Salivary Cortisol and Anxiety in Canadian Dentists over 1 Year of COVID-19.

The dental profession has endured unprecedented disruption amid COVID-19. Novel stressors have included a high risk of occupational exposure to COVID-19, financial losses, and stricter infection prevention and control requirements. The present study investigated the longitudinal impact of COVID-19 on the stress and anxiety levels of a cohort of Canadian dentists (N = 222) between September 2020 and October 2021. Salivary cortisol was selected as a biomarker of mental stress, and 10 sets of monthly saliva samples (2,131 in total) were self-collected, sent to our laboratory in prepaid courier envelopes, and analyzed by enzyme-linked immunosorbent assay. To assess COVID-19 anxiety, 9 monthly online questionnaires were administered, comprising a general COVID-19 anxiety instrument and 3 items regarding the impact of dentistry-related factors. Bayesian log-normal mixed effect models were fitted to estimate the longitudinal trajectory of salivary cortisol levels and their association with the disease burden of COVID-19 in Canada. After accounting for age, sex, vaccination status, and the diurnal rhythm of cortisol secretion, a modest positive association was found between dentists' salivary cortisol levels and the count of COVID-19 cases in Canada (96% posterior probability). Similarly, the self-reported impact of dentistry-related factors, such as fear of getting COVID-19 from a patient or coworker, was greatest during peaks of COVID-19 waves in Canada; however, general COVID-19 anxiety decreased consistently throughout the study period. Interestingly, at all collection points, the majority of participants were not concerned about personal protective equipment. Overall, participants reported relatively low rates of psychological distress symptoms in relation to COVID-19, a result that should be reassuring for the dental community. Our findings strongly suggest a link between self-reported and biochemical measurements of stress and anxiety in Canadian dentists during the COVID-19 pandemic.

Periodontal Inflammation Primes the Systemic Innate Immune Response.

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

The Biology of Social Adversity Applied to Oral Health.

Biological embodiment is a concept derived from Engel's biopsychosocial model to health, theorized as the process by which adverse social exposures trigger neuroendocrine and immune responses, leading to disease and/or increased disease susceptibility. This critical review discusses the biopsychosocial model as applied to oral health and its relevance to oral health policy while deciphering some of the pathobiological processes underlying social adversity. In periodontal disease, for example, such processes can occur via the activation of the hypothalamic-pituitary-adrenal axis and the consequent release of the chronic stress hormone cortisol. The latter contributes to a proinflammatory immune state that increases the risk for periodontal inflammation. Recent research shows that cortisol relates to an elevated oral inflammatory load, demonstrated as hyperactive neutrophils that are pivotal to periodontal tissue damage. Consistent with the biopsychosocial model, this relationship is amplified in those of lower income and higher financial stress. Similarly, among children from lower socioeconomic backgrounds, cortisol is linked to a higher cariogenic bacterial load. Such findings implicate the stress pathway as key in the oral pathogenic process, particularly under social/socioeconomic adversity. Collectively, this work emphasizes the importance of addressing social factors in alleviating oral disease burden and reducing the social gaps therein.

Adseverin modulates morphology and invasive function of MCF7 cells.

Adseverin (Ads) is a Ca2+-dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis.

Resolving Macrophages Counter Osteolysis by Anabolic Actions on Bone Cells.

Progression of inflammatory osteolytic diseases, including rheumatoid arthritis and periodontitis, is characterized by increased production of proinflammatory mediators and matrix-degrading enzymes by macrophages and increased osteoclastic activity. Phenotypic changes in macrophages are central to the healing process in virtually all tissues. Using a murine model of periodontitis, we assessed the timing of macrophage phenotypic changes and the impact of proresolving activation during inflammatory osteolysis and healing. Proinflammatory macrophage activation and TNF-α overproduction within 3 wk after induction of periodontitis was associated with progressing bone loss. Proresolving activation within 1 wk of stimulus removal and markers of resolving macrophages (IL-10, TGF-ß, and CD206) correlated strongly with bone levels. In vivo macrophage depletion with clodronate liposomes prevented bone resorption but impaired regeneration. Induction of resolving macrophages with rosiglitazone, a PPAR-γ agonist, led to reduced bone resorption during inflammatory stimulation and increased bone formation during healing. In vitro assessment of primary bone marrow-derived macrophages activated with either IFN-γ and LPS (proinflammatory activation) or IL-4 (proresolving activation) showed that IL-4-activated cells have enhanced resolving functions (production of anti-inflammatory cytokines; migration and phagocytosis of aged neutrophils) and exert direct anabolic actions on bone cells. Cystatin C secreted by resolving but not inflammatory macrophages explained, in part, the macrophage actions on osteoblasts and osteoclasts. This study supports the concept that therapeutic induction of proresolving functions in macrophages can recouple bone resorption and formation in inflammatory osteolytic diseases.

Oral and Blood Neutrophil Activation States during Experimental Gingivitis.

Polymorphonuclear neutrophils (PMNs) are the primary leukocytes present in the healthy and inflamed oral cavity. While unique PMN activation states have been shown to differentiate health and periodontitis, little is known about the changes in PMN activation states that occur during the transition from periodontal health to gingivitis. The objective of this study was to characterize oral and circulatory PMNs during induction and resolution of experimental gingivitis. Healthy volunteers were recruited to undergo experimental gingivitis. Clinical assessment of pocket depths, bleeding on probing, gingival index, and plaque index, as well as flow cytometric analysis of CD (cluster of differentiation) activation markers on blood and oral PMNs, was performed weekly. All clinical parameters increased significantly during the induction period and returned to baseline levels during the resolution phase. During the induction phase, while oral PMN counts increased, oral PMN activation state based on surface expression of CD63, CD11b, CD16, and CD14 was diminished compared to those seen in health and during the resolution phase. PMNs in circulation during onset showed increased activation based on CD55, CD63, CD11b, and CD66a. Using clinical parameters and oral PMN counts assessed at day 21, we noted 2 unique disease patterns where one-third of subjects displayed an exaggerated influx of oral PMNs with severe inflammation compared to the majority of the population who experienced a moderate level of inflammation and PMN influx. This supports the notion that PMN influx and severe inflammatory changes during gingivitis could identify subjects at risk for the development of severe gingival inflammation and progression toward destructive periodontitis. This study demonstrates that oral PMN activation states are reduced in gingivitis and suggest that only in periodontitis do PMNs become hyperactivated and tissue damaging. Knowledge Transfer Statement: Our article creates a paradigm for future studies of the evolution of essential oral and circulatory biomarkers to identify individuals at risk to develop periodontitis at an early stage of periodontal disease, which is reversible upon proper oral hygiene practices and dental treatments.

The Role of NrF2 in the Regulation of Periodontal Health and Disease.

Immune-related disease tolerance is an important defense strategy that facilitates the maintenance of health in organs and tissues that are commonly colonized by bacteria. Immune tolerance to dysbiotic, tooth-borne biofilms is a poorly understood yet clinically relevant concept in the immunopathological mechanisms that are involved in the pathogenesis of periodontitis, particularly those related to neutrophil and macrophage responses. In periodontal health, neutrophils and macrophages respond to the formation of pathogenic bacterial biofilms by the production of bactericidal reactive oxygen species (ROS). However, when released in excess, ROS cause tissue damage and exacerbate inflammation. To counter these destructive responses, many cell types, including neutrophils and macrophages, launch a dedicated antioxidant system that limits the cell and tissue-damaging effects of ROS. The expression of antioxidants is primarily regulated by genetic response elements in their promoters. Here we consider the roles of nuclear factor erythroid 2-related factor (NrF2), a transcription factor, and other key regulators of antioxidants. The concept of disease tolerance, neutrophil and macrophage-generated oxidative stress, and their relationship to the pathogenesis of periodontitis is reviewed. We focus on the regulation of NrF2 and recent evidence suggesting that NrF2 plays a central role in host protection against tissue destruction in periodontitis.

Heterogeneity of osteoclast activity and bone turnover in different skeletal sites.

OBJECTIVE: To compare osteoclasts and bone turnover in the cranial and appendicular skeletons of mice and determine whether estrogen depletion has an impact on these differences. DESIGN: In vitro osteoclastogenesis (OCG) was performed on osteoclasts precursors derived from calvarial, mandibular and femoral bone marrow. In vitro, mature osteoclasts were stained with TRAP in plastic petri dishes and with DAPI and Phalloidin on glass coverslips to identify mature osteoclasts and compare osteoclast surface area and nuclei number in the different bone sites, respectively. Quantification of osteoclast resorption pit (Rpit) volume and surface area from different bone sites was achieved using dentin slices stained with Picrosirius red and confocal microscopy. In vivo TRAP, static and dynamic histomorphometric analyses were performed on 5-month-old mouse calvarial, long bone and mandibular trabecular bone to compare bone resorption and formation rates, respectively. Mice were ovariectomized (OVX) at 5 months of age and sacrificed at 6 months of age to establish an osteoporosis model for differences in osteoclasts activity and to monitor the changes in bone turnover rates in the three bone sites upon estrogen depletion. RESULT: s Phalloidin stained calvarial osteoclasts were larger compared to long bone and mandibular osteoclasts. Rpits from osteoclasts derived from mandibular bone were smaller and had lower volume values compared to long bone and calvarial bone Rpits. In vivo analysis showed significant increases in bone formation rates in calvarial trabecular bone compared to long bone and mandibular trabecular bone. Turnover was enhanced upon estrogen depletion in calvarial trabecular bone. Resorption was increased without a corresponding increase in bone formation in the trabecular metaphysis of long bone. Mandibular trabecular bones do not appear to be affected by OVX. CONCLUSION: The cranial and appendicular skeletons differ from one another in that osteoclasts from calvarial bone have the highest resorptive capacity which is coupled to bone formation both pre and post-OVX. Mandibular bones show the lowest turnover rates and are not affected by OVX.

Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state.

Identification of quantitative trait loci influencing inflammation-mediated alveolar bone loss: insights into polygenic inheritance of host-biofilm disequilibria in periodontitis.

BACKGROUND AND OBJECTIVE: The relative contribution of genetic and environmental factors to the onset and progression of periodontitis is inconclusive. Despite the high prevalence, phenotypic heterogeneity and significant local and systemic implications of this disease, early detection and individualized therapy are problematic. Using a murine model of periodontitis in a panel of 17 recombinant inbred mice, the current study addressed the heritability of, and oral dysbiosis associated with, inflammation-mediated alveolar bone loss (iABL), the hallmark of periodontitis. MATERIAL AND METHODS: Quantitative trait locus (QTL) genomics and quantitative PCR for over 99% of known murine oral microbiota were used. RESULTS: It was found that iABL is a polygenic trait with 32.7% heritability. One suggestive QTL, nicknamed inflammation-mediated alveolar bone loss locus (iABLL), was identified on chromosome 2. Eleven genes involved in innate immune responses and bone metabolism, particularly related to macrophage and osteoblast function, namely Etl4, Pdss1, Cobll1, 9330158F14Rik, Xirp2, Stk39, Mettl5, Metapl1, Itga6, Pdk1 and Sp3, were found in the iABLL using cis expression QTL and nonsynonymous single nucleotide polymorphism analyses. Specific oral microbiome shifts in saliva and tongue mucosa are associated with disease in this model. CONCLUSION: Our results indicate that complex host-biofilm interactions generate pathogenic states that extend beyond subgingival biofilms and periodontal tissues. Although no temporal relationship between the onset of iABL and microbiome changes were established, our findings suggest that host factors may be responsible for pathogenic shifts in subgingival biofilms when persistent and undisturbed.

Factors Influencing Adoption of New Technologies into Dental Practice: A Qualitative Study.

The objective of this study was to explore factors affecting decisions to adopt new technologies into dental practice using a colorimetric rinse test for detection of periodontal disease as a model. Focus groups with key informants in Canadian dentistry and dental hygiene were conducted. A deductive approach used Rogers's diffusion of innovation theory as a framework for organizing codes and subcodes. Two members of the research team independently reviewed and analyzed the data using NVivo 10. The attributes of the technology itself emerged as primary influencers. Perceived relative advantages of the diagnostic mouth rinse over existing methods were potential time efficiency, low implementation cost, and utility of the tool. Low complexity, compatibility with existing routines/beliefs, and the potential for reinvention-the use of a technology for other than its intended purpose (i.e., patient education, monitoring of disease, screening tool in nondental settings)-were other important features enhancing adoption. An overarching concern was that any new technology benefit the patient. Contextual factors also play a role. Numerous communication channels, including opinion leaders, patients, marketing, continuing education courses, and strength of evidence, influenced clinicians, with peer interaction being a stronger influence than marketing. Similar themes arose from specialist, general dentist, and dental hygienist focus groups. Adopter characteristics also came into play: participants ranged in their self-reported innovativeness with many considering themselves "early adopters" of new technology. Findings of this study suggest that the innovation adoption process is not straightforward, but attributes of the innovation, contextual factors, and adopter characteristics play important roles in the process. Knowledge Transfer Statement: Various factors affect the adoption of new tools into clinical dental practice. These include attributes of the test or tool itself, the context of the settings in which the tool is introduced to practitioners, and the characteristics of the clinicians themselves. A qualitative study of dentists and dental hygienists investigated these factors. Situations in which dentists and hygienists interact with their peers and colleagues-through social networks, continuing education courses, conventions, or personal contact-were a major driver in the decision to adopt new technologies. However, even among "early adopters," most were reluctant to use new tests or tools unless they perceived a benefit to their patients or practice.

Deletion of filamin A in monocytes protects cortical and trabecular bone from post-menopausal changes in bone microarchitecture.

The objective of the study was to determine the in vivo role of Filamin A (FLNA) in osteoclast generation and function, through the assessment of trabecular bone morphology, bone turnover, and the resulting changes in mechanical properties of the skeleton in mice with targeted deletion of FLNA in pre-osteoclasts. Using a conditional targeted knockdown of FLNA in osteoclasts, we assessed bone characteristics in vivo including micro-computed tomography (micro-ct), histomorphometric analyses, and bone mechanical properties. These parameters were assessed in female mice at 5 months of age, in an aging protocol (comparing 5-month-old and 11-month-old mice) and an osteoporosis protocol [ovariectomized (OVX) at 5 months of age and then sacrificed at 6 and 11 months of age]. In vivo bone densitometry, mechanical and histomorphometric analyses revealed a mild osteoporotic phenotype in the FLNA-null 5-month and aging groups. The WT and FLNA-KO bones did not appear to age differently. However, the volumetric bone mineral density decrease associated with OVX in WT is absent in FLNA-KO-OVX groups. The skeleton in the FLNA-KO-OVX group does not differ from the FLNA-KO group both in mechanical and structural properties as shown by mechanical testing of femora and vertebrae and histomorphometry of vertebrae. Additionally, FLNA-KO femora are tougher and more ductile than WT femora. The result of this study indicates that while FLNA-KO bones are weaker than WT bones, they do not age differently and are protected from estrogen-mediated post-menopausal osteoporosis.

Quantifying oral inflammatory load: oral neutrophil counts in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Neutrophils are the primary white blood cells that are recruited to fight the initial phases of microbial infections. While healthy norms have been determined for circulating blood neutrophil counts in order to identify patients with suspected systemic infections, the levels of oral neutrophils (oPMNs) in oral health and in the presence of periodontal diseases have not been described. It is important to address this deficiency in our knowledge as neutrophils are the primary immune cell present in the crevicular fluid and oral environment and previous work has suggested that they may be good indicators of overall oral inflammation and periodontal disease severity. The objective of this study was to measure oPMN counts obtained in a standardized oral rinse from healthy patients and from those with chronic periodontal disease in order to determine if oPMN levels have clinical relevance as markers of periodontal inflammation. A parallel goal of this investigation was to introduce the concept of 'oral inflammatory load', which constitutes the inflammatory burden experienced by the body as a consequence of oral inflammatory disease. MATERIAL AND METHODS: Periodontal examinations of patients with a healthy periodontium and chronic periodontal disease were performed (n = 124). Two standardized consecutive saline rinses of 30 s each were collected before patient examination and instrumentation. Neutrophils were quantified in the rinse samples and correlated with the clinical parameters and periodontal diagnosis. RESULTS: Average oPMN counts were determined for healthy patients and for those with mild, moderate and severe chronic periodontal diseases. A statistically significant correlation was found between oPMN counts and deep periodontal probing, sites with bleeding on probing and overall severity of periodontal disease. CONCLUSIONS: oPMN counts obtained through a 30-s oral rinse are a good marker of oral inflammatory load and correlate with measures of periodontal disease severity.

The effect of bisphosphonate therapy on neutrophil function: a potential biomarker.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) occurs subsequent to intravenous and oral bisphosphonate exposure in a small subset of patients. The identification of the pathophysiologic mechanisms has not been fully elucidated. Evidence of concurrent bacterial colonization at sites of bone necrosis, previous reports of neutrophil-related complications in some patients taking some bisphosphonates, along with perturbed neutrophil function in bisphosphonate-treated mice, suggest an innate immune role in the development of BRONJ. This study investigated neutrophil function in BRONJ patients to determine if neutrophil functional defects may serve as a potential biomarker for BRONJ susceptibility. Two populations were studied: patients with BRONJ and those beginning intravenous pamidronate. Healthy control patients were used for comparison. Twenty-three patients with BRONJ and five patients who were beginning pamidronate therapy provided neutrophil samples from the mouth (oral rinses) and from blood. Neutrophils from the population of patients with BRONJ and from those post-pamidronate treatment showed lower reactive-oxygen species production and impaired chemotaxis relative to controls. These data suggest that a compromise in neutrophil function may be a potential biomarker for BRONJ susceptibility.

Zoledronate and pamidronate depress neutrophil functions and survival in mice.

BACKGROUND AND PURPOSE: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been identified as a severe complication of patients previously treated with i.v. bisphosphonates. It has been noted that necrotic bone from BRONJ sites display signs of bacterial infection suggesting that an immune defect may play a role in the pathophysiology of BRONJ. Here, we have examined the effect of two potent bisphosphonates, zoledronate and pamidronate, on neutrophil function, differentiation and survival. EXPERIMENTAL APPROACH: The effect of bisphosphonates on chemotaxis, NADPH oxidase activity and neutrophil survival were assessed in vitro using bone marrow-derived primary neutrophils or in vitro differentiated haematopoetic progenitors from mice. The same parameters and the number of circulating neutrophils were quantified in neutrophils isolated from mice treated in vivo with zoledronate. In vivo recruitment of neutrophils was assessed by sodium periodate-induced peritonitis. KEY RESULTS: Zoledronate and pamidronate inhibited in vitro neutrophil chemotaxis and NADPH oxidase activity in a dose-dependent manner. In vivo recruitment of neutrophils was also suppressed. Zoledronate did not affect in vitro differentiation of neutrophils but shortened their life span in a granulocyte-colony stimulating factor-dependent manner. fMLP-induced activation of RhoA activity was decreased by zoledronate treatment. CONCLUSIONS AND IMPLICATIONS: Our results show that bisphosphonate exposure leads to impaired neutrophil chemotaxis, neutrophil NADPH oxidase activity and reduced circulating neutrophil counts. This work suggests that bisphosphonates have the potential to depress the innate immune system for a prolonged time, possibly contributing to the pathogenesis of BRONJ.

Deleting Rac1 improves vertebral bone quality and resistance to fracture in a murine ovariectomy model.

SUMMARY: The roles of Rac1 and Rac2 in regulating osteoclast-mediated bone quality in postmenopausal osteoporosis were evaluated using an ovariectomized murine model. Animals' bone composition and architecture were evaluated. Our results demonstrate that the deletion of Rac1 increases vertebral bone quality compared to wild-type bones in an ovariectomized model. INTRODUCTION: To determine the roles of the Rho family small GTPases Rac1 and Rac2 in regulating osteoclast-mediated bone quality in a model of postmenopausal osteoporosis. METHODS: Twelve-month-old female mice from three genotypes-wild type (WT), Rac1 null (LysM.Rac1 KO), and Rac2 null (Rac2KO)--were studied in control and ovariectomized groups (mice previously ovariectomized at 4 months of age). Animals were sacrificed at 12 months of age, and the femora and vertebrae were harvested for mechanical testing, bone densitometry, micro-computed tomography, and histomorphometric analyses to evaluate bone mineralization and architecture. The results were compared between groups using ANOVA and LSD post-hoc tests. RESULTS: We observed that LysM.Rac1 KO mice showed higher vertebral bone mineral density compared to WT in both control and ovariectomized groups. Consistent with this finding, LysM.Rac1 KO vertebrae showed increased resistance to fracture and increased trabecular connectivity compared to WT in both groups. Micro-CT analysis revealed that Rac2KO ovariectomized vertebrae have more trabecular bone compared to WT and LysM.Rac1 KO, but this did not translate into increased fracture resistance. CONCLUSION: Our results demonstrate that the deletion of Rac1 increases vertebral bone quality compared to WT bones in a postmenopausal osteoporosis model.

Novel rinse assay for the quantification of oral neutrophils and the monitoring of chronic periodontal disease.

BACKGROUND AND OBJECTIVES: The aim of this study was to develop a single, rapid, noninvasive oral rinse assay to enable the accurate quantification of oral neutrophils. Products released by neutrophils are partly responsible for the destruction observed in periodontitis. Quantification of oral neutrophil levels is important for understanding their role in periodontal diseases. Previous studies have relied on time-consuming serial rinses and cumbersome counting techniques for the collection and quantification of oral neutrophils. MATERIAL AND METHODS: Patients with chronic periodontal disease provided rinse samples before and after phase I periodontal treatment. Cells in the rinse samples were stained with acridine orange, and neutrophil counts were carried out using a fluorescence microscope and a hemocytometer. RESULTS: This assay allowed us to detect a significant difference in pretreatment oral neutrophil counts between periodontal disease and healthy control groups (p < 0.001). Patients who responded favorably to phase I therapy demonstrated a 43% reduction in oral neutrophil counts compared with their pretreatment levels (p = 0.019). Patients who did not respond to phase I periodontal treatment showed no significant difference in oral neutrophil levels (p = 0.39). CONCLUSION: Oral neutrophil levels, as determined by a rapid oral rinse, reflect the severity of periodontal disease and treatment response. A single, rapid, oral rinse assay is an effective means of collecting and quantifying oral neutrophil levels and may serve as an excellent research tool for further study of the role of neutrophils in periodontal diseases.

A noninvasive oral rinse assay to monitor engraftment, neutrophil tissue delivery and susceptibility to infection following HSCT in pediatric patients.

The time interval between neutrophil tissue delivery and blood confirmed engraftment following hematopoietic stem cell transplantation (HSCT) may serve as an indicator of patient susceptibility to infection. Using an oral rinse protocol, we studied neutrophil tissue delivery kinetics and its relationship to clinical parameters post-HSCT in 29 pediatric patients. Oral neutrophil counts were compared to circulating neutrophil levels, oral mucositis scores and infection-related febrile episodes after engraftment. Blood engraftment (BE) is currently defined by a blood neutrophil count of > or =0.5 x 10(9)/l. We defined oral engraftment (OE) as the day neutrophils returned in the mouth post-HSCT (> or =0.25 x 10(4)/ml oral neutrophils in the rinse sample). We found that neutrophils reappeared 6.3+/-3.9 s.d. days earlier in the mouth than in the circulation enabling us to identify successful engraftment almost 1 week sooner than using blood count values alone. Furthermore, the time-span between OE and BE was inversely related to the number of infection-related febrile episodes post-BE. We conclude that monitoring the timing of neutrophil tissue delivery through a rapid oral rinse may yield important insights into the biology of neutrophil recovery during and after engraftment and the factors associated with neutrophil tissue recruitment.

Scaling the microrheology of living cells.

We report a scaling law that governs both the elastic and frictional properties of a wide variety of living cell types, over a wide range of time scales and under a variety of biological interventions. This scaling identifies these cells as soft glassy materials existing close to a glass transition, and implies that cytoskeletal proteins may regulate cell mechanical properties mainly by modulating the effective noise temperature of the matrix. The practical implications are that the effective noise temperature is an easily quantified measure of the ability of the cytoskeleton to deform, flow, and reorganize.

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.