Prevalence of oral mucosal lesions in patients with pemphigus vulgaris: A systematic review and meta-analysis.

OBJECTIVE: To assess the prevalence of oral mucosal lesions in patients with Pemphigus Vulgaris. METHODS: Observational studies reporting the prevalence of oral lesions in pemphigus vulgaris patients, without restriction to language and year of publication, were selected in a two-phase process. Search strategies were applied to PubMed, Scopus, Livivo, Web of Science, LILACS, Google Scholar, and OpenGrey databases. Articles assessing the prevalence of oral lesions in patients with conditions other than pemphigus vulgaris were excluded. Risk of bias analysis was performed using the Joanna Briggs Institute's Critical Appraisal Checklist for Studies Reporting Prevalence Data. Synthesis of results was calculated by the software R Statistics version 4.0.2 (The R Foundation). Confidence in cumulative evidence was assessed with the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RESULTS: From 1957 studies identified, 40 were included in qualitative synthesis and 38 in meta-analyses. The pooled prevalence of patients with oral lesions solely or concurrent with other mucocutaneous lesions was 90.3%. The prevalence of patients with exclusive oral mucosal lesions was 50.8%. Risk of bias was considered low, and the certainty of evidence was very low. CONCLUSION: Oral lesions were present in approximately nine out of 10 patients with pemphigus vulgaris. The oral mucosa was the most common site of disease onset. Further longitudinal studies are urged to assess the prevalence of oral lesions at different disease stages.

Prognostic and clinicopathological significance of podoplanin immunoexpression in oral and oropharyngeal squamous cell carcinoma: A systematic review.

BACKGROUND: Podoplanin (PDPN) is a glycoprotein associated with epithelial-mesenchymal transition, invasion, and metastasis in several types of malignancies, including oral and oropharyngeal squamous cell carcinoma (OSCC). The aim of this systematic review (SR) was to summarize and critically appraise the available evidence about the association between PDPN immunoexpression and clinicopathological features and its utility as a prognostic marker in OSCC. METHODS: Five electronic databases and three gray literature databases were searched for immunohistochemical studies, which were selected in a two-phase process. RESULTS: From 721 records identified, 22 cohort and seven analytical cross-sectional studies were included. Few studies reported that PDPN expression was associated with poorer survival rates in OSCC: overall survival = 4/12, disease-free survival = 4/7, and cancer-specific survival = 2/4 studies. Positive associations were most frequently reported on lymph node involvement, higher histopathological grade, and advanced clinical stages. CONCLUSION: Within limitations of this SR, PDPN may be associated with lymph node involvement, histopathological grade, and clinical stage of OSCC. Current evidence suggests that PDPN could be a useful prognostic marker for OSCC that needs further exploration.

Prevalence of oral mucosal disorders during pregnancy: A systematic review and meta-analysis.

OBJECTIVE: To assess the prevalence of oral mucosal disorders during pregnancy. METHODS: Observational studies were selected by two reviewers in a two-phase process. Search strategies were applied at CINAHL, LILACS, LIVIVO, PubMed, Scopus, Web of Science, Google Scholar, OpenGrey, and ProQuest. The risk of bias was assessed using the Joanna Briggs Institute's Critical Appraisal Checklist for Studies Reporting Prevalence Data. Synthesis of results was calculated by the software R Statistics version 3.5.1 (The R Foundation, Vienna, Austria). Confidence in cumulative evidence was assessed by using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) criteria. RESULTS: Fifteen studies met the eligibility criteria and were selected for qualitative synthesis and meta-analysis, of which 5935 participants were enrolled. The overall prevalence of oral mucosal disorders was 11.8%. Gingival hyperplasia (17.1%), morsicatio buccarum (10%), oral candidiasis (4.4%), pyogenic granuloma (3%), and benign migratory glossitis (2.8%) were the most prevalent lesions. The overall risk of bias was considered moderate, and the quality of evidence was very low. CONCLUSION: Disorders of the oral mucosa were present in approximately 1 out of 10 pregnant women. Gingival hyperplasia was the most prevalent lesion. Further studies should apply homogeneous methodology to improve the quality of evidence.

Inhibition of cancer stem cells promoted by Pimozide.

Over the past years, studies have described that users of antipsychotics are less likely to develop cancer than the population in general due to cytotoxic properties of this class of drugs on cancer cells. For this reason, Pimozide has been widely studied as a potential anticancer treatment, and satisfactory results in melanoma, central nervous system tumours, osteosarcoma, neuroblastoma, myeloproliferative neoplasms, breast, lung, prostate, ovarian, colorectal, pancreatic, and hepatocellular carcinoma have been showed. Moreover, advantages as clinical use approved by the Food and Drug Administration (FDA), high clinical safety, low side effects, and reasonable price have stimulated the treatment with Pimozide instead of other agents. The action mechanism remains unclear, but three vias associated to cancer stem cell (CSC) hypothesis show that Pimozide: (a) blocks CSC features, as epithelial-to-mesenchymal transition (EMT), through inhibition of Wnt-ß/catenin signalling; (b) acts as an inhibitor of signal transducer and activator of transcription (STAT-3 and 5), pathway which is activated and up-regulated in CSCs; (c) inhibits ubiquitine specific protease (USP1) and WD repeat-containing protein 48 (WDR48), that are proteins responsible to inhibit the differentiation and to maintain the cell in an undifferentiated state. Based on this perspective, the aim of this manuscript is to review the antineoplastic role of Pimozide during tumorigenesis and its potential to revert the process of undifferentiation and proliferation of CSC through different vias.

The synergistic effect of tobacco and alcohol consumption on oral squamous cell carcinoma: a systematic review and meta-analysis.

OBJECTIVES: This systematic review (SR) aimed to summarise and critically appraise available evidence about the association between the synergistic consumption of alcohol and tobacco and the occurrence of oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Observational studies investigating the association between the synergistic consumption of alcohol and tobacco and OSCC occurrence were included. Studies were selected in a two-phase process and searches were conducted on five main electronic databases, complemented by three grey literature databases. RESULTS: From 3260 records identified, 33 articles were included for qualitative analysis, of which 15 were included in the meta-analyses. Overall, the synergistic consumption was positively associated with the occurrence of OSCC (odds ratio [OR] = 5.37; 95% confidence interval [95%CI] = 3.54-8.14). Increased odds for OSCC occurrence were observed regarding the synergistic consumption of alcohol and smoked tobacco (OR = 4.74; 95%CI = 3.51-6.40), alcohol and smokeless tobacco (OR = 7.78; 95%CI = 2.86-21.14), and alcohol, smoked tobacco, and smokeless tobacco (OR = 16.17; 95%CI = 7.97-32.79). CONCLUSIONS: According to the results of this SR, the synergistic consumption of alcohol and tobacco (both smoked and smokeless) significantly increased the odds for the occurrence of OSCC. CLINICAL RELEVANCE: The knowledge provided by this SR may be useful for a better understanding of differences in the effect of synergistic consumption of alcohol and tobacco products in OSCC development. These data might aid healthcare authorities to develop measures for tobacco and alcohol control considering the needs of their population.

The role of FGF-2/HGF and fibronectin matrix on pleomorphic adenoma myoepithelial cell morphology and immunophenotype: an in vitro study.

Myoepithelial cells play a central role in glandular tumors, regulating the progression of in situ to invasive neoplasias, with the tumor microenvironment being shown to be involved in both initiation and progression. This study aimed to analyze the in vitro effects of fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor (HGF) in myoepithelial cells under the influence of the fibronectin matrix extracellular protein. Benign myoepithelial cells were obtained from pleomorphic adenoma and cultured on a fibronectin substratum. FGF-2 and HGF were supplemented at different concentrations and time intervals, in order to evaluate cell proliferation, morphology and immunophenotype. Individually, FGF-2 and HGF supplementation did not alter myoepithelial cell proliferation, morphology or immunophenotype. The fibronectin substratum provoked an increase in cell proliferation and immunopositivity for α-smooth muscle actin and FGF-2. The myoepithelial cell morphology changed when the fibronectin substratum and FGF-2 acted together, highlighting the importance of the fibronectin extracellular matrix protein on the behavior of these cells.

Tooth alterations in areas of bisphosphonate-induced osteonecrosis.

OBJECTIVE: Osteonecrosis of the jaw is a potential side effect when using bisphosphonates. Most studies on the effects of bisphosphonates on teeth have been conducted in vitro or in animal models of tooth development. Therefore, the aim of this study was to describe alterations found in human teeth extracted from areas of bisphosphonate-induced osteonecrosis. MATERIALS AND METHODS: Using a retrospective study design, 16 teeth from 13 patients were extracted from areas of bisphosphonate-induced osteonecrosis during surgical debridement. The specimens were decalcified and embedded in paraffin. A series of 5-µm sections were prepared, stained with hematoxylin and eosin (H&E) and observed under a light microscope. RESULTS: The majority of the patients were female (53.85 %), with a mean age of 60.23 ± 13.18 years. Zoledronate (IV) was the most common bisphosphonate used (92.3 %), over a mean period of 2 years. The commonest alteration observed was hypercementosis (87.5 %), followed by pulpar necrosis (81.25 %), pulp stones attached to the dentine and loose pulp stones in the pulp chamber and root canals in addition to linear calcifications (68.75 %), dentinoid/osteoid material formation (18.75 %), and dental ankylosis (6.25 %). CONCLUSIONS: Patients undergoing bisphosphonate therapy present diverse tooth alterations, which should be closely monitored by clinicians to prevent complications. CLINICAL RELEVANCE: It is paramount that the teeth involved in oral lesions are always examined. Attention should be drawn to the need to establish preventive measures, in terms of dental treatment, for patients prior to starting bisphosphonate therapy.

Peripheral odontogenic fibroma: an uncommonly overviewed lesion.

Peripheral odontogenic fibroma is considered a gingival tumor characterized by a proliferation of relatively cellular fibrous or fibromyxomatous connective tissue which exhibits variable amounts of odontogenic epithelium and sometimes foci of calcification in the form of dentinoid, cementicles, or bone. It is considered the extraosseous counterpart of central odontogenic fibroma. This lesion usually is presented as a focal swelling in the gingiva, occurring in a wide age range, and the anterior region of the gingiva is the most frequent anatomic site. Conservative local excision is the treatment frequently adopted and its recurrence rate varies widely, and its biologic behavior is still unknown. In this study, the authors discuss 3 cases of peripheral odontogenic fibroma, and present their clinical and histopathological features and management.

In vitro evaluation of the suppressor potential of conditioned medium from benign myoepithelial cells from pleomorphic adenoma in malignant cell invasion.

Tumoral invasion process is the result of a complex interaction between the tumor cells and microenvironment which plays an important role in modulating the growth and invasion of the cancer. The myoepithelial cells, present in glandular organs such as the breast and salivary glands, seem to exert paracrine effects on the glandular epithelium, acting as natural tumor suppressors. To verify the influence of the benign myoepithelial cells in the invasion of malignant cells, simulating an in situ carcinoma ex pleomorphic adenoma, we have cultured three different high-potential invasive malignant tumors (breast ductal adenocarcinoma, melanoma and oral squamous cell carcinoma) in conditioned medium of myoepithelial cells from salivary gland pleomorphic adenomas using transwell chambers with 8-µm pores membrane coated with matrigel. After 96 h, quantitative analyses of the results were performed by calculating the invasion index (number of cells that invaded in relation to the total number of cells). The results showed that there was a reduction of the invasion index mean for the three different malignant tumors. This study supports a tumoral suppressor function of the myoepithelial cells from pleomorphic adenoma in in vitro invasion process.

Anticancer effects of putative and validated BH3-mimetic drugs in head and neck squamous cell carcinomas: An overview of current knowledge.

The purpose of this review was to summarise available literature concerning the anticancer effects of both putative and validated BH3-mimetics in head and neck squamous cell carcinomas. A literature search was performed and studies assessing malignant cell lines, xenograft models, and/or humans were considered eligible. A total of 501 studies were identified, of which 40 were included. One phase-II clinical trial assessing gossypol (combined with docetaxel) was found. The remaining 39 preclinical studies investigated cell lines and/or xenograft models involving the use of six validated BH3-mimetics (A-1210477, A-1331852, ABT-737, navitoclax, S63845, venetoclax) and six putative BH3-mimetics (ApoG2, gossypol, obatoclax, sabutoclax, TW-37, and YC137). In preclinical settings, most validated BH3-mimetics were capable of inducing apoptosis (in-vitro) and tumour growth inhibition (in-vivo). The majority of putative BH3-mimetics were also capable of inducing cell death, although important off-target effects, such as autophagy induction, were also described. Combinations with conventional anticancer drugs, ionising radiation, or multiple BH3-mimetics generally resulted in enhanced anticancer effects, such as increased sensitivity to apoptotic stimuli, especially considering some cell lines that showed resistance to either treatment alone. In conclusion, although clinical data are still insufficient to evaluate the anticancer effects of BH3-mimetics in head and neck squamous cell carcinomas, promising results in preclinical settings were observed concerning induction of cell death and inhibition of tumour growth. Therefore, further clinical trials are highly encouraged.

Recurrent oral pyogenic granuloma in port-wine stain.

Pyogenic granuloma (PG) is a benign inflammatory lesion, nonneoplastic in nature, which occurs in the oral cavity and skin. This lesion arises in response to various stimuli such as low-grade local irritations, traumatic injury, or hormonal factors. Recently, in some cases, the occurrence of recurrent PGs in skin associated with vascular lesions, such as port-wine stains, has been described. It has been postulated that this association is promoted by arteriovenous anastomoses in the vascular lesions, leading to the development of PG. The authors discuss 2 cases of recurrent PG in patients with a port-wine stain, and the treatment options adopted.

Trabecular juvenile ossifying fibroma with aneurysmal bone cyst: a rare presentation.

Juvenile ossifying fibroma is an uncommon, benign, bone-forming neoplasm that is distinguished from other fibro-osseous lesions primarily by its age of onset, clinical presentation, and potential behavior. It mainly occurs in juveniles and has a slight male predilection and more aggressive behavior than a common ossifying fibroma. There are 2 distinct histopathological variants of this lesion: (1) psammomatoid pattern and (2) trabecular pattern. An aneurysmal bone cyst may occur in association with other bone lesions, such as fibrous dysplasia, ossifying fibroma, and giant cell lesion. The clinical management and prognosis of juvenile ossifying fibroma is somewhat uncertain, and this tumor has high rates of recurrence. Such behavior may be related to younger patient age and the concurrent development of aneurysmal bone cyst. The purpose of this paper was to report a case of trabecular juvenile ossifying fibroma in a 9-year-old girl associated with an aneurysmal bone cyst, presenting an aggressive behavior, and causing significant facial asymmetry.

Gardner syndrome with no clinical family history.

Gardner syndrome, a variant of familial adenomatous polyposis, is a hereditary disorder inherited as an autosomal dominant with high penetrance and variable expression that is caused by a mutation of the adenomatous polyposis coli gene. It is characterized by gastrointestinal polyps associated with multiple osteomas, dental anomalies, skin and soft tissue tumors, and congenital hypertrophy of the retinal pigment epithelium. Early diagnosis of Gardner syndrome is crucial and may be life saving because the polyps usually undergo malignant change by the fourth decade of life. The extraintestinal manifestations frequently precede gastrointestinal symptoms; consequently, the dentist may play an important role in the diagnosis of this syndrome. The authors report a case of Gardner syndrome with no clinical family history that was diagnosed by the dentist based on oral and maxillofacial findings.

The risk of osteonecrosis on alveolar healing after tooth extraction and systemic administration of antiresorptive drugs in rodents: a systematic review.

PURPOSE: There is much concern about the increasing number of patients with medication-related osteonecrosis of the jaw (MRONJ), and many studies have been published in an attempt to understand the pathophysiology of this condition. This study aimed to systematically review the literature on MRONJ arising in rodents under antiresorptive drug therapy after tooth extraction. METHODS: A search of electronic databases, including LILACS, PROQUEST, PubMed, SCOPUS, and the Web of Science. RESULTS: The search resulted in 2319 titles after removing the duplicates, and one paper was identified using the reference list. Ninety-eight full-text papers were then screened for eligibility, resulting in 20 for inclusion in the final qualitative synthesis. The quality of the articles was assessed using the 'ARRIVE' tool. CONCLUSION: Despite the wide heterogeneity of the methodologies used by the authors, the current available evidence suggests that the combination of bisphosphonate and/or denosumab therapy and tooth extraction is associated with osteonecrosis of the jaw in rodents.

Oral health related to quality of life in patients with stomatological diseases.

AIM: To describe the oral health-related quality of life (OHR-QoL) in patients with stomatological diseases. METHODS: All new patients seen in the Stomatology Clinic, from August 2008 to July 2009 were selected. OHR-QoL was measured using OHIP-14, in face-to-face interviews. The stomatological diseases were classified into groups according to their origin. OHIP-14 data were used to calculate two variables: prevalence and severity. RESULTS: The questionnaire was completed by 113 subjects, with a mean of 53.77 years; 63.7% women; 38% of the subjects reported one or more OHR-QoL impacts "fairly often" or "very often" in the last 6 months. The overall result of OHIP-14 index showed a mean of 14.35 (+/-12.01). There was no statistical significance between the stomatological disease groups and the prevalence scores (p=0.25) and mean severity scores (p=0.57). CONCLUSIONS: It seems that bad oral conditions affected quality of life of these patients, especially physical pain and psychological discomfort.

Myoepithelial cells from pleomorphic adenoma are not influenced by tumor conditioned media from breast ductal adenocarcinoma and melanoma cells: An in vitro study.

Myoepithelial cells have been implicated in the regulation of the transition from in situ to invasive neoplasia in salivary gland tumors. Considering the importance of the microenvironment of the tumor, the present in vitro study therefore analyzed the morphological and phenotypic changes undergone by benign myoepithelial cells from pleomorphic adenoma (PA) stimulated by tumor-conditioned medium. The benign myoepithelial cells were obtained from PA and were cultured with fibronectin extracellular matrix protein, supplemented with tumor-conditioned medium, which was harvested from breast ductal adenocarcinoma AU-565 and melanoma Hs 852.T cells. The morphological alterations were assessed by immunofluorescence analysis using vimentin antibody. The α-smooth muscle actin (α-SMA) and fibroblast growth factor (FGF)-2 proteins were analyzed by indirect immunofluorescence and quantitative polymerase chain reaction (qPCR). No morphological changes were observed in the myoepithelial cells cultured in fibronectin protein under stimulation from either tumor-conditioned medium. The immunofluorescence results, which were supported by qPCR analysis, revealed that only α-SMA was upregulated in the fibronectin substratum, with or without tumor-conditioned medium obtained from breast ductal adenocarcinoma and melanoma cells. No significant difference in FGF-2 mRNA expression was detected when the cells were cultured either in the tumor-conditioned medium or in the fibronectin substratum. The tumor-conditioned medium harvested from breast ductal adenocarcinoma and melanoma did not affect myoepithelial cell differentiation and function, which was reflected by the fact that there was no observed increase in α-SMA and FGF-2 expression, respectively.

Cellular senescence and autophagy of myoepithelial cells are involved in the progression of in situ areas of carcinoma ex-pleomorphic adenoma to invasive carcinoma. An in vitro model.

During tumor invasion, benign myoepithelial cells of carcinoma ex-pleomorphic adenoma (CXPA) surround malignant epithelial cells and disappear. The mechanisms involved in the death and disappearance of these myoepithelial cells were investigated via analysis of the expression of regulatory proteins for apoptosis, autophagy and cellular senescence in an in situ in vitro model. Protein expression relating to apoptosis (Bax, Bcl-2, Survivin), autophagy (Beclin-1, LC3B) and cellular senescence (p21, p16) was evaluated using indirect immunofluorescence. ß-galactosidase expression was assessed via histochemistry. Biopsies of CXPA (ex vivo) allowed immunhistochemical evaluation of p21 and p16, whilst LC3B, p21 and p16 protein expression was analyzed by western blotting. In the in vitro model, the myoepithelial cells were positive for LC3B (cytoplasm) and p21 (nucleus), whilst in vivo positivity for p21 and p16 was observed. In vitro, ß-galactosidase activity increased in the myoepithelial cells over time. Western blotting analysis revealed an increased LC3B, p16 and p21 expression in the myoepithelial cells with previous contact with the malignant cells when compared with those without contact. The investigation of behavior of benign myoepithelial cells in ductal areas of CXAP revealed that the myoepithelial cells are involved in the autophagy-senescence phenotype that subsequently leads to their disappearance.

Necrotizing sialometaplasia in a patient who is HIV positive: a case report.

Necrotizing sialometaplasia (NS) is a self-limiting, benign, inflammatory disease of the minor salivary glands of the hard palate. The main significance of the NS lesion lies in the fact that it may be mistaken for mucoepidermoid or squamous cell carcinoma. A case report is presented of a patient with NS who was HIV positive; the lesion was located in the minor salivary glands of the hard palate. Clinically, the lesion presented as a deep ulcer with slightly elevated irregular borders and a necrotic base in the hard palate. Histologically, the tissue was characterized by squamous metaplasia of ducts and acini, lobular coagulation necrosis, and pseudoepitheliomatous hyperplasia of the overlying epithelium. The lesion disappeared completely after 2 weeks.

Avaliação do potencial de invasão de células epiteliais malignas (carcinoma epidermoide) influenciadas por células mioepiteliais benignas / Evaluation of the potential invasion of malignant epithelial cells (epidermoid carcinoma) influenced by Benign myoepithelial cells

A invasão tumoral é um complexo processo biológico influenciado não só pelas células tumorais como também pelas células do microambiente. Dentre elas, as células mioepiteliais parecem modular negativamente o crescimento e a invasão tumoral por promover a regulação da progressão do ciclo celular além de secretar altos níveis de moléculas supressoras de tumor, como inibidores de proteinases e inibidores angiogênicos. O objetivo deste trabalho foi avaliar a influência das células mioepiteliais (CM) neoplásicas benignas do adenoma pleomórfico de glândula salivar no potencial de invasão das células epiteliais malignas do adenocarcinoma ductal (AD) por meio de ensaios de invasão in vitro utilizando a técnica de cultivo celular. Para isso, células do AD (HS578T, ATCC) foram cultivadas no compartimento superior de câmaras transwell sobre insertos com membranas (poros de 8µm) cobertas por Matrigel. No grupo controle, foi utilizado meio DMEM e nos grupos experimentais, meios condicionados de 3 culturas primárias de CM (AP3, AP4 e AP8). Após 48 horas, foi realizada a análise quantitativa do índice de invasão das células malignas e do número absoluto de células que invadiram. Os resultados evidenciaram que as células do AD apresentaram índice de invasão de 42,02% e, quando suplementadas com meio condicionado de CM, este foi de 25,77% (AP4), 26,77% (AP8) e 34,63% (AP3), com diferença estatisticamente significante (p<0,05) entre todos os grupos e o grupo controle, além de uma redução no número de células que invadiram de 523,66 no grupo controle para 233,5 (AP4), 183,5 (AP8) e 34,63 (AP3), porém esta redução não mostrou diferença estatística (p>0,05) entre todos os grupos. Concluiu-se que o meio condicionado de CM pode apresentar um papel supressor no processo de invasão in vitro das células do AD

Tumoral invasion is a complex biologic process influenced not only by tumoral cells but by cells in the microenvironment as well. Among these, the myoepithelial cells appear to negatively modulate tumor growth and invasion by promoting regulation of cell cycle progression, in addition to secreting high levels of tumor suppressor molecules, such as protease inhibitors and angiogenesis inhibitors. The aim of this study was to evaluate the influence of benign myoepithelial cells of pleomorphic adenoma of the salivary gland (MC) on the invasion potential of malignant epithelial cells of ductal adenocarcinoma (DA) by means of an in vitro invasion assay method using the cell culture technique. For this purpose, cells from the DA (HS578T, ATCC) were grown in DMEM medium in the top compartment of the Transwell chamber on inserts with filters (8µm pore size) covered with Matrigel. Conditioned medium of 3 strains of primary cultures of MC was added. After 48 hours, a quantitative analysis of the invasion rate of malignant cells and absolute number of cells that invaded was performed. The results showed that the DA cells presented an invasion rate of 42.02% and when supplemented with conditioned medium of the MC the rates were 25.77% (AP4), 26.77% (AP8) and 34.63% (AP3) and this difference being statistically significant (p<0,05) in all groups, beyond a reduction in the absolute number cells that invaded, from 523.63 in the control group to 233.4 (AP4), 183.5 (AP8) and 316.83 (AP3), however this reduction being not statistically significant (p>0,05) in all groups. It was concluded that conditioned medium of the MC could play a suppressive role in the invasion process in vitro of DA cells