Identifying patterns and predictors of lifestyle modification in electronic health record documentation using statistical and machine learning methods.

Just under half of the 85.7 million US adults with hypertension have uncontrolled blood pressure using a hypertension threshold of systolic pressure ≥ 140 or diastolic pressure ≥ 90. Uncontrolled hypertension increases risks of death, stroke, heart failure, and myocardial infarction. Guidelines on hypertension management include lifestyle modification such as diet and exercise. In order to improve hypertension control, it is important to identify predictors of lifestyle modification assessment or advice to tailor future interventions using these effective, low-risk interventions. Electronic health record data from 14,360 adult hypertension patients at an academic medical center were analyzed using statistical and machine learning methods to identify predictors and timing of lifestyle modification. Multiple variables were statistically significant in analysis of lifestyle modification documentation at multiple time points. Random Forest was the best machine learning method to classify lifestyle modification documentation at any time with Area Under the Receiver Operator Curve (AUROC) 0.831. Logistic regression was the best machine learning method for classifying lifestyle modification documentation at ≤3 months with an AUROC of 0.685. Analyzing narrative and coded data from electronic health records can improve understanding of timing of lifestyle modification and patient, clinic and provider characteristics that are correlated with or predictive of documentation of lifestyle modification for hypertension. This information can inform improvement efforts in hypertension care processes, treatment implementation, and ultimately hypertension control.

Development and validation of a pragmatic natural language processing approach to identifying falls in older adults in the emergency department.

BACKGROUND: Falls among older adults are both a common reason for presentation to the emergency department, and a major source of morbidity and mortality. It is critical to identify fall patients quickly and reliably during, and immediately after, emergency department encounters in order to deliver appropriate care and referrals. Unfortunately, falls are difficult to identify without manual chart review, a time intensive process infeasible for many applications including surveillance and quality reporting. Here we describe a pragmatic NLP approach to automating fall identification. METHODS: In this single center retrospective review, 500 emergency department provider notes from older adult patients (age 65 and older) were randomly selected for analysis. A simple, rules-based NLP algorithm for fall identification was developed and evaluated on a development set of 1084 notes, then compared with identification by consensus of trained abstractors blinded to NLP results. RESULTS: The NLP pipeline demonstrated a recall (sensitivity) of 95.8%, specificity of 97.4%, precision of 92.0%, and F1 score of 0.939 for identifying fall events within emergency physician visit notes, as compared to gold standard manual abstraction by human coders. CONCLUSIONS: Our pragmatic NLP algorithm was able to identify falls in ED notes with excellent precision and recall, comparable to that of more labor-intensive manual abstraction. This finding offers promise not just for improving research methods, but as a potential for identifying patients for targeted interventions, quality measure development and epidemiologic surveillance.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand genotype do not correlate with clinical outcome of renal cell carcinoma patients receiving high-dose IL2.

NK cells play a role in many cancer immunotherapies. NK cell activity is tightly regulated by killer immunoglobulin-like receptor (KIR) and KIR-ligand interactions. Inhibitory KIR-ligands have been identified as HLA molecules, while activating KIR-ligands are largely unknown. Individuals that have not inherited the corresponding KIR-ligand for at least one inhibitory KIR gene are termed the "KIR-ligand missing" genotype, and they are thought to have a subset of NK cells that express inhibitory KIRs for which the corresponding KIR-ligand is missing on autologous tissue, and thus will not be inhibited through KIR-ligand recognition. In some settings where an anticancer immunotherapeutic effect is likely mediated by NK cells, individuals with a KIR-ligand missing genotype have shown improved clinical outcome compared to individuals with an "all KIR-ligands present" genotype. In addition, patients receiving hematopoietic stem cell transplants for leukemia may do better if their donor has more activating KIR genes (i.e., KIR haplotype-B). In a recent multi-institution clinical trial of patients with metastatic renal cell carcinoma receiving high-dose IL2 (HD-IL2), 25 % of patients showed a complete or partial tumor response to this therapy. We genotyped KIR and KIR-ligand genes for these patients (n = 107) and tested whether KIR/KIR-ligand genotypes correlated with patient clinical outcomes. In these analyses, we did not find any significant association of KIR/KIR-ligand genotype (either KIR-ligand missing or the presence of KIR haplotype-B) with patient outcome in response to the HD-IL2 therapy.

Formation of nitrated naphthalene in the sulfate radical oxidation process in the presence of nitrite.

Polycyclic aromatic hydrocarbons (PAHs) have become a global environmental concern due to their potential hazardous implication for human health. In this study, we found that sulfate radical (SO4•-) could effectively degrade naphthalene (NAP), a representative PAH in groundwaters, generating 1-naphthol. This intermediate underwent further degradation, yielding ring-opening products including phthalic acid and salicylic acid. However, the presence of nitrite (NO2-), a prevalent ion in subsurface environments, was observed to compete with NAP for SO4•-, thus slowing down the NAP degradation. The reaction between NO2- and SO4•- generated a nitrogen dioxide radical (NO2•). Concurrently, in-situ formed 1-naphthol underwent further oxidization to the 1-naphthoxyl radical by SO4•-. The coupling of 1-naphthoxyl radicals with NO2• gave rise to a series of nitrated NAP, namely 2-nitro-1-naphthol, 4-nitro-1-naphthol, and 2,4-dinitro-1-naphthol. In addition, the in-situ formed phthalic acid and salicylic acid also underwent nitration, generating nitrophenolic products, although this pathway appeared less prominent than the nitration of 1-naphthol. When 10 µΜ NAP was subjected to heat activated peroxydisulfate oxidation in the presence of 10 µΜ NO2-, the total yield of nitrated products reached 0.730 µΜ in 120 min. Overall, the presence of NO2- dramatically altered the behavior of NAP degradation by SO4•- oxidation and contributed to the formation of toxic nitrated products. These findings raise awareness of the potential environmental risks associated with the application of SO4•--based oxidation processes for the remediation of PAHs-polluted sites in presence of NO2-.

An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling.

The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of 'kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.

Ambiguous and Incomplete: Natural Language Processing Reveals Problematic Reporting Styles in Thyroid Ultrasound Reports.

OBJECTIVE: Natural language processing (NLP) systems convert unstructured text into analyzable data. Here, we describe the performance measures of NLP to capture granular details on nodules from thyroid ultrasound (US) reports and reveal critical issues with reporting language. METHODS: We iteratively developed NLP tools using clinical Text Analysis and Knowledge Extraction System (cTAKES) and thyroid US reports from 2007 to 2013. We incorporated nine nodule features for NLP extraction. Next, we evaluated the precision, recall, and accuracy of our NLP tools using a separate set of US reports from an academic medical center (A) and a regional health care system (B) during the same period. Two physicians manually annotated each test-set report. A third physician then adjudicated discrepancies. The adjudicated "gold standard" was then used to evaluate NLP performance on the test-set. RESULTS: A total of 243 thyroid US reports contained 6,405 data elements. Inter-annotator agreement for all elements was 91.3%. Compared with the gold standard, overall recall of the NLP tool was 90%. NLP recall for thyroid lobe or isthmus characteristics was: laterality 96% and size 95%. NLP accuracy for nodule characteristics was: laterality 92%, size 92%, calcifications 76%, vascularity 65%, echogenicity 62%, contents 76%, and borders 40%. NLP recall for presence or absence of lymphadenopathy was 61%. Reporting style accounted for 18% errors. For example, the word "heterogeneous" interchangeably referred to nodule contents or echogenicity. While nodule dimensions and laterality were often described, US reports only described contents, echogenicity, vascularity, calcifications, borders, and lymphadenopathy, 46, 41, 17, 15, 9, and 41% of the time, respectively. Most nodule characteristics were equally likely to be described at hospital A compared with hospital B. CONCLUSIONS: NLP can automate extraction of critical information from thyroid US reports. However, ambiguous and incomplete reporting language hinders performance of NLP systems regardless of institutional setting. Standardized or synoptic thyroid US reports could improve NLP performance.

Calcium-mediated stress kinase activation by DMP1 promotes osteoblast differentiation.

Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.

Natural language processing of lifestyle modification documentation.

Lifestyle modification, including diet, exercise, and tobacco cessation, is the first-line treatment of many disorders including hypertension, obesity, and diabetes. Lifestyle modification data are not easily retrieved or used in research due to their textual nature. This study addresses this knowledge gap using natural language processing to automatically identify lifestyle modification documentation from electronic health records. Electronic health record notes from hypertension patients were analyzed using an open-source natural language processing tool to retrieve assessment and advice regarding lifestyle modification. These data were classified as lifestyle modification assessment or advice and mapped to a coded standard ontology. Combined lifestyle modification (advice and assessment) recall was 99.27 percent, precision 94.44 percent, and correct classification 88.15 percent. Through extraction and transformation of narrative lifestyle modification data to coded data, this critical information can be used in research, metric development, and quality improvement efforts regarding care delivery for multiple medical conditions that benefit from lifestyle modification.

Follicular lymphoma patients with KIR2DL2 and KIR3DL1 and their ligands (HLA-C1 and HLA-Bw4) show improved outcome when receiving rituximab.

BACKGROUND: The ECOG-ACRIN Cancer Research Group evaluated rituximab treatment schedules for patients with newly-diagnosed low-tumor-burden follicular-lymphoma (FL). All patients received 4-weekly rituximab treatments as induction therapy. Clinically-responding patients were randomized to receive rituximab every 13 weeks ("maintenance") vs. no additional rituximab until progression ("non-maintenance"). Based on "time-to-rituximab-failure (TTRF)", the study-committee reported there was no overall-benefit for maintenance rituximab in this setting. Tumor-reactive mAbs, like rituximab, trigger natural killer (NK) cells. NK-cell responses are regulated, in part, by interactions between killer immunoglobulin-like receptors (KIRs) on NK cells and their interactions with KIR-ligands. In a separate study of children with neuroblastoma treated with a different mAb, we found certain KIR/KIR-ligand genotypes associated with improved outcome. Here, we assessed whether a subset of FL patients show improved outcome from the maintenance rituximab based on these same KIR/KIR-ligand genotypes. METHODS: Genotypes for KIR/KIR-ligand were determined and assessed for associations with outcome [duration of response, TTRF and % tumor shrinkage] as a post-hoc analysis of this phase III trial. Our primary objective was to assess specific KIR/KIR-ligand genotype associations, followed by separate prespecified KIR/KIR-ligand genotype associations in follow-up analyses. Statistical analyses for association of genotype with clinical outcome included: Log-rank tests and Cox proportional hazards regression models to assess duration of response and TTRF; analysis of variance (ANOVA) was used for assessment of % tumor shrinkage. RESULTS: We found that patients inheriting KIR2DL2 and its ligand (HLA-C1) along with KIR3DL1 and its ligand (HLA-Bw4) had improved outcome over patients without this genotype. In addition, patients with KIR2DL2 and HLA-C1 along with KIR3DL1 and HLA-Bw4 also showed improved duration of response and tumor shrinkage if they received maintenance, while patients without this genotype showed no such improvement when receiving maintenance. CONCLUSIONS: The data presented here indicate that a subset of FL patients, identified by certain KIRs/KIR-ligands, have improved outcome and may benefit from additional rituximab treatment. Taken together, this suggests that the efficacy of tumor-reactive mAb treatment for some patients is influenced by KIRs on NK cells. However, prior to considering these genotypes in a clinically-actionable manner, these findings need independent validation in other studies.

Neuroblastoma Patients' KIR and KIR-Ligand Genotypes Influence Clinical Outcome for Dinutuximab-based Immunotherapy: A Report from the Children's Oncology Group.

Purpose: In 2010, a Children's Oncology Group (COG) phase III randomized trial for patients with high-risk neuroblastoma (ANBL0032) demonstrated improved event-free survival (EFS) and overall survival (OS) following treatment with an immunotherapy regimen of dinutuximab, GM-CSF, IL2, and isotretinoin compared with treatment with isotretinoin alone. Dinutuximab, a chimeric anti-GD2 monoclonal antibody, acts in part via natural killer (NK) cells. Killer immunoglobulin-like receptors (KIR) on NK cells and their interactions with KIR-ligands can influence NK cell function. We investigated whether KIR/KIR-ligand genotypes were associated with EFS or OS in this trial.Experimental Design: We genotyped patients from COG study ANBL0032 and evaluated the effect of KIR/KIR-ligand genotypes on clinical outcomes. Cox regression models and log-rank tests were used to evaluate associations of EFS and OS with KIR/KIR-ligand genotypes.Results: In this trial, patients with the "all KIR-ligands present" genotype as well as patients with inhibitory KIR2DL2 with its ligand (HLA-C1) together with inhibitory KIR3DL1 with its ligand (HLA-Bw4) were associated with improved outcome if they received immunotherapy. In contrast, for patients with the complementary KIR/KIR-ligand genotypes, clinical outcome was not significantly different for patients who received immunotherapy versus those receiving isotretinoin alone.Conclusions: These data show that administration of immunotherapy is associated with improved outcome for neuroblastoma patients with certain KIR/KIR-ligand genotypes, although this was not seen for patients with other KIR/KIR-ligand genotypes. Further investigation of KIR/KIR-ligand genotypes may clarify their role in cancer immunotherapy and may enable KIR/KIR-ligand genotyping to be used prospectively for identifying patients likely to benefit from certain cancer immunotherapy regimens. Clin Cancer Res; 24(1); 189-96. ©2017 AACRSee related commentary by Cheung and Hsu, p. 3.

IRB Process Improvements: A Machine Learning Analysis.

OBJECTIVE: Clinical research involving humans is critically important, but it is a lengthy and expensive process. Most studies require institutional review board (IRB) approval. Our objective is to identify predictors of delays or accelerations in the IRB review process and apply this knowledge to inform process change in an effort to improve IRB efficiency, transparency, consistency and communication. METHODS: We analyzed timelines of protocol submissions to determine protocol or IRB characteristics associated with different processing times. Our evaluation included single variable analysis to identify significant predictors of IRB processing time and machine learning methods to predict processing times through the IRB review system. Based on initial identified predictors, changes to IRB workflow and staffing procedures were instituted and we repeated our analysis. RESULTS: Our analysis identified several predictors of delays in the IRB review process including type of IRB review to be conducted, whether a protocol falls under Veteran's Administration purview and specific staff in charge of a protocol's review. CONCLUSIONS: We have identified several predictors of delays in IRB protocol review processing times using statistical and machine learning methods. Application of this knowledge to process improvement efforts in two IRBs has led to increased efficiency in protocol review. The workflow and system enhancements that are being made support our four-part goal of improving IRB efficiency, consistency, transparency, and communication.

FCGR Polymorphisms Influence Response to IL2 in Metastatic Renal Cell Carcinoma.

Purpose: Fc-gamma receptors (FCGRs) are expressed on immune cells, bind to antibodies, and trigger antibody-induced cell-mediated antitumor responses when tumor-reactive antibodies are present. The affinity of the FCGR/antibody interaction is variable and dependent upon FCGR polymorphisms. Prior studies of patients with cancer treated with immunotherapy indicate that FCGR polymorphisms can influence antitumor response for certain immunotherapies that act via therapeutically administered mAbs or via endogenous tumor-reactive antibodies induced from tumor antigen vaccines. The previously published "SELECT" trial of high-dose aldesleukin (HD-IL2) for metastatic renal cell carcinoma resulted in an objective response rate of 25%. We evaluated the patients in this SELECT trial to determine whether higher-affinity FCGR polymorphisms are associated with outcome.Experimental Design: SNPs in FCGR2A, FCGR3A, and FCGR2C were analyzed, individually and in combination, for associations between genotype and clinical outcome.Results: When higher-affinity genotypes for FCGR2A, FCGR3A, and FCGR2C were considered together, they were associated with significantly increased tumor shrinkage and prolonged survival in response to HD-IL2.Conclusions: Although associations of higher-affinity FCGR genotype with clinical outcome have been demonstrated with mAb therapy and with idiotype vaccines, to our knowledge, this is the first study to show associations of FCGR genotypes with outcome following HD-IL2 treatment. We hypothesize that endogenous antitumor antibodies may engage immune cells through their FCGRs, and HD-IL2 may enhance antibody-induced tumor destruction, or antibody-enhanced tumor antigen presentation, via augmented activation of innate or adaptive immune responses; this FCGR-mediated immune activity would be augmented through immunologically favorable FCGRs. Clin Cancer Res; 23(9); 2159-68. ©2016 AACR.

HLA-Bw4-I-80 Isoform Differentially Influences Clinical Outcome As Compared to HLA-Bw4-T-80 and HLA-A-Bw4 Isoforms in Rituximab or Dinutuximab-Based Cancer Immunotherapy.

Killer-cell immunoglobulin-like receptors (KIRs) are a family of glycoproteins expressed primarily on natural killer cells that can regulate their function. Inhibitory KIRs recognize MHC class I molecules (KIR-ligands) as ligands. We have reported associations of KIRs and KIR-ligands for patients in two monoclonal antibody (mAb)-based trials: (1) A Children's Oncology Group (COG) trial for children with high-risk neuroblastoma randomized to immunotherapy treatment with dinutuximab (anti-GD2 mAb) + GM-CSF + IL-2 + isotretinion or to treatment with isotretinoin alone and (2) An Eastern Cooperative Oncology Group (ECOG) trial for adults with low-tumor burden follicular lymphoma responding to an induction course of rituximab (anti-CD20 mAb) and randomized to treatment with maintenance rituximab or no-maintenance rituximab. In each trial, certain KIR/KIR-ligand genotypes were associated with clinical benefit for patients randomized to immunotherapy treatment (immunotherapy in COG; maintenance rituximab in ECOG) as compared to patients that did not receive the immunotherapy [isotretinoin alone (COG); no-maintenance (ECOG)]. Namely, patients with both KIR3DL1 and its HLA-Bw4 ligand (KIR3DL1+/HLA-Bw4+ genotype) had improved clinical outcomes if randomized to immunotherapy regimens, as compared to patients with the KIR3DL1+/HLA-Bw4+ genotype randomized to the non-immunotherapy regimen. Conversely, patients that did not have the KIR3DL1+/HLA-Bw4+ genotype showed no evidence of a difference in outcome if receiving the immunotherapy vs. no-immunotherapy. For each trial, HLA-Bw4 status was determined by assessing the genotypes of three separate isoforms of HLA-Bw4: (1) HLA-B-Bw4 with threonine at amino acid 80 (B-Bw4-T80); (2) HLA-B-Bw4 with isoleucine at amino acid 80 (HLA-B-Bw4-I80); and (3) HLA-A with a Bw4 epitope (HLA-A-Bw4). Here, we report on associations with clinical outcome for patients with KIR3DL1 and these separate isoforms of HLA-Bw4. Patients randomized to immunotherapy with KIR3DL1+/A-Bw4+ or with KIR3DL1+/B-Bw4-T80+ had better outcome vs. those randomized to no-immunotherapy, whereas for those with KIR3DL1+/B-Bw4-I80+ there was no evidence of a difference based on immunotherapy vs. no-immunotherapy. Additionally, we observed differences within treatment types (either within immunotherapy or no-immunotherapy) that were associated with the genotype status for the different KIR3DL1/HLA-Bw4-isoforms. These studies suggest that specific HLA-Bw4 isoforms may differentially influence response to these mAb-based immunotherapy, further confirming the involvement of KIR-bearing cells in tumor-reactive mAb-based cancer immunotherapy.

Msx1/Bmp4 genetic pathway regulates mammalian alveolar bone formation via induction of Dlx5 and Cbfa1.

In the developing mammalian tooth, the cranial neural crest derived dental mesenchyme consists of the dental papilla and dental follicle. The dental papilla gives rise to odontoblasts and dental pulp and the dental follicle gives rise to the periodontium, including the osteoblasts that contribute to the alveolar process. The alveolar process is a specialized intramembranous bone that forms the primary support structure for the dentition. The Msx1 gene controls many aspects of craniofacial development, as evidenced by craniofacial abnormalities seen in Msx1(-/-) mice, including the arrest of tooth development and the absence of the alveolar bone. Previous studies demonstrated that ectopic expression of Bmp4, a downstream target of Msx1, in the Msx1(-/-) dental mesenchyme rescued alveolar bone formation. Here we confirm an early requirement of BMP activity for alveolar bone formation. We show that the expression of Cbfa1 and Dlx5, two genes encode transcription factors that are critical for bone differentiation, overlaps with that of Msx1 and Bmp4 in the developing tooth and alveolar process. We have demonstrated that Dlx5 and Cbfa1 expression is down-regulated in Msx1(-/-) dental mesenchyme and that Msx1 and Bmp4 expression are unaltered in Cbfa1(-/-) mice. These data place Dlx5 and Cbfa1 downstream from the Msx1/Bmp4 in the genetic pathway that regulates tooth development. Ectopic expression of Bmp4 in Msx1 mutants restores the expression of Dlx5, but not Cbfa1, in the dental mesenchyme, and rescues the expression of both Dlx5 and Cbfa1 in the developing alveolar bone. Therefore, the early expression of Cfba1 in the dental mesenchyme appears dispensable for the development of the alveolar bone. Taken together with in vitro gene induction studies, our results demonstrate that BMP4 controls Dlx5 expression in dental mesenchyme, and functions upstream to both Dlx5 and Cbfa1 to regulate alveolar bone formation during tooth development.

Expression and function of NUMB in odontogenesis.

NUMB is a multifunctional protein implicated to function in self-renewal and differentiation of progenitors in several tissues. To characterize the transcripts and to analyze the expression pattern of NUMB in odontogenesis, we isolated 2 full-length clones for NUMB from mouse dental pulp mRNA. One novel sequence contained 200 bp insertion in the phosphotyrosine binding domain (PTB). Confocal microscopy analysis showed strong NUMB expression in human dental pulp stem cells (hDPSC) and preameloblasts. Western blot analysis indicated that NUMB isoforms were differentially expressed in various dental tissues. Immunohistochemical analysis showed that in postnatal mouse tooth germs, NUMB was differentially expressed in the preameloblasts, odontoblasts, cervical loop region, and in the dental pulp stem cells during development. Interestingly, overexpression of NUMB in HAT-7, a preameloblast cell line, had dramatic antagonizing effects on the protein expression level of activated Notch 1. Further analysis of the Notch signaling pathway showed that NUMB significantly downregulates sonic hedgehog (Shh) expression in preameloblasts. Therefore, we propose that NUMB maintains ameloblast progenitor phenotype at the cervical loop by downregulating the activated Notch1 protein and thereby inhibiting the mRNA expression of Shh.

Development of three-dimensional biomimetic scaffold to study epithelial-mesenchymal interactions.

Epithelial-mesenchymal interactions play a key role in the development of tissues such as tooth, lungs, and kidneys. To successfully engineer or repair such living tissues it is necessary to first understand the complex cell-cell and cell-matrix interactions underlying organogenesis. To mimic an in vivo setting it is necessary to assemble a three-dimensional matrix that would facilitate cell-cell interaction leading to proliferation and cellular differentiation. In this study, we have developed an in vitro three-dimensional multilayered coculture system using type I collagen and chitosan blends as matrices, to study epithelial-mesenchymal interactions that occur during tooth morphogenesis. Results from this study showed that the matrix composition influenced the migration, proliferation, and differentiation properties of the epithelial and mesenchymal cells. Specifically, the system supported the migration and differentiation of the HAT-7 epithelial cells and mesenchymal-derived dental pulp stem cells. Results from the in vivo implantation study of the coculture system in mice demonstrated a similar cellular migration and differentiation pattern that corroborates well with the in vitro model. Interestingly, the biopolymer matrix also permitted neovascularization in vivo.

Mouse embryonic diastema region is an ideal site for the development of ectopically transplanted tooth germ.

The anterior eye chamber and the kidney capsule of the mouse have been traditionally used for long-term culture of tooth germ grafts. However, although these sites provide an excellent growth environment, they do not represent real in situ sites for the development of a grafted tooth germ. Here, we describe a protocol to transplant a tooth germ into the mandibular diastema region of mouse embryos using exo utero surgery. Our results demonstrate that the mouse embryonic diastema region represents a normal physiological environment for the development of transplanted tooth germs. Transplanted tooth germs developed synchronically with and became indistinguishable from the endogenous ones. These ectopic teeth were vascularized and surrounded with nerve fibers, and were able to erupt normally. Thus, the exo utero transplantation approach will provide a new avenue to study tooth development and regeneration.

Application of lentivirus-mediated RNAi in studying gene function in mammalian tooth development.

RNA interference (RNAi) has recently become a powerful tool to silence gene expression in mammalian cells, but its application in assessing gene function in mammalian developing organs remains highly limited. Here we describe several unique developmental properties of the mouse molar germ. Embryonic molar mesenchyme, but not the incisor mesenchyme, once dissociated into single cell suspension and re-aggregated, retains its odontogenic potential, the capability of a tissue to instruct an adjacent tissue to initiate tooth formation. Dissociated molar mesenchymal cells, even after being plated in cell culture, retain odontogenic competence, the capability of a tissue to respond to odontogenic signals and to support tooth formation. Most interestingly, while dissociated epithelial and mesenchymal cells of molar tooth germ are mixed and re-aggregated, the epithelial cells are able to sort out from the mesenchymal cells and organize into a well-defined dental epithelial structure, leading to the formation of a well-differentiated tooth organ after sub-renal culture. These unique molar developmental properties allow us to develop a strategy using a lentivirus-mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development. We show that knockdown of Msx1 or Dlx2 expression in the dental mesenchyme faithfully recapitulates the tooth phenotype of their targeted mutant mice. Silencing of Barx1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Barx1 in tooth formation. Our studies have established a reliable and rapid assay that would permit large-scale analysis of gene function in mammalian tooth development.

Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate.

The short stature homeobox gene SHOX is associated with idiopathic short stature in humans, as seen in Turner syndrome and Leri-Weill dyschondrosteosis, while little is known about its close relative SHOX2. We report the restricted expression of Shox2 in the anterior domain of the secondary palate in mice and humans. Shox2-/- mice develop an incomplete cleft that is confined to the anterior region of the palate, an extremely rare type of clefting in humans. The Shox2-/- palatal shelves initiate, grow and elevate normally, but the anterior region fails to contact and fuse at the midline, owing to altered cell proliferation and apoptosis, leading to incomplete clefting within the presumptive hard palate. Accompanied with these cellular alterations is an ectopic expression of Fgf10 and Fgfr2c in the anterior palatal mesenchyme of the mutants. Tissue recombination and bead implantation experiments revealed that signals from the anterior palatal epithelium are responsible for the restricted mesenchymal Shox2 expression. BMP activity is necessary but not sufficient for the induction of palatal Shox2 expression. Our results demonstrate an intrinsic requirement for Shox2 in palatogenesis, and support the idea that palatogenesis is differentially regulated along the anteroposterior axis. Furthermore, our results demonstrate that fusion of the posterior palate can occur independently of fusion in the anterior palate.

Timing of odontogenic neural crest cell migration and tooth-forming capability in mice.

The mammalian tooth develops through sequential and reciprocal interactions between cranial neural crest (CNC)- derived ectomesenchymal cells and the stomadial epithelium. Classic tissue recombination studies demonstrated that premigratory CNC cells and CNC-derived ectomesenchymal cells possess odontogenic capacity and can respond to oral epithelial signals to form a tooth, suggesting that the CNC cells contributing to odontogenic tissue are not prespecified. Here we show that, in mice, CNC cells have populated the forming first branchial arch before the 9-somite stage and continue to migrate into the arch by the 13-somite stage. Grafts of the first arch from the 10-somite embryo or earlier yielded membranous bone and cysts but no teeth after subrenal culture. However, grafts of the first arch with its dorsally adjacent tissue containing migrating neural crest cells from the same age embryos gave rise to teeth. In contrast, teeth formed in first arch grafts that do not contain migrating neural crest cells from embryos with 12 or more somites. Interestingly, the acquisition of tooth forming capability in the first arch coincides with the onset of Fgf8 expression in the oral epithelium. These results suggest that there exists a population of odontogenic neural crest cells that migrates into the first arch between the 10- and 12-somite stages. These cells either possess odontogenic potential and are able to initiate tooth development, or can respond to odontogenic signals derived from the oral epithelium to support tooth formation.