The Murine Oral Metatranscriptome Reveals Microbial and Host Signatures of Periodontal Disease.

Periodontal disease is accompanied by alterations to cellular profiles and biological activities of both the subgingival microbiome and host tissues. Although significant progress has been made in describing the molecular basis of the homeostatic balance of host-commensal microbe interactions in health compared to the destructive imbalance in disease, particularly with respect to immune and inflammatory systems, few studies have attempted a comprehensive analysis in diverse host models. Here, we describe the development and application of a metatranscriptomic approach to analysis of host-microbe gene transcription in a murine periodontal disease model, based on oral gavage infection using Porphyromonas gingivalis in C57BL6/J mice. We generated 24 metatranscriptomic libraries from individual mouse oral swabs, representing health and disease. On average, 76% ± 11.7% reads in each sample belonged to the murine host genome and the remainder to the microbes. We found 3,468 (2.4% of the total) murine host transcripts differentially expressed between health and disease, of which 76% were overexpressed in periodontitis. Predictably, there were prominent alterations to genes and pathways linked with the host immune compartment in disease-the CD40 signaling pathway being the top enriched biological process in this data set. However, in addition, we observed significant alterations to other biological processes in disease, particularly cellular/metabolic processes and biological regulation. The number of differentially expressed microbial genes particularly indicated shifts in carbon metabolism pathways in disease with potential consequences for metabolic end-product formation. Together, these metatranscriptome data reveal marked changes between the gene expression patterns in both the murine host and microbiota, which may represent signatures of health and disease, providing the basis for future functional studies of prokaryotic and eukaryotic cellular responses in periodontal disease. In addition, the noninvasive protocol developed in this study will enable further longitudinal and interventionist studies of host-microbe gene expression networks.

The regional and cellular distribution of GABAA receptor subunits in the human amygdala.

GABAergic neurotransmission in the amygdala plays a crucial role in mediating emotional learning, fear, and memory. In this study, expression of five major GABAA receptor subunits (α1, α2, α3, ß2,3, and γ2) was investigated in the normal human amygdala using immunohistochemistry. At the regional level, the amygdala contains a highly heterogeneous distribution of all the subunits investigated. The most intense staining for α1, α2, ß2,3, and γ2 subunits was present in the lateral nucleus (LA), and α3 in the intercalated nuclei (ICM). Six distinct cell populations that express GABAA receptor subunits were identified throughout the amygdala: type 1 aspiny cells in the basolateral nuclear group (BLNG) and superficial cortical-like nuclear region (SCLR) express α1, ß2,3, and γ2; type 2 larger aspiny cells in the paralaminar nucleus (PL) express α1, ß2,3, and γ2; type 3 aspiny cells in the BLNG express α1, ß2,3, and γ2 as well as calcium-binding proteins including parvalbumin (PV), calbindin (CB), and calretinin (CR); type 4 pyramidal cells in the BLNG and SCLR express α2, α3, ß2,3, and γ2 subunits at high levels on proximal specialised spines; type 5 cells in the central nucleus (CE) express α2, α3, and ß2,3; type 6 cells are found closely packed in the intercalated cell masses (ICM) and express α3 and ß2,3. The α1 subunit rarely co-labelled with α2 and α3 in the same cell population, while the α2 and α3 were often expressed within the same type 4 or 5 cell though not at always at the same puncta. The predominant GABAA receptor subunit combinations expressed in the human amygdala are the α1ß2,3γ2 and α2ß2,3γ2. Cells classified as interneuron types (types 1-3) contained GAD and principally expressed α1ß2,3γ2. The major projection neurons of the BLNG (type 4) are non-GABAergic and mainly express α2ß2,3γ2. The α3 subunit was found intracellularly in type 5 cells and decorating the surface of type 6 cells but rarely co-labelled with the subunits investigated. The results reveal a complex and heterogeneous distribution of GABAA receptor subtypes throughout the amygdala as well as on a variety of cell types through which inhibitory processing is carried out to maintain emotional responses, and control anxiety and fear responses in the brain.

Loss of Neutrophil Homing to the Periodontal Tissues Modulates the Composition and Disease Potential of the Oral Microbiota.

Periodontal disease is considered to arise from an imbalance in the interplay between the host and its commensal microbiota, characterized by inflammation, destructive periodontal bone loss, and a dysbiotic oral microbial community. The neutrophil is a key component of defense of the periodontium: defects in their number or efficacy of function predisposes individuals to development of periodontal disease. Paradoxically, neutrophil activity, as part of a deregulated inflammatory response, is considered an important element in the destructive disease process. In this investigation, we examined the role the neutrophil plays in the regulation of the oral microbiota by analysis of the microbiome composition in mice lacking the CXCR2 neutrophil receptor required for recruitment to the periodontal tissues. A breeding protocol was employed that ensured that only the oral microbiota of wild-type (CXCR2+/+) mice was transferred to subsequent generations of wild-type, heterozygote, and homozygote littermates. In the absence of neutrophils, the microbiome undergoes a significant shift in total load and composition compared to when normal levels of neutrophil recruitment into the gingival tissues occur, and this is accompanied by a significant increase in periodontal bone pathology. However, transfer of the oral microbiome of CXCR2-/- mice into germfree CXCR2+/+ mice led to restoration of the microbiome to the wild-type CXCR2+/+ composition and the absence of pathology. These data demonstrate that the composition of the oral microbiome is inherently flexible and is governed to a significant extent by the genetics and resultant phenotype of the host organism.

The P. gingivalis Autocitrullinome Is Not a Target for ACPA in Early Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory disease affecting primarily the joints, is frequently characterized by the presence of autoimmune anticitrullinated protein antibodies (ACPA) during preclinical stages of disease and accumulation of hypercitrullinated proteins in arthritic joints. A strong association has been reported between RA and periodontal disease, and Porphyromonas gingivalis, a known driver of periodontitis, has been proposed as the microbial link underlying this association. We recently demonstrated P. gingivalis-mediated gut barrier breakdown and exacerbation of joint inflammation during inflammatory arthritis. In the present study, we investigated another potential role for P. gingivalis in RA etiopathogenesis, based on the generation of ACPA through the activity of a unique P. gingivalis peptidylarginine deiminase (PPAD) produced by this bacterium, which is capable of protein citrullination. Using a novel P. gingivalis W50 PPAD mutant strain, incapable of protein citrullination, and serum from disease-modifying antirheumatic drug-naïve early arthritis patients, we assessed whether autocitrullinated proteins in the P. gingivalis proteome serve as cross-activation targets in the initiation of ACPA production. We found no evidence for patient antibody activity specific to autocitrullinated P. gingivalis proteins. Moreover, deletion of PPAD did not prevent P. gingivalis-mediated intestinal barrier breakdown and exacerbation of disease during inflammatory arthritis in a murine model. Together, these findings suggest that the enzymatic activity of PPAD is not a major virulence mechanism during early stages of inflammatory arthritis.

Horizontal and Vertical Transfer of Oral Microbial Dysbiosis and Periodontal Disease.

One of the hallmark features of destructive periodontal disease, well documented over the last 50 y, is a change to the quantitative and qualitative composition of the associated microbiology. These alterations are now generally viewed as transformational shifts of the microbial populations associated with health leading to the emergence of bacterial species, which are only present in low abundance in health and a proportionate decrease in the abundance of others. The role of this dysbiosis of the health associated microbiota in the development of disease remains controversial: is this altered microbiology the driving agent of disease or merely a consequence of the altered environmental conditions that invariably accompany destructive disease? In this work, we aimed to address this controversy through controlled transmission experiments in the mouse in which a dysbiotic oral microbiome was transferred either horizontally or vertically into healthy recipient mice. The results of these murine studies demonstrate conclusively that natural transfer of the dysbiotic oral microbiome from a periodontally diseased individual into a healthy individual will lead to establishment of the dysbiotic community in the recipient and concomitant transmission of the disease phenotype. The inherent resilience of the dysbiotic microbial community structure in diseased animals was further demonstrated by analysis of the effects of antibiotic therapy on periodontally diseased mice. Although antibiotic treatment led to a reversal of dysbiosis of the oral microbiome, in terms of both microbial load and community structure, dysbiosis of the microbiome was reestablished following cessation of therapy. Collectively, these data suggest that an oral dysbiotic microbial community structure is stable to transfer and can act in a similar manner to a conventional transmissible infectious disease agent with concomitant effects on pathology. These findings have implications to our understanding of the role of microbial dysbiosis in the development and progression of human periodontal disease.

Imposed running exercise does not alter cell proliferation in the neurogenic niches of young lambs.

Neurogenesis, the process by which neurons are generated in the brain from progenitor cells, occurs in the subventricular zone (SVZ) and the subgranular zone (SGZ) in the adult human brain. Recently, rodent studies have demonstrated that exercise can increase neurogenesis in the SGZ; however, it is unclear if exercise also has this effect in more complex mammalian brains. The overarching aim of this study was to explore whether exercised-induced neurogenesis occurs in larger mammalian brains more representative of human brains and to explore the use of a model for exercising large animals such as sheep. For these studies, 6 male twin lambs had a structured exercise regime for 4 wk and 6 other twin male lambs were kept in an open field pen. All lambs were injected with bromodeoxyuridine (BrdU), a thymidine analog that is incorporated into the DNA of proliferating cells. Immunoperoxidase was used to visualize and quantify BrdU-positive cells in the SVZ and SGZ. Overall, no significant change in the number or distribution of BrdU-positive cells was observed in the lamb SVZ and SGZ with exercise or colabeling of BrdU with mature neuronal or glial markers in the exercised and nonexercised lamb SVZ and SGZ. Overall, this study provides a novel methodology to investigate the effects of imposed exercise on large animals and exercise-induced neurogenesis in animals with gyrencephalic brains.

Role of microbial communities in the pathogenesis of periodontal diseases and caries.

The microbiological characteristics of both caries and periodontal disease show significant change from those in health. In both instances, there is evidence of co-association of different organisms into consortia. AIM: We review and summarize a number of issues pertinent to the community organization and functional activity of the bacterial populations resident on supra- and subgingival tooth surface and the influence of these populations on disease. METHODS: A literature review was undertaken with a particular emphasis on recent publications involving high-throughput, deep sequencing approaches to the analysis of microbial populations and their functional activity. RESULTS: There is increasing evidence to suggest that both caries and periodontal disease represent dysbiotic states of the oral microbiome. The mode of acquisition of the oral microbial communities may be less passive than previously recognized but once established remains relatively stable within an individual although there are very significant site variations. A repertoire of stable dysbiotic states may occur in both caries and periodontitis involving different microbial community structures with potentially similar functional properties. CONCLUSIONS: The processes which underlie the development and stability of microbial populations in the healthy mouth are fundamental to understanding how these populations are transformed into a dysbiotic state in disease.

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A-LPS.

Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)-protoporphyrin IX] or hemin [Fe(III)-protoporphyrin IX-Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin-binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, µ-oxo-bisheme {[Fe(III)PPIX]2 O}, which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of µ-oxo-bisheme on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys-gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α-1, 3-mannosyltransferase) produce non-pigmented colonies. However, only those mutants lacking A-LPS showed reduced hemin-binding when cells in suspension were incubated with hemin. Using native, de-O-phosphorylated and de-lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin-binding to O-polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin-binding to A-PS. We conclude that A-LPS in the outer-membrane of P. gingivalis serves as a scaffold/anchor for the retention of µ-oxo-bisheme on the cell surface and pigmentation is dependent on the presence of A-LPS.

Site-Specific Neutrophil Migration and CXCL2 Expression in Periodontal Tissue.

The oral microbial community is the best-characterized bacterial ecosystem in the human host. It has been shown in the mouse that oral commensal bacteria significantly contribute to clinically healthy periodontal homeostasis by influencing the number of neutrophils that migrate from the vasculature to the junctional epithelium. Furthermore, in clinically healthy tissue, the neutrophil response to oral commensal bacteria is associated with the select expression of the neutrophil chemokine CXCL2 but not CXCL1. This preliminary study examined the contribution of commensal bacteria on neutrophil location across the tooth/gingival interface. Tissue sections from the root associated mesial (anterior) of the second molar to the root associated distal (posterior) of the second molar were examined for neutrophils and the expression of the neutrophil chemokine ligands CXCL1 and CXCL2. It was found that both the number of neutrophils as well as the expression of CXCL2 but not CXCL1 was significantly increased in tissue sections close to the interdental region, consistent with the notion of select tissue expression patterns for neutrophil chemokine expression and subsequent neutrophil location. Furthermore, mice gavaged with either oral Streptococcus or Lactobacillus sp. bacteria induced a location pattern of neutrophils and CXCL2 expression similar to the normal oral flora. These data indicate for the first time select neutrophil location and chemokine expression patterns associated with clinically healthy tissue. The results reveal an increased inflammatory load upon approaching the interproximal region, which is consistent with the observation that the interproximal region often reveals early clinical signs of periodontal disease.

Neurogenesis and progenitor cell distribution in the subgranular zone and subventricular zone of the adult sheep brain.

Progenitor cell proliferation is ubiquitous in the subventricular zone (SVZ) and subgranular zone (SGZ) of adult mammalian brains, however, the abundance and distribution of proliferation are surprisingly heterogeneous between species. In rodents, proliferation is high in both the SVZ and SGZ, while in humans proliferation is prominent in the SVZ but limited in the SGZ. To accurately study proliferation and how it changes in human disease, we should focus on animals in which the patterns of proliferation are consistent with the human brain. In this study, we characterized the neurogenic niches of the adult sheep, an animal model with a longer lifespan than rodents and a highly gyrencephalic brain, using 5-bromo-2'-deoxyuridine (BrdU) as a mitotic marker and neuronal nuclear antigen to identify neuronal lineage cells. Our study demonstrates that the sheep SVZ is organized into the same distinct layers that are comparable to what has been described in humans. The rate of maturation of new neurons was slower in sheep than in previous reports in rodents, with only 20% of BrdU-positive cells showing neuronal phenotype after 4 months survival following BrdU administration. Most importantly, as in the human, there was much greater proliferation in the sheep SVZ than in the SGZ. These results suggest that the sheep is a better basis for comparisons with human SVZ and SGZ neurogenesis than rodents.

The effects of chronic alcoholism on cell proliferation in the human brain.

Neurogenesis continues in the human subventricular zone and to a lesser extent in the hippocampal subgranular zone throughout life. Subventricular zone-derived neuroblasts migrate to the olfactory bulb where survivors become integrated as interneurons and are postulated to contribute to odor discrimination. Adult neurogenesis is dysregulated in many neurological, neurovascular and neurodegenerative diseases. Alcohol abuse can result in a neurodegenerative condition called alcohol-related brain damage. Alcohol-related brain damage manifests clinically as cognitive dysfunction and the loss of smell sensation (hyposmia) and pathologically as generalized white matter atrophy and focal neuronal loss. The exact mechanism linking chronic alcohol intoxication with alcohol-related brain damage remains largely unknown but rodent models suggest that decreased neurogenesis is an important component. We investigated this idea by comparing proliferative events in the subventricular zone and olfactory bulb of a well-characterized cohort of 15 chronic alcoholics and 16 age-matched controls. In contrast to the findings in animal models there was no difference in the number of proliferative cell nuclear antigen-positive cells in the subventricular zone of alcoholics (mean±SD=28.7±20.0) and controls (27.6±18.9, p=1.0). There were also no differences in either the total (p=0.89) or proliferative cells (p=0.98) in the granular cell layer of the olfactory bulb. Our findings show that chronic alcohol consumption does not affect cell proliferation in the human SVZ or olfactory bulb. In fact only microglial proliferation could be demonstrated in the latter. Therefore neurogenic deficits are unlikely to contribute to hyposmia in chronic alcoholics.

Porphyromonas gingivalis as a potential community activist for disease.

An extensive analysis of dental plaque samples over the years has led to the identification of "red" complex oral bacteria that have a strong association with each other and with disease. Consequently, these bacteria have been labeled 'periopathogens'. Studies with one of these bacteria, Porphyromonas gingivalis, have revealed that it contains several different mechanisms which either impede or modulate periodontal protective mechanisms. In a mouse model of periodontitis, it has been shown that modulation of complement function by P. gingivalis facilitates a significant change in both the amount and composition of the normal oral microbiotia. This altered oral commensal microbiota is responsible for pathologic bone loss in the mouse. Thus, P. gingivalis creates a dysbiosis between the host and dental plaque, and this may represent one mechanism by which periodontitis can be initiated. We have therefore termed P. gingivalis a keystone pathogen.

No change in progenitor cell proliferation in the hippocampus in Huntington's disease.

Increases in cell proliferation in the hippocampus have been robustly demonstrated in animal models of neurodegenerative diseases like Huntington's disease (HD). However, in the subventricular zone, animal models of HD have demonstrated no change in cell proliferation compared to wild types, while in humans there is a distinct increase in cell proliferation in HD cases. Interestingly, there have been no reports on cell proliferation in the human subgranular zone (SGZ) of the hippocampus in HD, despite numerous transgenic mouse models of HD showing decreased proliferation in the SGZ. Furthermore, HD can be divided into those with mainly mood and mainly motor symptomatology. We hypothesized that HD cases with mainly mood symptomatology would show a greater change in hippocampal proliferation, which has previously been implicated in mood disorders such as depression. Therefore, in the current study we examined and compared proliferation in the SGZ in normal vs. HD, HD mood, and HD motor affected cases. However, our results revealed no significant differences in SGZ proliferation between normal and HD cases, and no differences when divided into groups based on mood and motor symptomatology. Our results were confirmed using a range of cell-cycle protein markers and, overall, were comparable with previous studies of the human hippocampus, where very little proliferation was detected in the adult SGZ. These results demonstrate that in humans the SGZ is far less proliferative than the SVZ, and suggests that hippocampal plasticity in humans does not primarily involve cell proliferation.

Allelic imbalance of tissue-type plasminogen activator (t-PA) gene expression in human brain tissue.

We have identified a single-nucleotide polymorphism (SNP) in the t-PA enhancer (-7351C>T), which is associated with endothelial t-PA release in vivo. In vitro studies demonstrated that this SNP is functional at the level of transcription. In the brain, t-PA has been implicated in both physiologic and pathophysiologic processes. The aim of the present study was to examine the effect of the t-PA -7351C>T SNP on t-PA gene expression in human brain tissue. Allelic mRNA expression was measured in heterozygous post-mortem brain tissues using quantitative TaqMan genotyping assay. Protein-DNA interactions were assessed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Significantly higher levels of t-PA mRNA were generated from chromosomes that harboured the wild-type -7351C allele, as compared to those generated from the mutant T allele (for the hippocampus, C to T allelic ratio of ~1.3, p=0.010, n=12; and for the cortex, C to T allelic ratio of ~1.2, p=0.017, n=12). EMSA showed reduced neuronal and astrocytic nuclear protein binding affinity to the T allele, and identified Sp1 and Sp3 as the major transcription factors that bound to the -7351 site. ChIP analyses confirmed that Sp1 recognises this site in intact cells. In conclusion, the t-PA -7351C>T SNP affects t-PA gene expression in human brain tissue. This finding might have clinical implications for neurological conditions associated with enhanced t-PA levels, such as in the acute phase of cerebral ischaemia, and also for stroke recovery.

Characterization of the netrin/RGMa receptor neogenin in neurogenic regions of the mouse and human adult forebrain.

In the adult rodent forebrain, astrocyte-like neural stem cells reside within the subventricular zone (SVZ) and give rise to progenitors and neuroblasts, which then undergo chain migration along the rostral migratory stream (RMS) to the olfactory bulb, where they mature into fully functional interneurons. Neurogenesis also occurs in the adult human SVZ, where neural precursors similar to the rodent astrocyte-like stem cell and neuroblast have been identified. A migratory pathway equivalent to the rodent RMS has also recently been described for the human forebrain. In the embryo, the guidance receptor neogenin and its ligands netrin-1 and RGMa regulate important neurogenic processes, including differentiation and migration. We show in this study that neogenin is expressed on neural stem cells (B cells), progenitor cells (C cells), and neuroblasts (A cells) in the adult mouse SVZ and RMS. We also show that netrin-1 and RGMa are ideally placed within the neurogenic niche to activate neogenin function. Moreover, we find that neogenin and RGMa are also present in the neurogenic regions of the human adult forebrain. We show that neogenin is localized to cells displaying stem cell (B cell)-like characteristics within the adult human SVZ and RMS and that RGMa is expressed by the same or a closely apposed cell population. This study supports the hypothesis that, as in the embryo, neogenin regulates fundamental signalling pathways important for neurogenesis in the adult mouse and human forebrain.

Expression of ezrin radixin moesin proteins in the adult subventricular zone and the rostral migratory stream.

Continuous proliferation occurs in the adult subventricular zone (SVZ) of the lateral ventricles throughout life. In the SVZ, progenitor cells differentiate into neuroblasts, which migrate tangentially along the rostral migratory stream (RMS) to reach their final destination in the olfactory bulb. These progenitor cells mature and integrate into the existing neural network of the olfactory bulb. Long distance migration of neuroblasts in the RMS requires a highly dynamic cytoskeleton with the ability to respond to surrounding stimuli. Radixin is a member of the ERM (Ezrin, Radixin, Moesin) family, which connect the actin cytoskeleton to the extracellular matrix through transmembrane proteins. The membrane-cytoskeleton linker proteins of the ERM family may regulate cellular events with a high demand on cytoskeleton plasticity, such as cell motility. Recently, specific expression of the ERM protein ezrin was shown in the RMS. Radixin however has not been characterized in this region. Here we used immunohistochemistry and confocal microscopy to examine the expression of radixin in the different cell types of the adult subventricular zone niche and in the RMS. Our findings indicate that radixin is strongly expressed in neuroblasts of the adult RMS and subventricular zone, and also in Olig2-positive cells. We also demonstrate the presence of radixin in the cerebral cortex, striatum, cerebellum, thalamus, hippocampus as well as the granular and periglomerular layers of the olfactory bulb. Our studies also reveal the localization of radixin in neurosphere culture studies and we reveal the specificity of our labeling using Western blotting. The expression pattern demonstrated here suggests a role for radixin in neuronal migration and differentiation in the adult RMS. Understanding how adult neuronal migration is regulated is of importance for the development of new therapeutic interventions using endogenous repair for neurodegenerative diseases.

Ractopamine hydrochloride improves growth performance and carcass composition in immunocastrated boars, intact boars, and gilts.

The beta-agonist ractopamine is a dietary ingredient that improves growth and increases the lean mass with little change in fat mass in gilts and barrows. Limited data in boars indicate that dietary ractopamine may increase lean tissue and decrease fat deposition, whereas there are no data for immunocastrated boars. The aims of this investigation were 1) to assess whether the growth performance of all sexes could be maintained over 31 d by using a step-up dietary ractopamine feeding program of 5 mg/kg of ractopamine for the first 14 d, then increasing the dose to 10 mg/kg for a further 17 d, and 2) to determine if dietary ractopamine would increase lean mass in all sexes and decrease fat mass in boars and immunocastrated boars. The study involved 286 pigs randomized and proportionally allocated by breed into 24 groups of 11 or 12 pigs at 17 wk of age, with equal groups of boars, immunocastrated boars, and gilts. Dietary ractopamine decreased (P = 0.005) ADFI during the first 2 wk, particularly in the intact and immunocastrated boars, with the reduction in ADFI being maintained in the immunocastrated boars after the increment in dietary ractopamine. Daily BW gain was not altered by dietary ractopamine during the first 2 wk, but was increased (P < 0.001) after the increment in dietary ractopamine. Dietary ractopamine decreased (P < or = 0.033) feed conversion ratio in all sexes with the response being greater after the increase in dietary ractopamine. Carcass weight was increased (P < 0.001) by dietary ractopamine in all sexes, whereas back fat tended (P = 0.076) to be reduced in the immunocastrated boars. Dietary ractopamine increased (P = 0.018) lean tissue mass by 4.0, 4.8, and 6.5 kg in the intact boars, gilts, and immunocastrated boars, respectively. In the entire and immunocastrated boars, the increase in lean tissue was accompanied with a decrease (P = 0.004) in fat mass. There was little effect of dietary ractopamine on fat mass in gilts. However, carcass percent fat was decreased (P = 0.004) and percent lean increased (P = 0.006) in all sexes. Immunocastration caused a decrease in lean tissue mass and an increase in fat mass and an increase in ADFI in the last one-half of the study. Dietary ractopamine may decrease fat mass in intact and immunocastrated boars and offers an excellent means of maximizing the effects of immunocastration and minimizing the increase in fat mass sometimes observed in immunocastrated boars.

Expression and regulation of the NALP3 inflammasome complex in periodontal diseases.

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)-1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real-time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck-like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD-containing protein 2), IL-1beta and IL-18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono-Mac-6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL-1beta or IL-18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono-Mac-6 cells by enhancing NALP3 and down-regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine-signalling pathway by bacterial challenge.