A Simple and Efficient Device for Demonstrating Cross-sectional Anatomy of the Head

Described in this article is a novel device that facilitates study of the cross-sectional anatomy of the human head. In designing our device, we aimed to protect sections of the head from the destructive action of handling during anatomy laboratory while also ensuring excellent visualization of the anatomic structures. We used an electric saw to create 15-mm sections of three cadaver heads in the three traditional anatomic planes and inserted each section into a thin, perforated display box made of transparent acrylic material. The thin display boxes with head sections are kept in anatomical order in a larger transparent acrylic storage box containing formaldehyde solution, which preserves the specimens but also permits direct observation of the structures and their anatomic relationships to each other. This box-within-box design allows students to easily view sections of a head in its anatomical position as well as to examine internal structures by manipulating individual display boxes without altering the integrity of the preparations. This methodology for demonstrating cross-section anatomy allows efficient use of cadaveric material and technician time while also giving learners the best possible handling and visualization of complex anatomic structures. Our approach to teaching cross-sectional anatomy of the head can be applied to any part of human body, and the value of our device design will only increase as more complicated understandings of cross-sectional anatomy are required by advances and proliferation of imaging technology. Anat Sci Educ 3: 141-143, 2010. (C) 2010 American Association of Anatomists.

Imagem por ressonância magnética na investigação da cabeça de cães

A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não invasivo mais sensível para avaliar as partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. O método fundamenta-se no fenômeno da ressonância magnética nuclear que ocorre quando núcleos atômicos com propriedades magnéticas presentes no corpo são submetidos a um campo magnético intenso, sendo posteriormente excitados por energia de radiofrequência e gerando, por sua vez, um sinal de onda de radiofrequência capaz de ser captado por uma antena receptora, passando por um processo matemático, chamado Transformada de Fourier, para posterior formação da imagem. Esse estudo objetivou realizar 10 exames completos da cabeça em cadáveres de cães normais à IRM e confeccionar um Atlas com as estruturas identificadas. As imagens foram adquiridas em um aparelho de ressonância magnética Gyroscan S15/HP Philips com campo magnético de 1,5Tesla. Os cadáveres foram posicionados com a cabeça no interior de uma bobina de cabeça humana e foram submetidos a cortes iniciais sagitais a partir de onde se planejou os cortes transversais e dorsais nas sequências de pulso spin-eco T1, T2 e DP. Em T1 utilizou-se TR=400ms e TE=30ms, T2 utilizou-se TR=2000ms e TE=80ms e na DP utilizou-se TR=2000ms e TE=30ms. A espessura do corte foi de 4mm, o número de médias foi igual a 2, a matriz foi de 256x256, o fator foi igual a 1,0 e o campo de visão foi de 14cm. A duração do exame completo da cabeça foi de 74,5minutos. As imagens obtidas com as sequências utilizadas e com a bobina de cabeça humana foram de boa qualidade. Em T1 a gordura tornou-se hiperintensa e o líquido hipointenso. Em T2 a gordura ficou menos hiperintensa e o líquido hiperintenso. A cortical óssea e o ar foram hipointensos em todas as sequências utilizadas devido a baixa densidade de prótons. A sequência DP mostrou o melhor contraste entre a substância branca e cinzenta quando comparada a T2 e a T1. T2 evidenciou o líquido cefalorraquidiano tornando possível a distinção dos sulcos e giros cerebrais. Através do exame de IRM foi possível, pelo contraste, identificar as estruturas ósseas componentes da arquitetura da região, músculos, grandes vasos venosos e arteriais e estruturas do sistema nervoso central, além de elementos do sistema digestório, respiratório e estruturas dos olhos entre outras. Nesse estudo as IRM adquiridas nas sequências T1, DP e T2 foram complementares para o estudo dos aspectos anatômicos da cabeça de cães demonstrando-os com riqueza de detalhes. O tempo requerido para o exame completo da cabeça é compátivel para uso em animais vivos desde que devidamente anestesiados e controlados. Os resultados obtidos por esse trabalho abrem caminho em nosso meio, para o estudo de animais vivos e para o início da investigação de doenças, principalmente as de origem neurológica, visto ser esta técnica excelente para a visibilização do encéfalo.

Research Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGF beta 1, TGF beta 2, TGF beta 3 in oral SCC. Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2(-) and Smad6(+) oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2(+)/Smad6(-) subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGF beta isoforms, we found that Smad2 mRNA and TGF beta 1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGF beta 1(+) patients were Smad2(-). In larynx SCC, Smad7(-) patients did not reach mOS whereas mOS of Smad7(+) patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGF beta signaling should be better clarified in the future.

Low human papillomavirus prevalence in head and neck cancer: results from two large case-control studies in high-incidence regions

Background Recent studies support an important role for human papillomavirus (HPV) in a subgroup of head and neck squamous cell carcinomas (HNSCC). We have evaluated the HPV deoxyribonucleic acid (DNA) prevalence as well as the association between serological response to HPV infection and HNSCC in two distinct populations from Central Europe (CE) and Latin America (LA). Methods Cases (n = 2214) and controls (n = 3319) were recruited from 1998 to 2003, using a similar protocol including questionnaire and blood sample collection. Tumour DNA from 196 fresh tissue biopsies was analysed for multiple HPV types followed by an HPV type-specific polymerase chain reaction (PCR) protocol towards the E7 gene from HPV 16. Using multiplex serology, serum samples were analysed for antibodies to 17 HPV types. Statistical analysis included the estimation of adjusted odds ratios (ORs) and the respective 95% confidence intervals (CIs). Results HPV16 E7 DNA prevalence among cases was 3.1% (6/196), including 4.4% in the oropharynx (3/68), 3.8% in the hypopharynx/larynx (3/78) and 0% among 50 cases of oral cavity carcinomas. Positivity for both HPV16 E6 and E7 antibodies was associated with a very high risk of oropharyngeal cancer (OR = 179, 95% CI 35.8-899) and hypopharyngeal/laryngeal cancer (OR = 14.9, 95% CI 2.92-76.1). Conclusions A very low prevalence of HPV DNA and serum antibodies was observed among cases in both CE and LA. The proportion of head and neck cancer caused by HPV may vary substantially between different geographical regions and studies that are designed to evaluate the impact of HPV vaccination on HNSCC need to consider this heterogeneity.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Functional microarray analysis suggests repressed cell-cell signaling and cell survival-related modules inhibit progression of head and neck squamous cell carcinoma

Background: Cancer shows a great diversity in its clinical behavior which cannot be easily predicted using the currently available clinical or pathological markers. The identification of pathways associated with lymph node metastasis (N+) and recurrent head and neck squamous cell carcinoma (HNSCC) may increase our understanding of the complex biology of this disease. Methods: Tumor samples were obtained from untreated HNSCC patients undergoing surgery. Patients were classified according to pathologic lymph node status (positive or negative) or tumor recurrence (recurrent or non-recurrent tumor) after treatment (surgery with neck dissection followed by radiotherapy). Using microarray gene expression, we screened tumor samples according to modules comprised by genes in the same pathway or functional category. Results: The most frequent alterations were the repression of modules in negative lymph node (N0) and in non-recurrent tumors rather than induction of modules in N+ or in recurrent tumors. N0 tumors showed repression of modules that contain cell survival genes and in non-recurrent tumors cell-cell signaling and extracellular region modules were repressed. Conclusions: The repression of modules that contain cell survival genes in N0 tumors reinforces the important role that apoptosis plays in the regulation of metastasis. In addition, because tumor samples used here were not microdissected, tumor gene expression data are represented together with the stroma, which may reveal signaling between the microenvironment and tumor cells. For instance, in non-recurrent tumors, extracellular region module was repressed, indicating that the stroma and tumor cells may have fewer interactions, which disable metastasis development. Finally, the genes highlighted in our analysis can be implicated in more than one pathway or characteristic, suggesting that therapeutic approaches to prevent tumor progression should target more than one gene or pathway, specially apoptosis and interactions between tumor cells and the stroma.

Immune response to vaccination with DNA-hsp65 in a phase I clinical trial with head and neck cancer patients

DNA-hsp65, a DNA vaccine encoding the 65-kDa heat-shock protein of Mycobacterium leprae (Hsp65) is capable of inducing the reduction of established tumors in mouse models. We conducted a phase I clinical trial of DNA-hsp65 in patients with advanced head and neck carcinoma. In this article, we report on the vaccine`s potential to induce immune responses to Hsp65 and to its human homologue, Hsp60, in these patients. Twenty-one patients with unresectable squamous cell carcinoma of the head and neck received three doses of 150, 400 or 600 mu g naked DNA-hsp65 plasmid by ultrasound-guided intratumoral injection. Vaccination did not increase levels of circulating anti-hsp65 IgG or IgM antibody, or lead to detectable Hsp65-specific cell proliferation or interferon-gamma (IFN-gamma) production by blood mononuclear cells. Frequency of antigen-induced IL-10-producing cells increased after vaccination in 4 of 13 patients analyzed. Five patients showed disease stability or regression following immunization; however, we were unable to detect significant differences between these patients and those with disease progression using these parameters. There was also no increase in antibody or IFN-gamma responses to human Hsp60 in these patients. Our results suggest that although DNA-hsp65 was able to induce some degree of immunostimulation with no evidence of pathological autoimmunity, we were unable to differentiate between patients with different clinical outcomes based on the parameters measured. Future studies should focus on characterizing more reliable correlations between immune response parameters and clinical outcome that may be used as predictors of vaccine success in immunosuppressed individuals. Cancer Gene Therapy (2009) 16, 598-608; doi:10.1038/cgt.2009.9; published online 6 February 2009

Sexual behaviours and the risk of head and neck cancers: a pooled analysis in the International Head and Neck Cancer Epidemiology (INHANCE) consortium

Background Sexual contact may be the means by which head and neck cancer patients are exposed to human papillomavirus (HPV). Methods We undertook a pooled analysis of four population-based and four hospital-based case-control studies from the International Head and Neck Cancer Epidemiology (INHANCE) consortium, with participants from Argentina, Australia, Brazil, Canada, Cuba, India, Italy, Spain, Poland, Puerto Rico, Russia and the USA. The study included 5642 head and neck cancer cases and 6069 controls. We calculated odds ratios (ORs) of associations between cancer and specific sexual behaviours, including practice of oral sex, number of lifetime sexual partners and oral sex partners, age at sexual debut, a history of same-sex contact and a history of oral-anal contact. Findings were stratified by sex and disease subsite. Results Cancer of the oropharynx was associated with having a history of six or more lifetime sexual partners [OR = 1.25, 95% confidence interval (CI) 1.01, 1.54] and four or more lifetime oral sex partners (OR = 2.25, 95% CI 1.42, 3.58). Cancer of the tonsil was associated with four or more lifetime oral sex partners (OR = 3.36, 95 % CI 1.32, 8.53), and, among men, with ever having oral sex (OR = 1.59, 95% CI 1.09, 2.33) and with an earlier age at sexual debut (OR = 2.36, 95% CI 1.37, 5.05). Cancer of the base of the tongue was associated with ever having oral sex among women (OR = 4.32, 95% CI 1.06, 17.6), having two sexual partners in comparison with only one (OR = 2.02, 95% CI 1.19, 3.46) and, among men, with a history of same-sex sexual contact (OR = 8.89, 95% CI 2.14, 36.8). Conclusions Sexual behaviours are associated with cancer risk at the head and neck cancer subsites that have previously been associated with HPV infection.

Total Exposure and Exposure Rate Effects for Alcohol and Smoking and Risk of Head and Neck Cancer: A Pooled Analysis of Case-Control Studies

Although cigarette smoking and alcohol consumption increase risk for head and neck cancers, there have been few attempts to model risks quantitatively and to formally evaluate cancer site-specific risks. The authors pooled data from 15 case-control studies and modeled the excess odds ratio (EOR) to assess risk by total exposure (pack-years and drink-years) and its modification by exposure rate (cigarettes/day and drinks/day). The smoking analysis included 1,761 laryngeal, 2,453 pharyngeal, and 1,990 oral cavity cancers, and the alcohol analysis included 2,551 laryngeal, 3,693 pharyngeal, and 3,116 oval cavity cancers, with over 8,000 controls. Above 15 cigarettes/day, the EOR/pack-year decreased with increasing cigarettes/day, suggesting that greater cigarettes/day for a shorter duration was less deleterious than fewer cigarettes/day for a longer duration. Estimates of EOR/pack-year were homogeneous across sites, while the effects of cigarettes/day varied, indicating that the greater laryngeal cancer risk derived from differential cigarettes/day effects and not pack-years. EOR/drink-year estimates increased through 10 drinks/day, suggesting that greater drinks/day for a shorter duration was more deleterious than fewer drinks/day for a longer duration. Above 10 drinks/day, data were limited. EOR/drink-year estimates varied by site, while drinks/day effects were homogeneous, indicating that the greater pharyngeal/oral cavity cancer risk with alcohol consumption derived from the differential effects of drink-years and not drinks/day.

Marijuana Smoking and the Risk of Head and Neck Cancer: Pooled Analysis in the INHANCE Consortium

Background: Marijuana contains carcinogens similar to tobacco smoke and has been suggested by relatively small studies to increase the risk of head and neck cancer (HNC). Because tobacco is a major risk factor for HNC, large studies with substantial numbers of never tobacco users could help to clarify whether marijuana smoking is independently associated with HNC risk. Methods: We pooled self-reported interview data on marijuana smoking and known HNC risk factors on 4,029 HNC cases and 5,015 controls from five case-control studies within the INHANCE Consortium. Subanalyses were conducted among never tobacco users (493 cases and 1,813 controls) and among individuals who did not consume alcohol or smoke tobacco (237 cases and 887 controls). Results: The risk of HNC was not elevated by ever marijuana smoking [odds ratio (OR), 0.88; 95% confidence intervals (95% Cl), 0.67-1.16], and there was no increasing risk associated with increasing frequency, duration, or cumulative consumption of marijuana smoking. An increased risk of HNC associated with marijuana use was not detected among never tobacco users (OR, 0.93; 95% Cl, 0.63-1.37; three studies) nor among individuals who did not drink alcohol and smoke tobacco (OR, 1.06; 95% Cl, 0.47-2.38; two studies). Conclusion: Our results are consistent with the notion that infrequent marijuana smoking does not confer a risk of these malignancies. Nonetheless, because the prevalence of frequent marijuana smoking was low in most of the contributing studies, we could not rule out a moderately increased risk, particularly among subgroups without exposure to tobacco and alcohol. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1544-51)

Interaction between Tobacco and Alcohol Use and the Risk of Head and Neck Cancer: Pooled Analysis in the International Head and Neck Cancer Epidemiology Consortium

Background: The magnitude of risk conferred by the interaction between tobacco and alcohol use on the risk of head and neck cancers is not clear because studies have used various methods to quantify the excess head and neck cancer burden. Methods: We analyzed individual-level pooled data from 17 European and American case-control studies (11,221 cases and 16,168 controls) participating in the International Head and Neck Cancer Epidemiology consortium. We estimated the multiplicative interaction parameter (psi) and population attributable risks (PAR). Results: A greater than multiplicative joint effect between ever tobacco and alcohol use was observed for head and neck cancer risk (psi = 2.15; 95% confidence interval, 1.53-3.04). The PAR for tobacco or alcohol was 72% (95% confidence interval, 61-79%) for head and neck cancer, of which 4% was due to alcohol alone, 33% was due to tobacco alone, and 35% was due to tobacco and alcohol combined. The total PAR differed by subsite (64% for oral cavity cancer, 72% for pharyngeal cancer, 89% for laryngeal cancer), by sex (74% for men, 57% for women), by age (33% for cases < 45 years, 73% for cases > 60 years), and by region (84% in Europe, 51% in North America, 83% in Latin America). Conclusions: Our results confirm that the joint effect between tobacco and alcohol use is greater than multiplicative on head and neck cancer risk. However, a substantial proportion of head and neck cancers cannot be attributed to tobacco or alcohol use, particularly for oral cavity cancer and for head and neck cancer among women and among young-onset cases. (Cancer Epidemiol Biomarkers Prev 2009;18(2):541-50)

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Since the role of human papillomavirus (HPV) infection in oral carcinogenesis is still unclear, the purpose of this study was to verify the association between the expression of p27, mdm2 and cathepsin B and by HPV-related oral lesions. Fifty-five oral biopsies were studied and HPV detection and typing (6/11, 16, 18, 31 and 33) were performed using polymerase chain reaction techniques. The distribution p27, mdm2 and cathepsin B was determined by immunohistochemistry. Twenty-one (38%) out of the 55 oral lesions tested positive for HPV, of which 6(33%) were HPV 6/11, 1 (5%) was HPV 16,14 (72%) were HPV 18 and none was HPV 33/31. Among the 55 biopsies, immunopostivity for p27, mdm2 and cathepsin B was observed in 17 (30.9%), 37 (67.2%) and 37 (67.2%), respectively. Among 21 HPV-positive oral lesions, immunopostivity of mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 benign lesions (BL), 4(22%) out of 18 potential malignant epithelial lesions (PMEL) and 11(57.9%) out of 19 malignant lesions (ML). High-risk HPV types may be associated with oral carcinoma, by cell-cycle control dysregulation, contributing to oral carcinogenesis and the overexpression of mdm2, p27 and cathepsin B. (C) 2009 Elsevier GmbH. All rights reserved.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Oral squamous Cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, all important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also it significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence oil BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC. but not in benign oral lesions.

Laser Phototherapy as Topical Prophylaxis Against Head and Neck Cancer Radiotherapy-Induced Oral Mucositis: Comparison Between Low and High/Low Power Lasers

Background and Objective: Oral mucositis is a dose-limiting and painful side effect of radiotherapy (RT) and/or chemotherapy in cancer patients. The purpose of the present study was to analyze the effect of different protocols of laser phototherapy (LPT) on the grade of mucositis and degree of pain in patients under RT. Patients and Methods: Thirty-nine patients were divided into three groups: G1, where the irradiations were done three times a week using low power laser; G2, where combined high and low power lasers were used three time a week; and G3, where patients received low power laser irradiation once a week. The low power LPT was done using an InGaAlP laser (660 nm/40 mW/6 J cm(-2)/0.24 J per point). In the combined protocol, the high power LPT was done using a GaAlAs laser (808 nm, 1 W/cm(2)). Oral mucositis was assessed at each LPT session in accordance to the oral-mucositis scale of the National Institute of the Cancer-Common Toxicity criteria (NIC-CTC). The patient self-assessed pain was measured by means of the visual analogue scale. Results: All protocols of LPT led to the maintenance of oral mucositis scores in the same levels until the last RT session. Moreover, LPT three times a week also maintained the pain levels. However, the patients submitted to the once a week LPT had significant pain increase; and the association of low/high LPT led to increased healing time. Conclusions: These findings are desired when dealing with oncologic patients under RT avoiding unplanned radiation treatment breaks and additional hospital costs. Lasers Surg.Med. 41:264-270,2009. (C) 2009Wiley-Liss, Inc.

Correlation between c-Jun and human papillomavirus in oral premalignant and malignant lesions

c-Jun, one of the components of the transcription factor activating protein-1 (AP-1), is suggested as a factor in malignant progression of oral lesions. c-Jun and other AP-1 components relationships with human papillomavirus (HPV) infection have been investigated, but not yet focusing on oral carcinogenesis. The aim of this study was to verify whether c-Jun immunohistochemical expression is related to HPV DNA detection in oral premalignant and malignant lesions. Fifty cases diagnosed as oral leukoplakias, with different degrees of epithelial dysplasia, and as oral squamous cell carcinomas (OSCC) were submitted to immunohistochemistry to detect c-Jun and to in situ hybridization with signal amplification to assess HPV DNA. It was verified that c-Jun nuclear expression increased according to the degree of dysplasia within the lesion, with the greatest expression in OSCC. The same did not happen concerning HPV infection - a discrete proportional relation was observed in indexes found in leukoplakia with no dysplasia, leukoplakia with dysplasia and OSCC, but statistically insignificant. When separating the group of leukoplakia by degrees of dysplasia, this relation of proportion was not observed. Nevertheless, the overall prevalence of HPV infection was 24% and the high-risk HPV types were the most frequently identified, which does not allow excluding HPV as a risk factor in oral carcinogenesis. When relating c-Jun expression and HPV infection, no statistically significant relationship is observed. Results suggest then that malignant progression mediated by c-Jun is independent of the presence of HPV in oral carcinogenesis. (C) 2007 Elsevier Ltd. All rights reserved.