be differentially regulated at various time points in mineralising DPC cultures treated with SAHA or 5-AZA-CdR. These data validated the RNA sequencing analysis and highlighted an increased and dynamic interaction between miRNA and epigenetic modifiers during the DPC reparative processes.

Early chronotype with metabolic syndrome favours resting and exercise fat oxidation in relation to insulin-stimulated non-oxidative glucose disposal.

NEW FINDINGS: What is the central question of this study? Chronotype reflects differences in circadian-mediated metabolic and hormonal profiles. But, does resting and/or exercise fuel use differ in early versus late chronotype as it relates to insulin sensitivity? What are the main finding and its importance? Early chronotypes with metabolic syndrome utilized more fat during rest and exercise independent of aerobic fitness when compared with late chronotypes. Early chronotypes were also more physically active throughout the day. Greater fat use was related to non-oxidative glucose disposal. These findings suggest that early chronotypes have differences in fuel selection that associate with type 2 diabetes risk. ABSTRACT: Early chronotypes (ECs) are often insulin-sensitive, in part, due to physical activity behaviour. It is unclear, however, if chronotypes differ in resting and/or exercise fuel oxidation in relation to insulin action. Using the Morningness-Eveningness Questionnaire (MEQ), adults with metabolic syndrome (ATP III criteria) were classified as EC (MEQ = 63.7 ± 0.9, n = 24 (19F), 54.2 ± 1.2 years) or late chronotype (LC; MEQ = 47.2 ± 1.4, n = 27 (23F), 55.3 ± 1.5 years). Carbohydrate (CHO) and fat oxidation (FOX, indirect calorimetry) were determined at rest, 55% and 85% V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ , along with heart rate and rating of perceived exertion. Physical activity patterns (accelerometers), body composition (DXA) and insulin sensitivity (clamp, 40 mU/m2 /min, 90 mg/dl) with an indirect calorimetry for non-oxidative glucose disposal (NOGD) were also determined. While demographics were similar, ECs had higher V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ (P = 0.02), NOGD (P < 0.001) and resting FOX (P = 0.02) than LCs. Both groups increased CHO reliance during exercise at 55% and 85% V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ (test effect, P < 0.01) from rest, although ECs used more fat (group effect, P < 0.01). ECs had lower sedentary behaviour and more physical activity during morning/midday (both, P < 0.05). FOX at 55% V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ correlated with V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ (r = 0.425, P = 0.004) whereas FOX at 85% V ̇ O 2 max ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{max}}}$ related to NOGD (r = 0.392, P = 0.022). ECs with metabolic syndrome used more fat in relation to insulin-stimulated NOGD.

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.

A pilot study of a pharmacist-led prescribing program for final-year medical students.

BACKGROUND: Junior doctors undertake a significant amount of prescribing; however, they are not well prepared for this, and report they would like more training in their undergraduate courses. To address this we tested a pharmacist-led prescribing program for final-year medical students. METHODS: Sixteen final-year students took part in the program. The program involved students writing prescriptions and getting feedback from clinical pharmacists, undertaking prescribing and calculation tutorials, and spending time in the pharmacy department. Evaluation included a pre- and post-assessment of their confidence and skills in prescribing, and a feedback session discussing the strengths and weakness of the program, and their perceptions about the role of pharmacists. Changes in the pre- and post-assessment of confidence and skills were examined with permutation and Mann-Whitney U tests. RESULTS: There was a significant improvement in students' confidence in prescribing, and a small but consistent improvement in prescribing skills. Of note, no student prescribed inappropriately and potentially harmfully after the program. Participants were positive about the program, and indicated a better understanding about the pharmacists' role and their ability to support them as junior doctors. CONCLUSIONS: This study has shown the potential effect of a pharmacist-led prescribing program on the skills and confidence in prescribing by medical students. It provided an interprofessional teaching opportunity, preparing students for a team-based approach to patient management.

Mucosal Humoral Immune Response to SIVmac239∆nef Vaccination and Vaginal Challenge.

Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine.

An in vitro screening assay for dental stain cleaning.

BACKGROUND: The present study aimed to develop an in vitro model for stain removal from natural enamel for the assessment and comparison of oral hygiene products. METHODS: Bovine teeth (n = 8 per group) were ground/polished to provide flat enamel specimens and ferric-tannate deposits were precipitated onto the enamel surfaces. The ferric-tannate stained enamel specimens were brushed using an in vitro tooth-brushing simulator with slurries containing commercially available toothpaste products, dental abrasive particles, and sodium tripolyphosphate (STP) solutions of different concentrations. The colour of the enamel surfaces was measured using a spectrophotometer before and after stain application as well as after the brushing treatments. RESULTS: Differences in stain removal efficacy were found between the toothpastes categorised as whitening and non-whitening comprising of different types of dental abrasives (hydrated silica and alumina). A mean value of 27% for stain removal was detected for the three non-whitening toothpastes and 59% of stain removal was detected for the three whitening toothpastes after 1000 strokes. Compared with the slurry with Zeodent 113 abrasive alone, the addition of STP provided better performance for stain removal under the same brushing conditions (mean value of 62% for Zeodent 113 abrasive alone and 72% with the addition of 5% (w/w) STP after 1000 strokes). No difference was evident between the STP concentration of 5% (w/w) and 10% (w/w). CONCLUSIONS: The ferric-tannate/bovine enamel model reported here provides good stain retention, is rapidly and easily prepared, and is shown to be progressively and reproducibly sensitive to toothbrushing using different toothpastes and surfactant/chelating agent solutions. Importantly, it provides good discrimination between various oral hygiene products. The stain removal assay reported here has considerable potential to enable comparative assessments of different toothpaste types in terms of their cleaning capabilities.

The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity.

Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (valproic acid, trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 µM); while low concentrations (1 µM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14 days) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14 days. 59% of genes were down-regulated at 24 h and 36% at 14 days, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7 d, 14 days) and enzyme activity (48 h, 14 days). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. J. Cell. Physiol. 231: 798-816, 2016. © 2015 Wiley Periodicals, Inc.

Live simian immunodeficiency virus vaccine correlate of protection: immune complex-inhibitory Fc receptor interactions that reduce target cell availability.

Principles to guide design of an effective vaccine against HIV are greatly needed, particularly to protect women in the pandemic's epicenter in Africa. We have been seeking these principles by identifying correlates of the robust protection associated with SIVmac239Δnef vaccination in the SIV-rhesus macaque animal model of HIV-1 transmission to women. We identified one correlate of SIVmac239Δnef protection against vaginal challenge as a resident mucosal system for SIV-gp41 trimer Ab production and neonatal FcR-mediated concentration of these Abs on the path of virus entry to inhibit establishment of infected founder populations at the portal of entry. In this study, we identify blocking CD4(+) T cell recruitment to thereby inhibit local expansion of infected founder populations as a second correlate of protection. Virus-specific immune complex interactions with the inhibitory FcγRIIb receptor in the epithelium lining the cervix initiate expression of genes that block recruitment of target cells to fuel local expansion. Immune complex-FcγRIIb receptor interactions at mucosal frontlines to dampen the innate immune response to vaginal challenge could be a potentially general mechanism for the mucosal immune system to sense and modulate the response to a previously encountered pathogen. Designing vaccines to provide protection without eliciting these transmission-promoting innate responses could contribute to developing an effective HIV-1 vaccine.

NK cell responses to simian immunodeficiency virus vaginal exposure in naive and vaccinated rhesus macaques.

NK cell responses to HIV/SIV infection have been well studied in acute and chronic infected patients/monkeys, but little is known about NK cells during viral transmission, particularly in mucosal tissues. In this article, we report a systematic study of NK cell responses to high-dose vaginal exposure to SIVmac251 in the rhesus macaque female reproductive tract (FRT). Small numbers of NK cells were recruited into the FRT mucosa following vaginal inoculation. The influx of mucosal NK cells preceded local virus replication and peaked at 1 wk and, thus, was in an appropriate time frame to control an expanding population of infected cells at the portal of entry. However, NK cells were greatly outnumbered by recruited target cells that fuel local virus expansion and were spatially dissociated from SIV RNA+ cells at the major site of expansion of infected founder populations in the transition zone and adjoining endocervix. The number of NK cells in the FRT mucosa decreased rapidly in the second week, while the number of SIV RNA+ cells in the FRT reached its peak. Mucosal NK cells produced IFN-γ and MIP-1α/CCL3 but lacked several markers of activation and cytotoxicity, and this was correlated with inoculum-induced upregulation of the inhibitory ligand HLA-E and downregulation of the activating receptor CD122/IL-2Rß. Examination of SIVΔnef-vaccinated monkeys suggested that recruitment of NK cells to the genital mucosa was not involved in vaccine-induced protection from vaginal challenge. In summary, our results suggest that NK cells play, at most, a limited role in defenses in the FRT against vaginal challenge.

Live simian immunodeficiency virus vaccine correlate of protection: local antibody production and concentration on the path of virus entry.

We sought design principles for a vaccine to prevent HIV transmission to women by identifying correlates of protection conferred by a highly effective live attenuated SIV vaccine in the rhesus macaque animal model. We show that SIVmac239Δnef vaccination recruits plasma cells and induces ectopic lymphoid follicle formation beneath the mucosal epithelium in the rhesus macaque female reproductive tract. The plasma cells and ectopic follicles produce IgG Abs reactive with viral envelope glycoprotein gp41 trimers, and these Abs are concentrated on the path of virus entry by the neonatal FcR in cervical reserve epithelium and in vaginal epithelium. This local Ab production and delivery system correlated spatially and temporally with the maturation of local protection against high-dose pathogenic SIV vaginal challenge. Thus, designing vaccines to elicit production and concentration of Abs at mucosal frontlines could aid in the development of an effective vaccine to protect women against HIV-1.

Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro.

The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs.

LPS promote the odontoblastic differentiation of human dental pulp stem cells via MAPK signaling pathway.

Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram-negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose-dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS-mediated differentiation of hDPSCs. The inhibition of JNK and NF-κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF-κB p65, IκB-α, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) in DPSCs in a time-dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF-κB signaling.

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.

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.

Histone deacetylase inhibitors epigenetically promote reparative events in primary dental pulp cells.

Application of histone deacetylase inhibitors (HDACi) to cells epigenetically alters their chromatin structure and induces transcriptional and cellular reparative events. This study investigated the application of two HDACi, valproic acid (VPA) and trichostatin A (TSA) on the induction of repair-associated responses in primary dental pulp cell (DPC) cultures. Flow cytometry demonstrated that TSA (100 nM, 400 nM) significantly increased cell viability. Neither HDACi was cytotoxic, although cell growth analysis revealed significant anti-proliferative effects at higher concentrations for VPA (>0.5 mM) and TSA (>50 nM). While high-content-analysis demonstrated that HDACi did not significantly induce caspase-3 or p21 activity, p53-expression was increased by VPA (3 mM, 5 mM) at 48 h. HDACi-exposure induced mineralization per cell dose-dependently to a plateau level (VPA-0.125 mM and TSA-25 nM) with accompanying increases in mineralization/dentinogenic-associated gene expression at 5 days (DMP-1, BMP-2/-4, Nestin) and 10 days (DSPP, BMP-2/-4). Both HDACis, at a range of concentrations, significantly stimulated osteopontin and BMP-2 protein expression at 10 and 14 days further supporting the ability of HDACi to promote differentiation. HDACi exert different effects on primary compared with transformed DPCs and promote mineralization and differentiation events without cytotoxic effects. These novel data now highlight the potential in restorative dentistry for applying low concentrations of HDACi in vital pulp treatment.

Mtb-Selective 5-Aminomethyl Oxazolidinone Prodrugs: Robust Potency and Potential Liabilities.

Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of action─linezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.

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.

The immunosuppressive role of IL-32 in lymphatic tissue during HIV-1 infection.

One pathological hallmark of HIV-1 infection is chronic activation of the immune system, driven, in part, by increased expression of proinflammatory cytokines. The host attempts to counterbalance this prolonged immune activation through compensatory mediators of immune suppression. We recently identified a gene encoding the proinflammatory cytokine IL-32 in microarray studies of HIV-1 infection in lymphatic tissue (LT) and show in this study that increased expression of IL-32 in both gut and LT of HIV-1-infected individuals may have a heretofore unappreciated role as a mediator of immune suppression. We show that: 1) IL-32 expression is increased in CD4(+) T cells, B cells, macrophages, dendritic cells, and epithelial cells in vivo; 2) IL-32 induces the expression of immunosuppressive molecules IDO and Ig-like transcript 4 in immune cells in vitro; and 3) in vivo, IL-32-associated IDO/Ig-like transcript 4 expression in LT macrophages and gut epithelial cells decreases immune activation but also may impair host defenses, supporting productive viral replication, thereby accounting for the correlation between IL-32 levels and HIV-1 replication in LT. Thus, during HIV-1 infection, we propose that IL-32 moderates chronic immune activation to avert associated immunopathology but at the same time dampens the antiviral immune response and thus paradoxically supports HIV-1 replication and viral persistence.

Dentin matrix component solubilization by solutions of pH relevant to self-etching dental adhesives.

PURPOSE: To evaluate the potential for phosphoric acid solutions - common constituents of dental adhesive systems - of varying pH to solubilize dentin matrix components (DMCs) from human dentin. MATERIALS AND METHODS: Human dentin chips were ground under liquid nitrogen to a powder (ca 100 µm) and incubated at 4°C with agitation in phosphoric acid of pH 1, 2, 3, 4, 5, and 6 (1 g/4 ml; n = 4) for six days with solution changes each day. Estimates of daily protein release were made by UV spectrophotometry at 280 nm. Extract solutions were dialyzed for 7 days in reverse osmosis water, lyophilized, and weighed. Non-collagenous proteins (NCPs) and glycosaminoglycans (GAGs) were quantitated by dye-binding assays. 1D-PAGE for preliminary protein characterization and sandwich ELISA for presence of TGF-ß1 were performed. The results were analyzed by ANOVA and regression (α <= 0.05). RESULTS: Protein release was drastically reduced after the first few days, with the highest amounts obtained from pH 1. There was no significant difference in the quantity of DMCs solubilized by the different pH levels, but there was a significant logarithmic relation between release and pH, suggesting that greater DMC solubilization occurs with higher hydrogen ion concentrations. Dye binding assays confirmed the release of NCPs and GAGs at all pH levels. There were only subtle differences in protein bands observed between the different pH levels (1D-PAGE). Significant levels of TGF-ß1 were identified from extraction at all pHs. CONCLUSION: Acids at pH levels relevant to those used in commercial dentin adhesives are capable of solubilizing human DMCs, with release being related to hydrogen ion concentration.