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The advancement of 3D digital scanners has allowed educators to uphold the quality of education whilst implementing novel methods of assessment.The purpose of this study was to compare two evaluation methods, conventional and digital scanned assessments, on student cavity preparations and students and faculty's perceptions of these assessment methods.Predoctoral students performed Class II amalgam and Class III resin composite preparations on simulated teeth for their preclinical competency examinations. Two calibrated faculty evaluated the students' preparations using dental instruments for conventional assessments and 3D scanned images for digital assessments. Faculty scores from both assessment methods were compared and statistically analysed (p .05). Students also self-assessed their performance using the digital scanned images and filled out a perception survey.There was no statistically significant difference between the conventional (Class II; 73.3 ± 12.2 and Class III; 82.5 ± 8.9) and the digital assessment with scanned images (Class II; 71.3 ± 11.0 and Class III; 82.2 ± 8.2). Faculty scores from both methods have shown a trend of comparability. Most students agreed that digital ideal preparation models were useful (84.2%) and almost all students agreed that digital technology helped them learn operative dentistry (91.1%). In contrast, most students agreed that conventional assessment should be the main self-evaluation system (82.2%) and disagreed that digital assessment is more effective than conventional self-assessment (68.3%).Our study demonstrated that 3D digital technology could be an alternative and/or supplementary educational tool to aid in upholding the quality of dental education.

The incisive branch of the inferior alveolar nerve is a vital anatomical structure within the anterior mandible that has not been thoroughly defined and outlined in reports in the literature until recent years. Advances in radiological imaging, particularly the widespread use of cone-beam computed tomography has allowed for accurate visualization of the mandibular incisive canal (MIC) and its associated incisive branch of the inferior alveolar nerve. Surgical damage to the MIC, which could result in hemorrhage and sensory disturbance, may occur in commonly practiced oral and maxillofacial procedures, such as chin bone harvesting, implant placement, fracture repair and removal of pathologic entities of the anterior mandible. Knowledge of both the presence, dimensions and location of the incisive branch is a vital component to pre and peri-operative planning of oral and maxillofacial surgeries performed within the mandible, particularly within the interforaminal zone. In this article, the terminology, anatomy, imaging, surgical consideration, and pathology of the incisive branch will be discussed.

Purpose/objectives Self-assessment is an essential skill for dental professionals. Understanding global trends in self-assessment can highlight the learning needs of students across a diversity of cultural backgrounds. The aim of this study is to compare the self-assessment ability of dental students in the United States and Japan, where cultural backgrounds may differ. Methods Students in the United States (n = 176) completed a typodont premolar and anterior Class II and Class III preparation and restoration. Students in Japan (n = 175) completed a typodont premolar crown preparation. Students and faculty then evaluated the student performance using rubrics for each respective procedure. The difference between the student's self-assessment score and the average faculty score (S-F gap) was calculated and the data were analyzed. Results The mean S-F gap was 2.8% in Japan and 7.6% in the United States. This indicates that Japanese students tended to assess themselves closer to their faculty graders than students in the United States. On average, students in both countries scored themselves higher than their faculty graders. Students in the United States more frequently overestimated their performance and students in Japan more frequently underestimated their performance. For students in the lower quartile, the mean S-F gap was 5.1% in Japan and 14.6% in the United States, indicating a large cultural discrepancy in the lower quartile groups. Conclusions Although different preclinical procedures were compared, our findings demonstrated that Japanese students may score themselves more closely to their faculty assessors than students in the United States. Further investigation with more students completing the same preclinical activity will be needed.

Summary: This study proposed the development of a protocol for class-II preparations with demineralized gingival margins for the improvement of the longevity of restorations. Evidence sources such as location/color/surface hardness/width of demineralized gingival margin with enamel/demineralized enamel (DE)/dentin/cementum were reviewed based on methodological studies and systematic reviews. A decision tree protocol was developed with criteria (i) lesion location: demineralized gingival margins in enamel must be removed, but if close to cementoenamel-junction, color should be evaluated. (ii) Color: yellow/brown lesions must be removed, but if white/opaque, then the surface hardness should be evaluated. (iii) Surface hardness: soft/demineralized gingival margin must be removed, but if adequately hard, width should be evaluated. (iv) Width: lesions less than half-enamel thickness and impenetrable by an explorer, remineralization is possible and the lesion does not need to be removed. A decision tree protocol was set up with the current available literature. Further continued investigations will be needed for the appropriate protocol updates.

There are many reported anatomical variations of the mandibular canal. Consequently, there is great variation in the retromolar area, such as the quantity, size, and location of the retromolar foramen (RMF), the bony entrance of the retromolar canal (RMC). These variations allow for different accessory innervations to the mandibular molars and their adjacent buccal tissue because the RMC contains neurovascular bundles. Consideration of these anatomical variations is crucial for avoiding complications in anesthesia, implant placement, and surgery. However, the rarer canal types are often only imaged by computed tomography (CT) or cone beam computed tomography (CBCT). We present a rare case with bilateral RMF and a unilateral trifid mandibular canal in a cadaver.

Admission to dental school is based on various factors including academic achievements in undergraduate coursework and the Dental Admission Test (DAT) scores. Students' success in an operative course requires fundamental knowledge, hand skills, spatial awareness, and self-assessment ability. The goal of this study is to evaluate how admissions factors, such as Grade Point Average (GPA) and DAT, including the Perceptual Ability Test (PAT), relate to students' academic and preclinical performance and self-assessment skills in preclinical operative dentistry.A total of 239 students were included from seven class years (2016-2022). Third-year dental students participated in a preclinical operative dentistry course. At the end of the course, they took the final multiple-choice exam and performed four competency examination procedures: Class II amalgam preparation and restoration and Class III resin-composite preparation and restoration. Calibrated faculty graded students' work independently and students also self-assessed their performance using the same rubrics as faculty. Linear regressions were performed to estimate the association between the admission factors with the mean faculty scores (measuring preclinical performance), student-faculty (S-F) gap scores (evaluating self-assessment skills), and their final didactic exam scores.Overall, students' self-assessment was higher compared to faculty score. Linear regression analysis demonstrated positive correlations between the PAT and students' preclinical performance as well as between the DAT and their didactic exam scores. In general, S-F gap score decreased as PAT score increased, and it was statistically significant lower for the Class III preparation, indicating a better self-assessment skill. No correlations were observed between student performance and GPA scores.The findings from the association between student performance and admission factors may play an important role in the dental school admissions process and assist students who may benefit from early faculty intervention and support.

Introduction During the COVID‐19 pandemic, dental schools were required to reformat their curricula to accommodate regulations mandated to protect the health of students and faculty. For students enrolled in the Operative Dentistry preclinical courses at the Harvard School of Dental Medicine (HSDM), this modified curriculum included frontloading the course with lectures delivered remotely, followed by in‐person laboratory exercises of learned concepts. The aim of this article was to determine the impact that the modifications had on student performance and student self‐evaluation capabilities. Materials and methods Thirty‐eight students were introduced to this restructured course. Their performance in a final multiple‐choice (MC) examination, four preclinical laboratory competency assessments (class II amalgam preparation and restoration, class III composite preparation and restoration) and their self‐assessment of these preclinical competency assessments were then compared with the pre‐COVID pandemic (P‐CP) classes from years 2014 to 2019 (n = 216 students). Linear regressions were performed to determine differences in mean faculty scores, self‐assessment scores, student‐faculty score gaps (S‐F gaps) and absolute S‐F gaps seen between the class impacted by the pandemic and the P‐CP classes. Results The results demonstrated that students during the COVID‐19 pandemic (D‐CP) had a higher average faculty score in all four preclinical laboratory competency assessments and in the final MC examination. In addition, the S‐F gap was smaller in this cohort as compared with the P‐CP classes. Conclusion Despite the challenges of restructuring the preclinical curricula, D‐CP students performed better than their P‐CP predecessors in multiple facets of this Operative Dentistry course including self‐assessment accuracy.

A 63-year-old healthy female presented in 2002 for implant replacement of her maxillary right premolars following failure of a three-unit bridge replacing the second premolar. In a single appointme...

BACKGROUND: Globally, the direct cost of dental caries is approximately $298B yearly, consuming 5–10% of national healthcare budgets. Bitewing radiographs (BWR) are the standard method of diagnosing interproximal dental caries. In Japan, bitewing radiographs are rarely used. This retrospective observational study was conducted to measure the potential economic impact of carious lesions left undiagnosed and untreated due to this omission of bitewing radiographs. METHODS: The total number of existing carious lesions, the number of undiagnosed lesions, and costs of treating these lesions were calculated from the national database of Ministry of Health, Labor and Wellness in Japan between June 2013 and 2017. The number of affected teeth was estimated using prevalence data and undiagnosed lesions were estimated. The expense associated with treating progressed lesions was calculated using the standard Japanese fee structure. BWR trends were assessed, and analyses were performed to understand the differences between states and populations over time. RESULTS: The average number of BWR taken monthly per office was 48.3±1.1 (average ± SD). It was calculated that an average of 6,429,155 lesions went undiagnosed per month, 93.5 teeth per practice, and 1.6 teeth per patient. The cost of treating lesions that went undiagnosed and then progressed into more invasive restorations was estimated to be between $57M–$218M more (difference between NaF varnish and class II restorations), and $150M–$443M more (difference between Class II restoration and crown or crown with RCT). CONCLUSIONS: BWRs are crucial in diagnosing a significant number of carious lesions. There is considerable impact on health and cost to the national health system due to undiagnosed lesions. Practitioners need to be educated on reading and understanding BWR, and policy should be changed to cover BWR.

PURPOSE/OBJECTIVE Self-assessment is a fundamental skill for dentists and other health care providers. It enables these professionals' ability to critically evaluate the quality of their clinical work and improve through self-directed learning. Researchers have investigated how gender affects self-assessment skills and have shown that male students tend to overestimate their performance while female students tend to underestimate theirs as compared to peer or faculty assessment. The goal of this study was to evaluate how the self-assessment skills of dental students differ by gender in operative preclinical dentistry. METHODS Third-year dental students (N = 208, Class of 2016-2021) self-assessed their work using the same rubrics as faculty on four operative dentistry competency examination procedures: Class II amalgam preparation and restoration and Class III resin-composite preparation and restoration. Two calibrated full-time faculty graded all procedures independently. The Student self-assessment-Faculty grade (S-F) gap scores were calculated and statistically analyzed. RESULTS Overall, both genders overestimated their self-assessment for all four procedures as compared to actual scores given by faculty. Males overestimated more significantly than females for Class II Amalgam preparation, but not for the other three procedures. However, when the S-F gap for all procedures was combined and analyzed together, male students significantly overestimated their self-assessments compared to female students. When female and male students grading scores were stratified into quartiles, there were significantly larger differences between males and females in the lower quartiles, which suggest that difference in self-assessment abilities was more prominent among the lower performing students. CONCLUSION The results of this study demonstrate that gender may affect the accuracy of self-assessment in operative preclinical dentistry.

OBJECTIVE: In this article we review the literature on the inferior labial gland from a clinical and anatomical perspective. BACKGROUND: Regardless of its importance in clinical practice, there are no medical literature that comprehensively reviewed the inferior labial gland. METHODS: A database search using PubMed and Google Scholar was conducted. The following keywords were used in the search: “lower labial salivary gland”, “lower labial gland”, “inferior labial salivary gland”, AND “inferior labial gland”. CONCLUSIONS: The human labial glands are types of minor salivary gland that continuously secrete small amounts of mucous and serous substances to maintain oral health. The inferior labial glands are innervated by the inferior labial branch of the mental nerve, and the inferior labial branch of the facial artery is the main arterial supply to the lower lip. Although they only have an auxiliary role in saliva production compared to the major salivary glands, minor salivary glands provide a certain amount of lubrication in the oral cavity by the continuous outflow of saliva. The inferior labial gland not only promotes moisturization in the oral cavity but also secretes substances with antibacterial effects, which is important for the function of the oral cavity. A recent study showed that the rate of salivary secretion from the inferior labial glands does not change with age, and in some cases the inferior labial glands are used for diagnosing intractable diseases such as Sjogren’s syndrome and cystic fibrosis. In addition, since the inferior labial glands themselves can be the site of cyst and/or neoplasia development, we should be careful to distinguish them from other diseases. Elucidation of the anatomy, physiology, and pathology of the inferior labial glands, is important for understanding human health and diseases.

Objectives The aim of this study is to assess the accuracy of bitewing radiograph interpretation of predoctoral students, residents and general dentists who work under different core curriculum of dental education and healthcare policy. Methods A total of 118 examiners including predoctoral dental students, residents and general dentists from USA and Japan were tasked with evaluating a series of bitewing radiographs and diagnosing interproximal carious lesions. This study was approved by the Harvard Medical School Institutional Review Board (IRB). Participants graded interproximal aspects of those images and categorized the following criteria; “intact”, “enamel caries 1/2 width” or “caries into dentin”. The gold-standard was determined by the consensus of two HSDM full-time faculty. Results There was significant difference in the sensitivity for all three caries levels between the two groups but there was no significant difference on specificity. The positive-predictive and negative-predictive values of the USA group for the enamel caries (Conclusion Teaching and adopting BW radiographs for diagnosis of interproximal caries is integral for dental providers to accurately and efficiently use them in their practices. It is critical that all dental educators approach policymakers to explain the importance of BW radiographs and promote their efficacy for prevention and early diagnosis of interproximal caries.

During the routine dissection of a cadaveric specimen, the left mylohyoid muscle was found to be innervated by both the trigeminal and hypoglossal nerves. This variation was found unilaterally. To our knowledge this dual innervation of the mylohyoid muscle is an extremely rare variation. The possibility of these variants may lead to clinical consequences such as anesthesia failure and iatrogenic injury during surgical procedures in this region. We discuss this anatomical variation and possible developmental etiologies.

Introduction A theory-practice gap in pre-doctoral dental education is a common source of stress for dental students. An interactive, small-group, case-based activity was designed to bridge the gap between pre-clinical and clinical experiences. The aim of our study was to assess the effectiveness of the case-based activity by evaluating students' comfort level in operative procedures. Materials and methods Over 5 years, a total of 172 second-year students from the classes of 2017 through 2021 participated in the case-based activity delivered after the completion of the core operative dentistry course. The exercise included a pre-activity online quiz, an in-class case-based session and a laboratory exercise. Students' self-reported comfort levels in performing operative procedures were collected by surveys at three different times. They included the post-course survey distributed after the completion of the core operative dentistry course, the post-activity survey distributed after the completion of the case-based activity, and the follow-up survey distributed after students completed their first operative procedures in clinic. Results There was a 93% response rate. The average rating of all eight statements revealed statistically significant increase in students' comfort level after completing the case-based activity and after performing their first operative procedures in the teaching practice. Conclusion This observation suggests that the case-based activity was effective in raising students' comfort levels. The activity may serve as an important tool in bridging the theory-practice gap between pre-clinical and clinical operative experiences.

Self-assessment is a critical skill for health care professionals. Because these professionals must be lifelong, self-directed learners and self-regulating practitioners, the ability to self-assess is essential for them. The aims of this study were to measure how dental students self-assessed and peer-assessed their performance in preclinical operative procedures and to evaluate any relationship between their assessment ability and preclinical skills or academic performance. The study was conducted from 2015 to 2016. Third-year students at Harvard School of Dental Medicine self-assessed their work on four preclinical practical exams: Class II amalgam preparation, Class II amalgam restoration, Class III composite preparation, and Class III composite restoration. Three faculty members graded the same preparations and restorations. The difference between the students' self-assessment and the mean faculty assessment was calculated as the student-faculty (S-F) gap. An absolute S-F gap was also calculated with absolute values of differences. A total of 71 students completed these practical exams: 36 in the Class of 2016 and 35 in the Class of 2017. All 71 self-assessments for each of the four practical exams were collected for a response rate of 100%. The results showed that the mean S-F gap ranged from 2% to 8%, and the absolute S-F gap ranged from 7% to 12%. Preclinical performance was correlated with self-assessment accuracy. Low-performing students significantly overestimated their self-assessments compared to the rest of the class. High-performing students had more accurate self-assessments and tended to underestimate themselves. Overall, these results showed that the students had room for improving the accuracy of their self-assessments.

OBJECTIVES: The purpose of this study was to assess the diagnostic property of intraoral bitewing radiographs (BTW) for early stage interproximal caries, and to compare them with periapical radiographs (PA) at different levels of caries progression. METHODS: A total of 241 interproximal surfaces of BTW and corresponding PAs were used. Seven teaching faculty consisting of three oral and maxillofacial radiologists, two operative faculty, and two prosthodontists evaluated the images. The observers graded images as either “intact”, “enamel caries 1/2 width”, or “caries into dentin”. The gold-standard was established by consensus of two experienced faculty with 35 years and 27 years of experience. Specificity, sensitivity, positive-predictive value, and negative-predictive value were calculated for the different level of caries progression. Furthermore, receiver operating curves) of BTW and PAs of each evaluator were made and the area under the curve of BTW and PAs were compared. RESULTS: There was no significant difference in the specificity of BTW and PAs. BTW showed significantly higher sensitivity than PAs in all levels of caries progression (p

The aim of this study was to determine how dental student self-assessment and faculty assessment of operative preparations compared for conventional visual assessment versus assessment of scanned digital 3D models. In 2016, all third-year students in the Class of 2018 (N=35) at Harvard School of Dental Medicine performed preclinical exams of Class II amalgam preparations (C2AP) and Class III composite preparations (C3CP) and completed self-assessment forms; in 2017, all third-year students in the Class of 2019 (N=34) performed the same exams. Afterwards, the prepared typodont teeth were digitally scanned. Students self-assessed their preparations digitally, and four faculty members graded the preparations conventionally and digitally. The results showed that, overall, the students assessed their preparations higher than the faculty assessments. The mean student-faculty gaps for C2AP and C3CP in the conventional assessments were 11% and 5%, respectively. The mean digital student-faculty gap for C2AP and C3CP were 8% and 2%, respectively. In the conventional assessments, preclinical performance was negatively correlated with the student-faculty gap (r=-0.47, p

Genetic variants at multiple loci have been shown to be associated with susceptibility to periodontitis. To better assess the genetic risk factors for periodontitis, we performed a case-control study in 319 Japanese individuals with periodontitis (172 aggressive and 147 chronic disease) and 303 race-matched healthy control individuals. Thirty-five functional gene polymorphisms that had been previously associated with immune responses were genotyped. For all gene polymorphisms tested, no significant differences were observed in the allele frequencies of persons with aggressive, chronic, and combined (aggressive and chronic) periodontitis, compared with control individuals. Multiple logistic regression analysis revealed a significant association of the vitamin D receptor +1056 T/C polymorphism with susceptibility to chronic periodontitis, after adjustment for age, gender, and smoking status (P = 0.002). These results suggest that none of the polymorphisms tested was strongly associated with periodontitis in a Japanese population. However, the vitamin D receptor +1056 polymorphism may be related to chronic periodontitis.

OBJECTIVE: The aim of this study is to compare the in vitro proliferative capacity of periodontal ligament (PDL) cells from aged and juvenile donors. MATERIALS AND METHODS: Flow-cytometric analysis of the cell cycle was used to compare the length of each cell cycle, and the ratio of the cells progressing through the cycles between four PDL cells from juvenile donors and four cells from aged donors. Then, replicative capacity of the PDL cells from three juvenile and three aged donors was compared by serial cultureS. Finally, expression of c-fos was compared between cells proliferating and cells which had reached senescent. RESULTS: Flow-cytometric analysis of the cell cycle had revealed that although there were no differences in the length of each phase of the cell cycle, significant differences were found in the ratio of the cells entering from Gap 1 to DNA synthesis phase of the cell cycle (P< 0.025).Replicative capacity was much longer in two cells from juvenile donors (about 20 population doublings), while all cells from aged donors showed short dividing abilities (less than eight population doublings), hence entered senescent phases shortly. Additionally, no c-fos was detected in cells which had reached senescence upon stimulation with serum. CONCLUSIONS: It is generally believed that aged humans have an impaired wound healing ability. We believe that more fibrotic PDL tissues seen in aged humans might be the reason for this, and suggest that this phenomena might be due to the progressive accumulation of senescent cell populations.

Human DOC-1/CDK2AP1 gene encodes a growth suppressor protein of 12 kDa (p12 DOC-1/CDK2AP1 ). Recently, p12 DOC-1/CDK2AP1 has been shown to associate with cell cycle proteins including CDK2 and DNA polymerase α/primase. It negatively regulates CDK2 activities and suppresses DNA replication. Therefore, identification of other p12 DOC-1/CDK2AP1 interacting proteins might clarify its role in the cell cycle regulation and carcinogenesis. The purpose of this study was to identify additional p12 DOC-1/CDK2AP1 interacting proteins using the yeast two-hybrid system. Using human p12 DOC-1/CDK2AP1 as a bait in a liver cDNA library screening, cDNA clones identical to human DOC-1R transcript were identified. The interaction between p12 DOC-1/CDK2AP1 and p14 DOC-1R was verified in vitro and in cells. GST pull-down assay and immunoprecipitation experiments confirmed the interaction between the two proteins. The critical region for p12 DOC-1/CDK2AP1 ’s interaction with p14 DOC-1R was defined to amino acids 20–25 by using a series of deletion mutants as baits in the yeast two-hybrid system. Our data indicated that p12 DOC-1/CDK2AP1 could associate with its homologous protein, p14 DOC-1R .

Genome-wide and high-throughput functional genomic tools offer the potential of identifying disease-associated genes and dissecting disease regulatory patterns. There is a need for a set of systematic bioinformatic tools that handles efficiently a large number of variables for extracting biological meaning from experimental outputs. We present well-characterized statistical tools to discover genes that are differentially expressed between malignant oral epithelial and normal tissues in microarray experiments and to construct a robust classifier using the identified discriminatory genes. Those tools include Wilks' lambda score, error rate estimated from leave-one out cross-validation (LOOCV) and Fisher Discriminant Analysis (FDA). High Density DNA microarrays and Real Time Quantitative PCR were employed for the generation and validation of the transcription profile of the oral cancer and normal samples. We identified 45 genes that are strongly correlated with malignancy. Of the 45 genes identified, six have been previously implicated in the disease, and two are uncharacterized clones.

Intraosseous squamous cell carcinomas of the mandible arise de novo or secondary to a tumor or transformed cyst epithelium. Current diagnostic tests frequently fail to distinguish between these tumors, leading to confusing classification schemes. We report the functional genomic analysis of a mandibular odontogenic carcinoma. Malignant keratinocytes from the lesion were isolated using laser capture microdissection. Target sample generated from the total RNA of the LCM-procured cells was used to hybridize high-density oligonucleotide arrays. Functional genomic analysis of the odontogenic carcinoma database compared with four oral mucosal squamous cell carcinoma gene expression databases was performed. Preliminary results suggest a small subset of genes distinguish this odontogenic carcinoma from oral mucosal epidermoid carcinomas.

Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oral cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected. Biotinylated cRNA was generated and hybridized to the human Test 1 GeneChip® probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis. Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%–33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.

p12DOC-1 is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12DOC-1 in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12DOC-1 associates with DNA polymerase α/primase (pol-α:primase) in vitro and in cells. The pol-α:primase binding domain in p12DOC-1 is mapped to the amino-terminal six amino acid (MSYKPN). The biological effect of p12DOC-1 on pol-α:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12DOC-1 suppresses DNA replication, leveling at ∼50%. Similar results were obtained using the M13 single-stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12DOC-1 affects the initiation step, not the elongation phase. The p12DOC-1 suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol-α:primase or by its effect on cycl...

TGFs play important roles in wound healing and carcinogenesis. We have previously demonstrated that eosinophils infiltrating into different pathologic processes elaborate TGF-α and TGF-β1. Eosinophils infiltrating hamster cutaneous wounds were found to express TGFs sequentially. In this study, we examined the biologic mediators that may regulate the expression of TGF-α and -β1 by eosinophils. Eosinophils were isolated from the peripheral blood of healthy donors and cultured in the absence or presence of IL-3, IL-4, and IL-5. Cells were analyzed by in situ hybridization and immunohistochemistry. Supernatants from these cultures were assayed for secreted TGF-α and TGF-β1 using TGF-specific ELISAs. IL-3, IL-4, and IL-5 independently up-regulated TGF-β1 mRNA and product expression by eosinophils in all donors. Interestingly, TGF-α production by eosinophils was up-regulated by IL-3 and IL-5 but was down-regulated by IL-4. Consistent with the ability of IL-4 to regulate eosinophil responses, IL-4 signaling molecules are present in human eosinophils. The observation that IL-4 can differentially regulate the expression of TGF-α and TGF-β1 suggests that IL-4 may serve as a physiologic molecular switch of TGF expression by the infiltrating eosinophils in wound healing and carcinogenesis.

Eosinophils are emerging as an increasingly important cell in the immunoregulatory network of normal and pathological processes. No studies has yet described optimized experimental strategies to transfect DNA into human eosinophils. Using a frequently employed in vitro model of human eosinophil, the EoL-1 cells, we now described the optimal transfection of DNA into these cells by electroporation. Our results indicate that electroporation can efficiently and reproducibly transfect DNA into EoL-1 cells. Optimal electroporation conditions consist of the use of 1 X RPMI medium 1640 with 10% FBS, voltage setting at 275 V, 1150 microF capacitance, 40 mg of DNA and 4.0 X 10(7) cells/ml per electroporation in a total volume of 0.5 ml in 0.4 cm gap cuvettes. These conditions may be a useful protocol for transfecting eosinophil cell lines.

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1β (IL-1β) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1β and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10−5M norepinephrine at 37°C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1β and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1β ( P < 0.03) and IL-6 ( P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1β and IL-6 were significantly higher ( P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1β and IL-6 and release these cytokines from their granules after α- and β-adrenergic stimulation.