Distinct Murine Mucosal Langerhans Cell Subsets Develop from Pre-dendritic Cells and Monocytes.

Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state.

Membrane barriers for guided bone regeneration: An overview of available biomaterials.

Dental implants revolutionized the treatment options for restoring form, function, and esthetics when one or more teeth are missing. At sites of insufficient bone, guided bone regeneration (GBR) is performed either prior to or in conjunction with implant placement to achieve a three-dimensional prosthetic-driven implant position. To date, GBR is well documented, widely used, and constitutes a predictable and successful approach for lateral and vertical bone augmentation of atrophic ridges. Evidence suggests that the use of barrier membranes maintains the major biological principles of GBR. Since the material used to construct barrier membranes ultimately dictates its characteristics and its ability to maintain the biological principles of GBR, several materials have been used over time. This review, summarizes the evolution of barrier membranes, focusing on the characteristics, advantages, and disadvantages of available occlusive barrier membranes and presents results of updated meta-analyses focusing on the effects of these membranes on the overall outcome.

Excessive inflammatory response to infection in experimental peri-implantitis: Resolution by Resolvin D2.

AIM: The aetiology and pathogenesis of peri-implantitis are currently under active research. This study aimed to dissect the pathogenesis of murine experimental peri-implantitis and assess Resolvin D2 (RvD2) as a new treatment modality. MATERIALS AND METHODS: Four weeks following titanium implant insertion, mice were infected with Porphyromonas gingivalis using single or multiple oral lavages. RvD2 was administrated following infection, and tissues were analysed using flow cytometry, quantitative RT-PCR, taxonomic profiling, and micro-computed tomography. RESULTS: Repeated infections with Pg resulted in microbial dysbiosis and a higher influx of innate and adaptive leukocytes to the peri-implant mucosa (PIM) than to gingiva surrounding the teeth. This was accompanied by increased expression levels of IFN-α, IL-1ß, and RANKL\OPG ratio. Interestingly, whereas repetitive infections resulted in bone loss around implants and teeth, a single infection induced bone loss only around implants, suggesting a higher susceptibility of the implants to infection. Treatment with RvD2 prevented Pg-driven bone loss and reduced leukocyte infiltration to the PIM. CONCLUSIONS: Murine dental implants are associated with dysregulated local immunity and increase susceptibility to pathogen-induced peri-implantitis. However, the disease can be prevented by RvD2 treatment, highlighting the promising therapeutic potential of this treatment modality.

Mutual interplay between IL-17-producing γδT cells and microbiota orchestrates oral mucosal homeostasis.

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6+ cells, and to a lesser extent Vγ1+ and Vγ4+ cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6+ cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6+ and Vγ4+ cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.

Cell-intrinsic regulation of murine epidermal Langerhans cells by protein S.

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.

GAS6 is a key homeostatic immunological regulator of host-commensal interactions in the oral mucosa.

The oral epithelium contributes to innate immunity and oral mucosal homeostasis, which is critical for preventing local inflammation and the associated adverse systemic conditions. Nevertheless, the mechanisms by which the oral epithelium maintains homeostasis are poorly understood. Here, we studied the role of growth arrest specific 6 (GAS6), a ligand of the TYRO3-AXL-MERTK (TAM) receptor family, in regulating oral mucosal homeostasis. Expression of GAS6 was restricted to the outer layers of the oral epithelium. In contrast to protein S, the other TAM ligand, which was constitutively expressed postnatally, expression of GAS6 initiated only 3-4 wk after birth. Further analysis revealed that GAS6 expression was induced by the oral microbiota in a myeloid differentiation primary response gene 88 (MyD88)-dependent fashion. Mice lacking GAS6 presented higher levels of inflammatory cytokines, elevated frequencies of neutrophils, and up-regulated activity of enzymes, generating reactive nitrogen species. We also found an imbalance in Th17/Treg ratio known to control tissue homeostasis, as Gas6-deficient dendritic cells preferentially secreted IL-6 and induced Th17 cells. As a result of this immunological shift, a significant microbial dysbiosis was observed in Gas6-/- mice, because anaerobic bacteria largely expanded by using inflammatory byproducts for anaerobic respiration. Using chimeric mice, we found a critical role for GAS6 in epithelial cells in maintaining oral homeostasis, whereas its absence in hematopoietic cells synergized the level of dysbiosis. We thus propose GAS6 as a key immunological regulator of host-commensal interactions in the oral epithelium.

Oral infection with Porphyromonas gingivalis induces peri-implantitis in a murine model: Evaluation of bone loss and the local inflammatory response.

AIM: Peri-implantitis is a major health concern, with unclear pathogenesis, and with no accessible animal models. Our aim was to establish a mouse model for peri-implantitis and to investigate mediators of inflammation. MATERIALS AND METHODS: Mice were divided into implanted versus non-implanted groups. Implants were inserted immediately following the extraction of the upper first molar. Four weeks following implantation, implanted and non-implanted mice were challenged with either Porphyromonas gingivalis or vehicle (eight mice in each subgroup, 32 mice in total). Alveolar bone loss and expression of inflammatory mediators in the soft tissue were assessed 42 days following infection. RESULTS: Porphyromonas gingivalis infection induced greater bone loss around implants than around teeth. In non-infected animals, the presence of the implant correlated with elevated expression of Il-10, Foxp3 and Rankl/Opg ratio, while Tnf-α levels were decreased relative to tissue around teeth. Six weeks following infection, Tnf-α increased significantly while the expression of Foxp3 decreased in the tissue around the implants. No significant differences in anti- or pro-inflammatory mediators were found around teeth of infected, relative to non-infected mice. CONCLUSIONS: Oral infection with P. gingivalis of mice with implants induced bone loss and a shift in gingival cytokine expression. This mouse model enables exploration of the pathogenesis of peri-implantitis and testing of novel treatments.

Langerhans cells down-regulate inflammation-driven alveolar bone loss.

Excessive bone resorption is frequently associated with chronic infections and inflammatory diseases. Whereas T cells were demonstrated to facilitate osteoclastogenesis in such diseases, the role of dendritic cells, the most potent activators of naive T cells, remains unclear. Using a model involving inflammation-driven alveolar bone loss attributable to infection, we showed that in vivo ablation of Langerhans cells (LCs) resulted in enhanced bone loss. An increased infiltration of B and T lymphocytes into the tissue surrounding the bone was observed in LC-ablated mice, including receptor activator of NF-κB ligand (RANKL)-expressing CD4(+) T cells with known capabilities of altering bone homeostasis. In addition, the absence of LCs significantly reduced the numbers of CD4(+)Foxp3(+) T-regulatory cells in the tissue. Further investigation revealed that LCs were not directly involved in presenting antigens to T cells. Nevertheless, despite their low numbers in the tissue, the absence of LCs resulted in an elevated activation of CD4(+) but not CD8(+) T cells. This activation involved elevated production of IFN-γ but not IL-17 or IL-10 cytokines. Our data, thus, reveal a protective immunoregulatory role for LCs in inflammation-induced alveolar bone resorption, by inhibiting IFN-γ secretion and excessive activation of RANKL(+)CD4(+) T cells with a capability of promoting osteoclastogenesis.

Periodontitis and Metabolic Syndrome: Statistical and Machine Learning Analytics of a Nationwide Study.

This study aimed to analyze the associations between periodontitis and metabolic syndrome (MetS) components and related conditions while controlling for sociodemographics, health behaviors, and caries levels among young and middle-aged adults. We analyzed data from the Dental, Oral, and Medical Epidemiological (DOME) record-based cross-sectional study that combines comprehensive sociodemographic, medical, and dental databases of a nationally representative sample of military personnel. The research consisted of 57,496 records of patients, and the prevalence of periodontitis was 9.79% (5630/57,496). The following parameters retained a significant positive association with subsequent periodontitis multivariate analysis (from the highest to the lowest OR (odds ratio)): brushing teeth (OR = 2.985 (2.739-3.257)), obstructive sleep apnea (OSA) (OR = 2.188 (1.545-3.105)), cariogenic diet consumption (OR = 1.652 (1.536-1.776)), non-alcoholic fatty liver disease (NAFLD) (OR = 1.483 (1.171-1.879)), smoking (OR = 1.176 (1.047-1.322)), and age (OR = 1.040 (1.035-1.046)). The following parameters retained a significant negative association (protective effect) with periodontitis in the multivariate analysis (from the highest to the lowest OR): the mean number of decayed teeth (OR = 0.980 (0.970-0.991)); North America as the birth country compared to native Israelis (OR = 0.775 (0.608-0.988)); urban non-Jewish (OR = 0.442 (0.280-0.698)); and urban Jewish (OR = 0.395 (0.251-0.620)) compared to the rural locality of residence. Feature importance analysis using the eXtreme Gradient Boosting (XGBoost) machine learning algorithm with periodontitis as the target variable ranked obesity, OSA, and NAFLD as the most important systemic conditions in the model. We identified a profile of the "patient vulnerable to periodontitis" characterized by older age, rural residency, smoking, brushing teeth, cariogenic diet, comorbidities of obesity, OSA and NAFLD, and fewer untreated decayed teeth. North American-born individuals had a lower prevalence of periodontitis than native Israelis. This study emphasizes the holistic view of the MetS cluster and explores less-investigated MetS-related conditions in the context of periodontitis. A comprehensive assessment of disease risk factors is crucial to target high-risk populations for periodontitis and MetS.

Myeloid cell-derived PROS1 inhibits tumor metastasis by regulating inflammatory and immune responses via IL-10.

Stimulation of TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases promotes tumor progression through numerous cellular mechanisms. TAM cognate ligands GAS6 and PROS1 (for TYRO3 and MERTK) are secreted by host immune cells, an interaction which may support tumor progression. Here, we revealed an unexpected antimetastatic role for myeloid-derived PROS1: suppressing metastatic potential in lung and breast tumor models. Pros1 deletion in myeloid cells led to increased lung metastasis, independent of primary tumor infiltration. PROS1-cKO bone marrow-derived macrophages (BMDMs) led to elevated TNF-α, IL-6, Nos2, and IL-10 via modulation of the Socs3/NF-κB pathway. Conditioned medium from cKO BMDMs enhanced EMT, ERK, AKT, and STAT3 activation within tumor cells and promoted IL-10-dependent invasion and survival. Macrophages isolated from metastatic lungs modulated T cell proliferation and function, as well as expression of costimulatory molecules on DCs in a PROS1-dependent manner. Inhibition of MERTK kinase activity blocked PROS1-mediated suppression of TNF-α and IL-6 but not IL-10. Overall, using lung and breast cancer models, we identified the PROS1/MERTK axis within BMDMs as a potent regulator of adaptive immune responses with a potential to suppress metastatic seeding and revealed IL-10 regulation by PROS1 to deviate from that of TNF-α and IL-6.

Dancing Dorsal Quadrilaterals: A Novel Peripherally Induced Movement Disorder.

Importance: Recognized peripherally induced movement disorders include the painful legs moving toes syndrome, postamputation dyskinesias, and belly dancer dyskinesias. Objective: To introduce and characterize the dancing dorsal quadrilaterals, a novel peripherally induced movement disorder that predominantly affects dorsal quadrilateral muscles (trapezius and rhomboids) after upper spine instrumentation. Design, Setting, and Participants: Between 1990 and 2015, a total of 4 patients who developed abnormal movements of the dorsal quadrilateral muscles after upper spine instrumentation were referred to movement disorders clinics at 3 academic medical centers in the United States, Canada, and Argentina. A prospective and retrospective analysis of the clinical and electrophysiologic characteristics of their abnormal movements is presented in this brief report. Data were analyzed between July 2015 and January 2018. Exposures: Extensive upper spine instrumentation complicated with misalignment and prolonged postsurgical neuropathic pain. Main Outcomes and Measures: Video documentation of clinical and electrophysiologic characteristics of dancing dorsal quadrilaterals. Results: Four patients with upper spine disease (2 women and 2 men, ranging in age from early 30s to early 70s) required extensive surgical manipulation and instrumentation that was complicated by misalignment, prolonged dorsal neuropathic pain, and unusual abnormal movements. These movements consisted of semirhythmic, repetitive writhing, and jerky movements of the scapular region with distinctive rotatory motions. They are referred to as the dancing dorsal quadrilaterals because they predominantly affected the bilateral trapezius and rhomboids (dorsal quadrilateral muscles) but could spread to adjacent muscles, and they are similar in appearance and possibly pathogenesis to "belly dancer" dyskinetic movements. The movements of the dancing dorsal quadrilaterals occur when upright but not when lying down or during voluntary muscle activation. Sensory stimulation also diminishes the movements. Long-duration bursts of normal motor unit potentials with normal recruitment pattern were evidenced. Conclusions and Relevance: The dancing dorsal quadrilaterals syndrome represents a further example of a peripherally induced movement disorder characterized by neuropathic pain preceding a regional movement disorder following soft-tissue or nerve injury.

Resolvin D2 Restrains Th1 Immunity and Prevents Alveolar Bone Loss in Murine Periodontitis.

Periodontitis is an infectious inflammatory disease of the supporting structures of the teeth. Resolvins are part of a large family of specialized pro-resolving lipid mediators that enhance active resolution of inflammation and return of inflammatory lesions to homeostasis. In this paper, we demonstrate that resolvin D2 (RvD2), a product of docosahexaenoic acid (DHA) metabolism, prevents alveolar bone loss in Porphyromonas gingivalis-induced experimental periodontitis. Investigations of the immune mechanism of RvD2 actions reveal that 6 weeks after infection, the gingiva of RvD2-treated mice exhibit decreased CD4+ T-cells as well as lower RANKL expression levels and higher osteoprotegerin expression levels. Systemically, RvD2 prevents chronic secretion of IFN-γ and rapidly restores IFN-α levels, without dampening the P. gingivalis-specific immune response. In the gingiva, immediately after P. gingivalis inoculation, RvD2 regulates the mRNA expression of IFN-γ, IL-1ß, TNF-α, and IL-10, hence contributing to maintaining local homeostasis. Moreover, RvD2 treatment reduces local neutrophil numbers, whereas pro-resolving macrophage counts were increased. These findings suggest that RvD2 resolves innate inflammatory responses, inhibiting systemic and gingival Th1-type adaptive responses that are known to mediate alveolar bone loss in this model.

Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen.

Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6-/- mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.

Impaired Differentiation of Langerhans Cells in the Murine Oral Epithelium Adjacent to Titanium Dental Implants.

Peri-implantitis is a destructive inflammatory process affecting tissues surrounding dental implants and it is considered a new global health concern. Human studies have suggested that the frequencies of Langerhans cells (LCs), the main antigen-presenting cells (APCs) of the oral epithelium, are dysregulated around the implants. Since LCs play a role in regulating oral mucosal homeostasis, we studied the impact of dental titanium implants on LC differentiation using a novel murine model. We demonstrate that whereas the percentage of LC precursors (CD11c+MHCII+) increased in the peri-implant epithelium, the frequencies of LCs (CD11c+MHCII+EpCAM+langerin+) were significantly reduced. Instead, a population of partially developed LCs expressing CD11c+MHCII+EpCAM+ but not langerin evolved in the peri-implant mucosa, which was also accompanied by a considerable leukocyte infiltrate. In line with the increased levels of LC precursors, expression of CCL2 and CCL20, chemokines mediating their translocation to the epithelium, was elevated in the peri-implant epithelium. However, expression of TGF-ß1, the major cytokine driving final differentiation of LCs, was reduced in the epithelium. Further analysis revealed that while the expression of the TGF-ß1 canonical receptor activing-like kinase (ALK)5 was upregulated, expression of its non-canonical receptor ALK3 was decreased. Since titanium ions releasing from implants were proposed to alter APC function, we next analyzed the impact of such ions on TGF-ß1-induced LC differentiation cultures. Concurring with the in vivo studies, the presence of titanium ions resulted in the generation of partially developed LCs that express CD11c+MHCII+EpCAM+ but failed to upregulate langerin expression. Collectively, these findings suggest that titanium dental implants have the capacity to impair the development of oral LCs and might subsequently dysregulate immunity in the peri-implant mucosa.

Analysis of Leukocytes in Oral Mucosal Tissues.

The oral mucosa is constantly exposed to an immense amount of microorganisms, while some colonize the various anatomical niches existing in the oral cavity. To deal with such a complex challenge, the oral mucosal immune system must tolerate commensal microorganisms but prevent invasion of pathogens. Such activity is likely to be achieved by a wide range of mechanisms that could be similar or different to those employed by other mucosal tissues. The dental biofilm represents a unique challenge to the mucosal immune system, and inadequate immune responses might lead to periodontal diseases and the associated adverse systemic complications. It is thus crucial to study the mechanisms by which the oral mucosal immune system maintains homeostasis, and also induces protective immune responses against pathogens. To facilitate probing oral mucosal immunity, we describe here methods allowing immunological analysis of murine oral tissues using flow cytometry and immunofluorescence techniques.

Decreased Palpebral Fissure in Patients with Parkinson's Disease.

BACKGROUND: The diagnosis of Parkinson's disease (PD) is based exclusively on clinical criteria established by the Queen Square Brain Bank. On occasion, these clinical symptoms may be subtle and inconclusive; therefore, it becomes necessary to appeal to contributory criteria to establish a correct diagnosis. The authors propose the observation of palpebral fissure (PF) asymmetry as an additional criterion for the diagnosis of PD. OBJECTIVES: The objectives of this study were to determine whether decreased PF (DPF) is more prevalent in patients with PD than in the general population and whether DPF in PD coincides with the side of onset of parkinsonian symptomatology. METHODS: In total, 112 consecutive patients with a diagnosis of PD and 112 control participants without PD were selected between April and June 2014. At the office visit, it was established through clinical observation whether DPF was present. In patients with PD, it was determined whether the DPF was consistent with the side of onset of parkinsonian symptomatology. RESULTS: Of 112 patients with PD, 39 (35%) had DPF clinically evident DPF, and, in 34 (87%), the DPF was consistent with the laterality of parkinsonian signs. In the control group, only 12% (14 of 112 controls) had PF asymmetry. The difference in prevalence of DPF between these groups was statistically significant (P < 0.0001), with an odds ratio of 3.7 (95% confidence interval, 1.8-7.3). Twenty-eight of the 39 patients with PD who had PF asymmetry were treated with levodopa. CONCLUSIONS: Although the data are purely observational, it may be concluded that DPF coincidental with the side of initial parkinsonian symptomatology in patients with probable PD is an additional sign worth considering.

Porphyromonas gingivalis Promotes Unrestrained Type I Interferon Production by Dysregulating TAM Signaling via MYD88 Degradation.

Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ T cells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.

HER2-Targeted Polyinosine/Polycytosine Therapy Inhibits Tumor Growth and Modulates the Tumor Immune Microenvironment.

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688-97. ©2016 AACR.

Isolation, processing and analysis of murine gingival cells.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously (1). Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.