Biofilm and cell adhesion strength on dental implant surfaces via the laser spallation technique.

OBJECTIVE: The aims of this study are to quantify the adhesion strength differential between an oral bacterial biofilm and an osteoblast-like cell monolayer to a dental implant-simulant surface and develop a metric that quantifies the biocompatible effect of implant surfaces on bacterial and cell adhesion. METHODS: High-amplitude short-duration stress waves generated by laser pulse absorption are used to spall bacteria and cells from titanium substrates. By carefully controlling laser fluence and calibration of laser fluence with applied stress, the adhesion difference between Streptococcus mutans biofilms and MG 63 osteoblast-like cell monolayers on smooth and rough titanium substrates is obtained. The ratio of cell adhesion strength to biofilm adhesion strength (i.e., Adhesion Index) is determined as a nondimensionalized parameter for biocompatibility assessment. RESULTS: Adhesion strength of 143 MPa, with a 95% C.I. (114, 176), is measured for MG 63 cells on smooth titanium and 292 MPa, with a 95% C.I. (267, 306), on roughened titanium. Adhesion strength for S. mutans on smooth titanium is 320 MPa, with a 95% C.I. (304, 333), and remained relatively constant at 332 MPa, with a 95% C.I. (324, 343), on roughened titanium. The calculated Adhesion Index for smooth titanium is 0.451, with a 95% C.I. (0.267, 0.622), which increased to 0.876, with a 95% C.I. (0.780, 0.932), on roughened titanium. SIGNIFICANCE: The laser spallation technique provides a platform to examine the tradeoffs of adhesion modulators on both biofilm and cell adhesion. This tradeoff is characterized by the Adhesion Index, which is proposed to aid biocompatibility screening and could help improve implantation outcomes. The Adhesion Index is implemented to determine surface factors that promote favorable adhesion of cells greater than biofilms. Here, an Adhesion Index ≫ 1 suggests favorable biocompatibility.

Fluorescence based detection of polychlorinated biphenyls (PCBs) in water using hydrophobic interactions.

Despite increasing controls in their production and disposal, persistent organic pollutants in water, even at concentrations below parts per million, represent an ongoing environmental health risk. Despite this concern, the detection of these compounds in water sources rely upon expensive, time consuming approaches that do not permit frequent monitoring and evaluation. In this work, a new fluorescence-based technique is presented for the detection of polychlorinated biphenyls (PCBs) in water. Benzopyrene (BaP) fluorescence was shown to increase with trace concentrations of aromatic organic pollutants. BaP forms a hydrophobic complex with PCBs, which has allowed for the successful detection of pollutants including PCB-126, PCB-153 and PCB-118. To determine the selectivity and robustness of this response, the impact of pH, ionic strength and humic acid to mimic surface water conditions is explored. While suppression of the signal was seen, these factors' impact on the detection of PCBs was minor, suggesting that a potential sensing strategy can be developed through this interaction. It is seen that the number and location of chlorine atoms are important along with the geometric orientation of molecule's structure.

Comparative analysis of microbial sensing molecules in mucosal tissues with aging.

Host-bacterial interactions at mucosal surfaces require recognition of the bacteria by host cells enabling targeted responses to maintain tissue homeostasis. It is now well recognized that an array of host-derived pattern recognition receptors (PRRs), both cell-bound and soluble, are critical to innate immune engagement of microbes via microbial-associated molecular patterns (MAMP). This report describes the use of a nonhuman primate model to evaluate changes in the expression of these sensing molecules related to aging in healthy gingival tissues. Macaca mulatta aged 3-24 years were evaluated clinically and gingival tissues obtained, RNA isolated and microarray analysis conducted for gene expression of the sensing pattern recognition receptors (PRRs). The results demonstrated increased expression of various PRRs in healthy aging gingiva including extracellular (CD14, CD209, CLEC4E, TLR4), intracellular (NAIP, IFIH1, DAI) and soluble (PTX4, SAA1) PRRs. Selected PRRs were also correlated with both bleeding on probing (BOP) and pocket depth (PD) in the animals. These findings suggest that aged animals express altered levels of various PRRs that could affect the ability of the tissues to interact effectively with the juxtaposed microbial ecology, presumably contributing to an enhanced risk of periodontitis even in clinically healthy oral mucosal tissues with aging.

Increased birth weight is associated with altered gene expression in neonatal foreskin.

Elevated birth weight is linked to glucose intolerance and obesity health-related complications later in life. No studies have examined if infant birth weight is associated with gene expression markers of obesity and inflammation in a tissue that comes directly from the infant following birth. We evaluated the association between birth weight and gene expression on fetal programming of obesity. Foreskin samples were collected following circumcision, and gene expression analyzed comparing the 15% greatest birth weight infants (n=7) v. the remainder of the cohort (n=40). Multivariate linear regression models were fit to relate expression levels on differentially expressed genes to birth weight group with adjustment for variables selected from a list of maternal and infant characteristics. Glucose transporter type 4 (GLUT4), insulin receptor substrate 2 (IRS2), leptin receptor (LEPR), lipoprotein lipase (LPL), low-density lipoprotein receptor-related protein 1 (LRP1), matrix metalloproteinase 2 (MMP2), plasminogen activator inhibitor-1 (PAI-1) and transcription factor 7-like 2 (TCF7L2) were significantly upregulated and histone deacetylase 1 (HDAC1) and thioredoxin (TXN) downregulated in the larger birth weight neonates v. CONTROLS: Multivariate modeling revealed that the estimated adjusted birth weight group difference exceeded one standard deviation of the expression level for eight of the 10 genes. Between 25 and 50% of variation in expression level was explained by multivariate modeling for eight of the 10 genes. Gene expression related to glycemic control, appetite/energy balance, obesity and inflammation were altered in tissue from babies with elevated birth weight, and these genes may provide important information regarding fetal programming in macrosomic babies.

Effects of aging in the expression of NOD-like receptors and inflammasome-related genes in oral mucosa.

The molecular changes underlying the higher risk of chronic inflammatory disorders during aging remain incompletely understood. Molecular variations in the innate immune response related to recognition and interaction with microbes at mucosal surfaces could be involved in aging-related inflammation. We developed an ontology analysis of 20 nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) and seven inflammasome-related genes (IRGs) in healthy and inflamed/periodontitis oral mucosal tissues from young, adolescent, adult, and aged non-human primates (Macaca mulatta) using the GeneChip(®) Rhesus Macaque Genome array. Validation of some of the significant changes was done by quantitative reverse transcription-polymerase chain reaction. The expression of NLRB/NAIP, NLRP12, and AIM2 increased with aging in healthy mucosa whereas NLRC2/NOD2 expression decreased. Although higher expression levels of some NLRs were generally observed with periodontitis in adult mucosal tissues (e.g. NLRB/NAIP, NLRP5, and NLRX1), various receptors (e.g. NLRC2/NOD2 and NLRP2) and the inflammasome adaptor protein ASC, exhibited a significant reduction in expression in aged periodontitis tissues. Accordingly, the expression of NLR-activated innate immune genes, such as HBD3 and IFNB1, was impaired in aged but not adult periodontitis tissues. Both adult and aged tissues showed significant increase in interleukin-1ß expression. These findings suggest that the expression of a subset of NLRs appears to change with aging in healthy oral mucosa, and that aging-related oral mucosal inflammation could involve an impaired regulation of the inflammatory and antimicrobial response associated with downregulation of specific NLRs and IRGs.

Factors affecting live foal rates of Thoroughbred mares that undergo manual twin elimination.

REASONS FOR PERFORMING STUDY: Mares diagnosed with twin vesicles at 13-17 days after ovulation commonly have one of 2 vesicles manually reduced. It is not known whether vesicle location (adjacent vs. nonadjacent), mare age, mare reproductive status, parity, month of breeding or mare plasma progesterone concentration affects live foal rates. OBJECTIVES: To determine factors associated with a positive outcome (live foal) in mares undergoing manual twin reduction between 13 and 17 days post ovulation when performed by a single operator. STUDY DESIGN: Retrospective case-control study. METHODS: Breeding records and the Jockey Club records of registered Thoroughbreds were used to evaluate factors affecting the outcome of pregnancies in mares undergoing twin elimination and mares with singleton pregnancies. Thoroughbred mares with twin pregnancies (n = 129) were matched by age, parity, farm location and month bred with mares diagnosed with a singleton pregnancy (n = 127). The effects of location of embryonic vesicles, mare age, reproductive status, parity, month of breeding, vesicle size and plasma progesterone concentration at pregnancy diagnosis on live foal rate were examined. RESULTS: Position of embryonic vesicles at time of manual elimination, parity and month bred had no effect on live foal rate. Live foal rates in mares >9 years of age were lower (71.8%) than in all mares ≤9 years (87.1%; P<0.05). Mares >9 years of age that had a twin reduced lost more pregnancies (34.8%) than age-matched control mares (20.0%; P<0.005). Mean plasma progesterone concentration of twin-reduced mares was greater than in control mares when compared on the same day post ovulation. Plasma progesterone concentrations did not differ between mares that lost their pregnancy and those that delivered a live foal. CONCLUSIONS: Mare age of >9 years is associated with decreased pregnancy rate after twin reduction. POTENTIAL RELEVANCE: Furthering the understanding of factors that affect live foal outcome following manual twin elimination in mares.

Identification of differentially expressed thyroid hormone responsive genes from the brain of the Mexican Axolotl (Ambystoma mexicanum).

The Mexican axolotl (Ambystoma mexicanum) presents an excellent model to investigate mechanisms of brain development that are conserved among vertebrates. In particular, metamorphic changes of the brain can be induced in free-living aquatic juveniles and adults by simply adding thyroid hormone (T4) to rearing water. Whole brains were sampled from juvenile A. mexicanum that were exposed to 0, 8, and 18 days of 50 nM T4, and these were used to isolate RNA and make normalized cDNA libraries for 454 DNA sequencing. A total of 1,875,732 high quality cDNA reads were assembled with existing ESTs to obtain 5884 new contigs for human RefSeq protein models, and to develop a custom Affymetrix gene expression array (Amby_002) with approximately 20,000 probe sets. The Amby_002 array was used to identify 303 transcripts that differed statistically (p<0.05, fold change >1.5) as a function of days of T4 treatment. Further statistical analyses showed that Amby_002 performed concordantly in comparison to an existing, small format expression array. This study introduces a new A. mexicanum microarray resource for the community and the first lists of T4-responsive genes from the brain of a salamander amphibian.

Apoptotic genes are differentially expressed in aged gingival tissue.

Cellular and molecular changes of the periodontium associated with a higher prevalence of oral diseases (e.g., chronic periodontitis) in aged populations have received little attention. Since impaired apoptosis during aging appears to be related to chronic inflammatory disorders, we hypothesized that the expression of genes associated with apoptotic processes are altered in aged healthy and periodontitis-affected gingival tissue. Ontology analysis of 88 genes related to apoptotic pathways was performed in gingival biopsies of healthy and periodontitis sites from young, adult, and aged non-human primates (Macaca mulatta), using the GeneChip® Rhesus Macaque Genome Array. Lower expression of anti-apoptotic and higher expression of pro-apoptotic genes were associated with healthy gingival tissue from young compared with aged animals. Few differences in gene expression were observed in healthy gingival tissue between adult and aged animals. Comparison between healthy and periodontitis gingival tissues showed that the up- or down-regulated apoptotic genes in diseased gingival tissue are different in adults compared with aged animals. These results suggest that apoptotic events normally occurring in gingival tissues could be reduced in aging,and unique aspects of apoptotic pathways are potentially involved in the pathophysiology of periodontal disease in adult vs. aged gingival tissues.

Macrophage-mediated neuroprotection and neurogenesis in the olfactory epithelium.

Resident and recruited olfactory epithelial macrophages participate in the regulation of the survival, degeneration, and replacement of olfactory sensory neurons (OSNs). We have reported that liposome-encapsulated clodronate (Lip-C) induced selective and statistically significant depletion of macrophages in the OE of sham and 48 h OBX mice (38 and 35%, respectively) that resulted in increased OSN apoptosis and decreased numbers of mature OSNs and proliferating basal cells compared to controls (Lip-O). The aim of this study was to identify molecular mechanisms by which the selective depletion of macrophages in the OE resulted in these cellular changes by using a microarray expression pattern analysis. A 2x2 ANOVA identified 4,085 overall significantly (P < 0.01) regulated genes in the OE of Lip-O and Lip-C sham and 48 h OBX mice, and further statistical analysis using pairwise comparisons identified 4,024 genes that had either a significant (P < 0.01) treatment main effect (n = 2,680), group main effect (n = 778), or interaction effect (n = 980). The mean hybridization signals of immune response genes, e.g., Cxcr4, and genes encoding growth factors and neurogenesis regulators, e.g., Hdgf and Neurod1, respectively, were primarily lower in Lip-C mice compared with Lip-O mice. Apoptosis genes, e.g., Bak1, were also differentially regulated in Lip-C and/or OBX mice. Expression patterns of selected genes were validated with real-time RT-PCR; immunohistochemistry was used to localize selected gene products. These results identified the differential regulation of several novel genes through which alternatively activated macrophages regulate OSN progenitor cell proliferation, differentiation, and maturation, and the survival of OSNs.

Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice.

Endometrial cytology and culture specimens (n=2123) were collected concurrently with a guarded uterine culture instrument from 970 mares (738 barren, 1230 foaling and 155 maiden mares) during three breeding seasons (2001-2004). Results were compared to the 28-d pregnancy rate for the cycle from which the samples were taken. Cytological smears were evaluated for inflammation at x100 and graded as: not inflammatory (0-2 neutrophils/field), moderate inflammation (2-5 neutrophils/field), severe inflammation (>5 neutrophils/field), or hypocellular (scant epithelial cells and no neutrophils). Uterine culture swabs were plated within 6h, incubated for 72 h and results determined at 24, 48, and 72 h. Approximately, 20% (n=423) cytology samples were positive for inflammation (>2 neutrophils), whereas approximately 11% (n=231) of cultures had microorganisms recovered. A majority (64%) of the positive cultures (147/231) had inflammation on cytology smears. Streptococcus equi subsp. zooepidemicus was associated with more positive cytology results than coliforms (P<0.01). Mares with positive cytology or culture had lower pregnancy rates than mares with normal findings (P<0.01). Lowest pregnancy rates were recorded for mares with severe endometrial inflammation (21%, versus moderate inflammation 48%). Isolation of a microorganism from mares with endometrial inflammation was not associated with a further reduction in pregnancy rates. In barren, foaling and maiden mares, cytology was positive in 28, 17, and 5%, respectively, and culture was positive in 12.2, 11.1, and 3.2%. Foaling and maiden mares had higher pregnancy rates than barren mares (62, 69, and 44%, respectively, P<0.001). In conclusion, a positive cytology was twice as common as a positive culture, and isolation of microorganisms was associated with reduced pregnancy rates, even in the apparent absence of inflammation.

Temporal gene expression profiles of target-ablated olfactory epithelium in mice with disrupted expression of scavenger receptor A: impact on macrophages.

Target ablation [removal of the olfactory bulb (OBX)] induces apoptotic death of olfactory sensory neurons (OSNs) and an immune response in which activation and recruitment of macrophages (ms) into the olfactory epithelium (OE) occupy a central role. Ms phagocytose apoptotic neurons and secrete cytokines/growth factors that regulate subsequent progenitor cell proliferation and neurogenesis. Scavenger receptor A (SR-A) is a pattern recognition receptor that mediates binding of ms to apoptotic cells and other relevant immune response functions. The aim of this study was to determine the impact of the absence of SR-A on the immune response to OBX. The immune response to OBX was evaluated in mice in which functional expression of the m scavenger receptor (MSR) was eliminated by gene disruption (MSR-/-) and wild-type (wt) mice of the same genetic background. OBX induced significant apoptotic death of mature OSNs in the two strains. However, subsequent m infiltration and activation and progenitor cell proliferation were significantly reduced in MSR-/- vs. wt mice. Gene expression profiling at short intervals after OBX demonstrated significant differences in temporal patterns of expression of several gene categories, including immune response genes. Many immune response genes that showed different temporal patterns of expression are related to m function, including cytokine and chemokine secretion, phagocytosis, and m maturation and activation. These studies suggest that impairment of the immune response to OBX in the OE of MSR-/- mice most likely resulted from decreased m adhesion and subsequent reduced infiltration and activation, with a resultant decrease in neurogenesis.

Harnessing the power of gene microarrays for the study of brain aging and Alzheimer's disease: statistical reliability and functional correlation.

During normal brain aging, numerous alterations develop in the physiology, biochemistry and structure of neurons and glia. Aging changes occur in most brain regions and, in the hippocampus, have been linked to declining cognitive performance in both humans and animals. Age-related changes in hippocampal regions also may be harbingers of more severe decrements to come from neurodegenerative disorders such as Alzheimer's disease (AD). However, unraveling the mechanisms underlying brain aging, AD and impaired function has been difficult because of the complexity of the networks that drive these aging-related changes. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important new research tool that potentially can provide the investigative power needed to address the complexity of brain aging/neurodegenerative processes. However, along with this new analytic power, microarrays bring several major bioinformatics and resource problems that frequently hinder the optimal application of this technology. In particular, microarray analyses generate extremely large and unwieldy data sets and are subject to high false positive and false negative rates. Concerns also have been raised regarding their accuracy and uniformity. Furthermore, microarray analyses can result in long lists of altered genes, most of which may be difficult to evaluate for functional relevance. These and other problems have led to some skepticism regarding the reliability and functional usefulness of microarray data and to a general view that microarray data should be validated by an independent method. Given recent progress, however, we suggest that the major problem for current microarray research is no longer validity of expression measurements, but rather, the reliability of inferences from the data, an issue more appropriately redressed by statistical approaches than by validation with a separate method. If tested using statistically defined criteria for reliability/significance, microarray data do not appear a priori to require more independent validation than data obtained by any other method. In fact, because of added confidence from co-regulation, they may require less. In this article we also discuss our strategy of statistically correlating individual gene expression with biologically important endpoints designed to address the problem of evaluating functional relevance. We also review how work by ourselves and others with this powerful technology is leading to new insights into the complex processes of brain aging and AD, and to novel, more comprehensive models of aging-related brain change.